阅读说明：本技术 罗汉果苷生物催化方法 (Biological catalysis method of mogroside ) 是由 因德拉·普拉卡什 佶·马 克里斯多佛·梅尔科利亚诺 卡罗尔·哈特利 马修·亚历山大·怀尔丁 于 2020-02-26 设计创作，主要内容包括：可以在特定反应条件下用酶处理含有罗汉果醇糖苷,例如罗汉果苷V的罗汉果提取物,以改变赛门苷I反应产物的分布。经修饰的酶也用于改变赛门苷I的分布,以提高赛门苷I的产率并减少反应污染物。还描述了纯化生物转化反应产物的方法。使用这些方法获得的赛门苷I是用于食物和饮料组合物等的有用的甜味剂和增味剂。(A luo han guo extract containing mogroside, e.g., mogroside V, may be treated with an enzyme under specific reaction conditions to alter the distribution of the siamenoside I reaction product. The modified enzyme is also used to alter the distribution of siamenoside I to increase siamenoside I yield and reduce reaction contaminants. Methods of purifying the bioconversion reaction product are also described. Siamenoside I obtained using these methods is a useful sweetener and flavor enhancer for food and beverage compositions and the like.)

1. A method for producing siamenoside I, the method comprising:

a) combining a solution comprising mogroside V with an effective amount of beta-galactosidase at a suitable pH and a suitable temperature to provide a beta-galactosidase/mogroside V solution;

b) incubating the beta-galactosidase/mogroside V solution for a suitable time to provide a solution comprising siamenoside I, and

c) purifying siamenoside I from the solution comprising siamenoside I,

wherein the siamenoside I has a purity of greater than about 90%.

2. The method according to claim 1, wherein the yield of siamenoside I produced by step b) is greater than 60%.

3. The method according to claim 1, wherein the purity of siamenoside I purified by step c) is greater than 95%.

4. The method of claim 1, wherein the suitable temperature is between about 45 ℃ and about 60 ℃ and the suitable pH is between about 6.1 and about 7.0.

5. The method of claim 1, wherein the β -galactosidase is wild-type aspergillus oryzae β -galactosidase (AoBG), or β -galactosidase has at least 50% identity to aspergillus oryzae β -galactosidase.

6. A modified β -galactosidase comprising one or more mutations in amino acid residues 200 to 212.

7. The modified beta-galactosidase of claim 5, wherein said beta-galactosidase having at least 50% identity to Aspergillus oryzae beta-galactosidase comprises one or more mutations selected from the group consisting of: G204G, C205R, V208E, or a combination thereof.

8. The modified β -galactosidase of claim 5, wherein said β -galactosidase having at least 50% identity to Aspergillus oryzae β -galactosidase comprises one or more mutations in the loop region selected from the group consisting of: E142A, G204G, C205R, and V208E.

9. The modified β -galactosidase of claim 7, wherein said β -galactosidase having at least 50% identity to Aspergillus oryzae β -galactosidase comprises said mutations G204G, C205R and V208E.

10. The modified β -galactosidase of claim 8, wherein said β -galactosidase having at least 50% identity to aspergillus oryzae β -galactosidase comprises said mutations E142A, G204G, C205R, and V208.

11. A process for purifying siamenoside I from a reaction mixture, the process comprising:

a) providing a mixture of low purity mogrosides;

b) dissolving the low-purity mogroside mixture in water or an aqueous alcohol solution to form an initial mogroside solution;

c) mixing the initial solution of mogrosides with an affinity adsorbent to bind mogrosides in the low purity mixture of mogrosides;

d) washing the affinity adsorbent with water to remove enzymes and impurities;

e) eluting the affinity adsorbent with a minimum volume of an organic solvent to obtain a mogroside/solvent solution;

f) distilling the mogroside/solvent solution to obtain a concentrated mogroside aqueous solution;

g) loading the concentrated aqueous mogroside solution onto a C18 resin;

h) eluting the C18 resin with a solvent/water mixture of increasing solvent concentration to produce one or more fractions containing siamenoside I;

i) distilling the one or more fractions containing siamenoside I to obtain a concentrated aqueous siamenoside I solution,

f) drying the concentrated aqueous solution of siamenoside I to obtain high purity siamenoside I, wherein said siamenoside I is greater than about 60% pure.

12. The method of claim 11, wherein the affinity adsorbent is selected from the group consisting of HP20 resin and C18 resin.

13. The method of claim 11, wherein the affinity adsorbent is added at 25x to 30x (w: w) of the mogroside content of the mixture.

14. The method of claim 11, wherein the organic solvent solution is selected from acetone, acetonitrile, ethanol, or methanol.

15. The method of claim 11, wherein step f) is preceded by a second cycle of steps c) -e).

16. The method of claim 11, wherein the aqueous mogroside solution is further concentrated and the unsaturated portion of the concentrated distillation product is loaded onto an affinity resin column.

17. The method of claim 11, wherein the organic solvent comprises between about 30-40% ethanol or between about 30-40% methanol.

18. The method of claim 11, wherein the organic solvent comprises between about 30-40% methanol and produces siamenoside I with a purity > 95%.

19. The method of claim 11, wherein the organic solvent comprises between about 50-100% methanol and produces mogroside IIIE with a purity > 95%.

20. A process for purifying siamenoside I from a reaction mixture, the process comprising:

a) providing a mixture of low purity mogroside and a reactive mixing reagent;

b) separating the mogroside from the reaction mix reagent by: (i) adjusting the pH of the mixture of a) to about 10 or higher, (ii) adding an alcohol to provide an alcohol solution and (iii) filtering the alcohol solution through a first ultrafiltration membrane to provide a first filtered solution;

c) adjusting the pH of the first filtered solution to between about 5 and about 7 and filtering through a second ultrafiltration membrane to provide a second filtered solution;

d) diafiltering the second filtered solution to concentrate the mogroside to provide a mogroside mixture, and then mixing the mogroside mixture with water/ammonia acetate to provide a mogroside/ammonium acetate solution;

e) contacting the mogroside/ammonia acetate solution with a fractionation column;

f) eluting and collecting fractions containing siamenoside I; and

g) drying the fractions containing siamenoside I to obtain high purity siamenoside I with a siamenoside content higher than about 60% (w/w).

21. The method of claim 20, wherein the mogroside mixture of step a) comprises at least 90% mogroside V.

22. The method of claim 20, wherein the mogroside mixture of step a) comprises at least 95% mogroside V.

23. A method for preparing siamenoside I, the method comprising:

a) combining a solution comprising mogroside V with an effective amount of beta-galactosidase at a suitable pH and a suitable temperature to provide a beta-galactosidase/mogroside V solution;

b) incubating the beta-galactosidase/mogroside V solution for a suitable time to provide a solution comprising siamenoside I;

c) mixing the solution comprising siamenoside I with HP20 resin;

d) washing the HP20 resin with water; and

e) eluting the HP20 resin with a minimal volume of an organic solvent to obtain a mogroside/solvent solution, thereby providing siamenoside I having a purity of at least about 90%.

FIELD

The present disclosure relates to methods useful for producing siamenoside I from luo han guo extracts. More particularly, the present disclosure relates to methods useful for producing high purity siamenoside I from mogroside V by biotransformation and purification, and enzymes used therein. Sweetener compositions comprising high purity siamenoside I are also disclosed, as well as foods and beverages containing these sweetener compositions.

Background

Lo Han Guo extract obtained from Lo Han Guo (Siraitia grosvenori) (Cucurbitaceae) is commercially used as a natural sweetener. However, Lo Han Guo extract may have taste characteristics that prevent it from being used as a replacement for caloric sweeteners (e.g., sugar) in food and beverage compositions. For example, the extract may have some off-taste or lingering aftertaste, or require a longer time to develop sweetness after consumption than expected (i.e., delayed sweetness onset).

There remains a need for sweeteners having improved taste characteristics, having low or no calories, and having reduced caloric content, as well as foods and beverages containing these sweeteners.

SUMMARY

In one aspect, a method for producing siamenoside I is disclosed, the method comprising:

a) combining a solution comprising mogroside V with an effective amount of beta-galactosidase at a suitable pH and a suitable temperature to provide a beta-galactosidase/mogroside V solution;

b) incubating the beta-galactosidase/mogroside V solution for a suitable time to provide a solution comprising siamenoside I; and

c) purifying siamenoside I from a solution comprising siamenoside I,

wherein the siamenoside I has a purity of greater than about 90%.

In one embodiment, the yield of siamenoside I produced by step b) is greater than 60%.

In one embodiment, the purity of siamenoside I purified by step c) is greater than 97%.

In one embodiment, the purity of siamenoside I purified by step c) is greater than 99%.

In one embodiment, a suitable temperature is between about 45 ℃ and about 60 ℃, and a suitable pH is between about 6.1 and about 7.0.

In one embodiment, a suitable temperature is between about 50 ℃ and about 60 ℃, and a suitable pH is between about 6.1 and about 7.0.

In one embodiment, a suitable temperature is between about 50 ℃ and about 55 ℃, and a suitable pH is between about 6.1 and about 7.0.

In one embodiment, a suitable temperature is between about 50 ℃ and about 55 ℃, and a suitable pH is between 6.3 and 7.0.

In one embodiment, a suitable temperature is between about 50 ℃ and about 55 ℃, and a suitable pH is between about 6.5 and about 7.0.

In one embodiment, a suitable temperature is between about 50 ℃ and about 55 ℃, and a suitable pH is between about 6.8 and about 7.0.

In one embodiment, the incubating step b) results in a yield of siamenoside I of greater than 60%. In certain embodiments, the yield of siamenoside I is greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, or greater than about 95%.

In one embodiment, the purification step c) results in siamenoside I with a purity of more than 90%. In certain embodiments, the purity of siamenoside I is greater than about 90%, greater than about 95%, or greater than about 99%.

In one embodiment, the methods disclosed herein increase the purity of the produced siamenoside I compared to conventional methods. In certain embodiments, the purity is increased by about 10%, about 20%, about 30%, about 40%, or about 50% or more compared to the purity of siamenoside I produced by conventional methods.

In one embodiment, the method produces siamenoside I having a purity between about 60% and about 90% purity.

In one embodiment, the method produces siamenoside I having a purity between about 60% and about 95% purity.

In one embodiment, the method produces siamenoside I with a purity between about 65% and about 90% purity.

In one embodiment, the method produces siamenoside I with a purity between about 65% and about 95% purity.

In one embodiment, the method produces siamenoside I with a purity between about 70% and about 90% purity.

In one embodiment, the method produces siamenoside I with a purity between about 70% and about 95% purity.

In one embodiment, the method produces siamenoside I having a purity between about 75% and about 90% purity.

In one embodiment, the method produces siamenoside I having a purity between about 75% and about 95% purity.

In one embodiment, the beta-galactosidase is wild-type aspergillus oryzae beta-galactosidase (AoBG) or a variant thereof.

In particular embodiments, the β -galactosidase is a variant of wild-type AoBG that is at least 50% identical to the AoBG, e.g., at least 60% identical, at least 70% identical, at least 80% identical, or at least 90% identical to the wild-type AoGB.

In a particular embodiment, the beta-galactosidase comprises the amino acid sequence of SEQ id No. 1 or a variant thereof. In one embodiment, the variant has at least 50% identity, at least 60% identity, at least 70% identity, at least 80% identity, or at least 90% identity to SEQ ID No. 1.

In another specific embodiment, the beta-galactosidase comprises the amino acid sequence of SEQ id No. 2 or a variant thereof. In one embodiment, the variant has at least 50% identity, at least 60% identity, at least 70% identity, at least 80% identity, or at least 90% identity to SEQ ID No. 2.

In one embodiment, the method increases the conversion of mogroside V to siamenoside I as compared to conventional methods. In certain embodiments, the conversion is increased by about 10%, about 20%, about 30%, about 40%, or about 50% or more as compared to the conversion of siamenoside I produced by conventional methods.

In a second aspect, modified β -galactosidases are disclosed comprising one or more mutations in the catalytic site or loop region.

In one embodiment, the modified β -galactosidase has at least 50% identity, at least 60% identity, at least 70% identity, at least 80% identity or at least 90% identity to SEQ ID No. 1.

In another embodiment, the modified β -galactosidase has at least 50% identity, at least 60% identity, at least 70% identity, at least 80% identity or at least 90% identity to SEQ ID No. 2.

In one embodiment, the one or more mutations comprises at least one substitution of an amino acid residue corresponding to any one of amino acids 142, 204, 205, or 208 of SEQ ID No. 1.

In one embodiment, the one or more mutations in the catalytic site are selected from E142, D219, E200, D258, E298, or E804, and are mutated to alanine (a) or glutamine (Q).

In one embodiment, the one or more mutations in the loop region are selected from N160, G165, C166, V169, S201, D219, E259, Y303, H316, Y323, a141, N199, G204, C205, V208, S240, D258, E298, Y342, H355, Y362, or E804, and are mutated to alanine (a) or glutamine (Q).

In one embodiment, the one or more mutations are selected from D258E, E804A, E142Q, E142A, E200A, D258A, D258Q, E200A/E298A, E200Q/E298Q, E298A, E298Q, or D258A/E298A.

In one embodiment, the one or more mutations are selected from E803A, E142Q, E142A, E298A, W298Q, or D258A/E298A.

In one embodiment, the one or more mutations have the effect of increasing the conversion rate and/or specificity of the conversion from mogroside V to siamenoside I.

In certain embodiments, one or more mutations have the effect of increasing the conversion by at least about 5%, at least about 10%, at least about 15%, at least about 20%, or at least about 25% or more.

In certain embodiments, the one or more mutations have the effect of increasing the specificity of transformation (siamenoside I yield) by at least about 5%, at least about 10%, at least about 15%, at least about 20%, or at least about 25% or more.

In one embodiment, the mutation changes the distribution to an increase in siamenoside I production.

In certain embodiments, the profile is altered to increase siamenoside I by at least about 5%, at least about 10%, at least about 15%, at least about 20%, or at least about 25% or more.

In a third aspect, a process for purifying siamenoside I from a reaction mixture is disclosed, the process comprising:

a) providing a mixture of low purity mogrosides;

b) dissolving the low purity mogroside mixture in water or an aqueous alcohol solution to form an initial mogroside solution;

c) mixing the initial solution of mogrosides with an affinity adsorbent to bind mogrosides in the low purity mixture of mogrosides;

d) washing the affinity adsorbent with water to remove enzymes and impurities;

e) eluting the affinity adsorbent with a minimum volume of an organic solvent to obtain a mogroside/solvent solution;

f) distilling the mogroside/solvent solution to obtain a concentrated mogroside aqueous solution;

g) loading the concentrated aqueous mogroside solution onto a C18 resin;

h) eluting the C18 resin with a solvent/water mixture of increasing solvent concentration to produce one or more fractions containing siamenoside I;

i) distilling the one or more fractions containing siamenoside I to obtain a concentrated aqueous siamenoside I solution,

j) drying the concentrated aqueous solution of siamenoside I to obtain high purity siamenoside I, wherein the siamenoside I is greater than about 60% pure.

In one embodiment, the mogroside mixture of step a) comprises at least 85% mogroside V.

In one embodiment, the low purity mogroside mixture of step a) comprises at least 90% mogroside V.

In one embodiment, the low purity mogroside mixture of step a) comprises at least 95% mogroside V.

In one embodiment, the affinity adsorbent is HP20 resin.

In one embodiment, the affinity adsorbent is a C18 resin.

In one embodiment, the affinity adsorbent/solvent mixture is combined and the solvent is removed by distillation.

In one embodiment, the affinity adsorbent is added at 25x to 30x (w: w) of the mogroside content of the mixture.

In one embodiment, the organic solvent is a 100% organic solvent selected from the group consisting of: acetone, acetonitrile, ethanol, or methanol.

In one embodiment, the organic solvent is 100% methanol.

In one embodiment, the organic solvent is 100% ethanol.

In one embodiment, another cycle of steps c) -e) is performed before step f).

In another embodiment, the organic solvent is an aqueous alcohol solution comprising water and an alcohol selected from the group consisting of: methanol, ethanol, n-propanol, 2-propanol, 1-butanol, and 2-butanol.

In one embodiment, the minimum volume of organic solvent is about 2 vol/wt of resin.

In one embodiment, the distillation occurs at a temperature between about 45 ℃ and about 50 ℃ to provide the aqueous mogroside solution.

In one embodiment, the affinity resin column is a C18 resin (Chromatorex SMB 150, 20-45 μm) column.

In one embodiment, the organic solvent comprises between about 30% -40% ethanol.

In one embodiment, the organic solvent comprises between about 30% -40% methanol.

In one embodiment, the organic solvent comprises between about 30-40% methanol and produces siamenoside I with a purity > 95%.

In one embodiment, the organic solvent comprises between about 50% and 100% methanol.

In one embodiment, the organic solvent comprises between about 50% -100% methanol and produces mogroside IIIE with a purity > 95%.

In another embodiment of the process, the adsorbent is a macroporous polymeric adsorbent resin capable of adsorbing mogroside.

In a fourth aspect, a process for purifying siamenoside I from a reaction mixture is disclosed, the process comprising:

a) providing a mixture of low purity mogroside and a reactive mixing reagent;

b) separating mogroside from the reaction mix by: (i) adjusting the pH of the low purity mogroside mixture to about 10 or greater, (ii) adding an alcohol to provide an alcohol solution and (iii) filtering the alcohol solution through a first ultrafiltration membrane to provide a first filtered solution;

c) adjusting the pH of the first filtered solution to between about 5 and about 7 and filtering through a second ultrafiltration membrane to provide a second filtered solution;

d) diafiltering the second filtered solution to concentrate the mogroside to provide a mogroside mixture, and then mixing the mogroside mixture with water/ammonia acetate to provide a mogroside/ammonium acetate solution;

e) contacting the mogroside/ammonia acetate solution with a fractionating column;

f) eluting and collecting fractions containing siamenoside I; and

g) the fractions containing siamenoside I are dried to obtain high purity siamenoside I with a siamenoside I content higher than about 60% (w/w).

In one embodiment, the reactive mixing reagent is an enzyme and a salt.

In one embodiment, the ultrafiltration membrane is a 10kDa nominal filtration membrane. In one embodiment, the ultrafiltration membrane is a 10kDa nominal filtration membrane.

In one embodiment, the fractionation column is a C18 resin column.

In one embodiment, the mogroside mixture of step a) comprises at least 85% mogroside V.

In one embodiment, the mogroside mixture of step a) comprises at least 90% mogroside V.

In one embodiment, the mogroside mixture of step a) comprises at least 95% mogroside V.

In a fifth aspect, sweetener mixtures are disclosed comprising high purity siamenoside I; wherein the high purity siamenoside I is blended with another sweetener.

Drawings

FIG. 1 provides a general schematic showing the pathway by which mogroside V is converted to mogroside IIIe by mogroside IV or siamenoside I.

FIG. 2 provides an HPLC plot showing negative control (FIG. 2A) samples and AoBG reaction expressed in Pichia pastoris (96 hour induction)

Mogroside concentration in samples reacted for 48 hours (FIG. 2B).

Figures 3A-3C provide a series of graphs showing mogroside production at different temperatures and pH.

Fig. 4 provides a table showing the gBlock gene fragments and restriction endonuclease sites for target mutations for cloning into the β -galactosidase sequence (UniProt B7VU 80).

FIG. 5 provides a diagram showing a vector map for transforming target cells and plasmids expressing mutant enzymes.

FIG. 6 provides a schematic showing a general production scheme for a scale-up bioconversion process.

FIG. 7 provides a graph showing the response spectrum of the 5kg amplification reaction.

FIG. 8 provides a flow chart showing the hydrolysis of mogroside V to siamenoside I: a downstream procedure for generating a semi-purified crude reaction mixture from the crude reaction mixture and secondary purifying the semi-purified crude mixture into a highly pure siamenoside I component (by Chromorex SMB C18, distillation and freeze-drying).

Figure 9 provides a graph showing HPLC analysis of pure fractions. Fig. 9a. general view of HPLC traces showing purity over 98% by peak area. Fig. 9b close-up view of small amount of impurities.

Figure 10 provides a process flow diagram for post-reaction purification.

FIG. 11 provides a diagram showing the general flow of a scale-up bioconversion process.

Figure 12 provides a process flow diagram for chromatography.

FIG. 13 provides a process flow diagram for diafiltration and finishing.

FIG. 14 shows the domains of AoBG.

Detailed Description

Methods and compositions are provided for increasing the yield and mogroside distribution from mogroside V catalyzed siamenoside I. In certain embodiments, the method is a biocatalytic method utilizing an engineered enzyme. In certain embodiments, the methods relate to process control, such as temperature and pH.

There are certain methods to catalyze the conversion of mogroside V to the reaction products mogroside IV, siamenoside I, and mogroside III, but conventional processes result in low yields of siamenoside I and increased contaminating mogroside reaction products. The present methods described herein provide improvements in increased production yields and/or reduced contaminating mogrosides. The purification process provides siamenoside I useful for sweetener applications in beverages and foodstuffs, etc.

The reaction of mogroside V to mogroside III can proceed via 2 pathway pathways, each involving 2 subreactions in the pathway. Since each substrate or intermediate involves a different chemistry/binding, process conditions are used to alter the rate and specificity of the enzyme to perform each of these sub-reactions. The general pathway for producing mogroside III from mogroside V is shown in figure 1.

Definition of

As used herein, the term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are subsequently modified, e.g., hydroxyproline, γ -carboxyglutamic acid, and O-phosphoserine.

As used herein, the term "amino acid analog" refers to a compound that has the same basic chemical structure (i.e., carbon, carboxyl group, amino group, and R group bound to a hydrogen, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium) as a naturally occurring amino acid. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.

As used herein, the term "amino acid difference" or "residue difference" refers to a difference in amino acid residues at a position in a polypeptide sequence relative to the amino acid residues at the corresponding position in a reference sequence. The difference may be, for example, a conservative substitution, a non-conservative substitution, a deletion, or an insertion.

As used herein, the term "amino acid mimetic" is a chemical compound that differs in structure from the general chemical structure of an amino acid, but functions in a manner similar to a naturally occurring amino acid.

As used herein, the term "β -galactosidase" or "β -gal" refers to a glycoside hydrolase that catalyzes the hydrolysis of β -galactosides to monosaccharides by the cleavage of glycosidic bonds.

As used herein, the term "β -galactosidase variant" comprises an amino acid sequence derived from the amino acid sequence of a "precursor β -galactosidase". The precursor β -galactosidase can include naturally occurring β -galactosidase and recombinant β -galactosidase. The amino acid sequence of the variant β -galactosidase can be derived from the amino acid sequence of the precursor β -galactosidase by substitution, deletion or insertion of one or more amino acids of the amino acid sequence of the precursor β -galactosidase.

As used herein, the term "biocatalysis" refers to a chemical process in which an enzyme or other biocatalyst performs a reaction between organic components.

As used herein, the term "bioconversion" refers to the process of converting a substrate into a product within a living organism (e.g., bacteria, fungi), which includes any modification of the chemical and/or biological properties and/or characteristics of the substrate that occurs within the living organism and results in the production of the product. Providing a single or multiple precursor molecules to a living system and allowing a period of metabolism followed by isolation of one or multiple products consisting of single or minor enzymatic modifications of one or multiple precursor molecules from the culture medium. In alternative embodiments, bioconversion may refer to the process of converting a substrate to a product by an isolated enzyme.

As used herein, the term "conservative amino acid substitution" means that one amino acid is substituted with another amino acid having a side chain with similar properties. Amino acid residues are classified into several families according to their side chains, such as basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), B-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, and histidine). Preferably, a conservative amino acid substitution is a substitution between amino acid residues in a family.

As used herein, the term "conventional method" with respect to the method of purifying siamenoside I includes the methods described in: for example, WO 2014140634A 1 and Chiu, Chun-Hui, et al, "Biotransformation of Mogriides from Siraitia grosvenorii Swingle by Saccharomyces cerevisiae ] [ Biotransformation of mogrosides in Lo Han Guo Swingle ]" Journal of Agricultural and Food Chemistry 61.29(2013):7127 and 7134.

As used herein, the term "conversion" or "bioconversion" refers to the enzymatic conversion (or biotransformation) of one or more substrates to the corresponding one or more products. "percent conversion" refers to the percentage of substrate that is converted to product under specified conditions over a period of time. Thus, the "enzymatic activity" or "activity" of a given polypeptide can be expressed as the "percent conversion" of substrate to product over a particular period of time.

As used herein, the term "engineered" with respect to a subject polypeptide/enzyme means that the subject has been modified from its native state. The engineered polypeptide/enzyme may differ from the native sequence by one or more amino acids, and/or be fused to a heterologous sequence. The term "engineered" is used interchangeably herein with the term "recombinant".

As used herein, the term "enzyme" refers to any substance that catalyzes or facilitates one or more chemical or biochemical reactions, which generally includes enzymes composed in whole or in part of polypeptides, but may also include enzymes composed of different molecules, including polynucleotides.

As used herein, the term "fragment" in reference to a polypeptide refers to a full-length polypeptide or a shorter portion of a protein, ranging in size from two amino acid residues to the entire amino acid sequence minus one amino acid residue. In certain embodiments of the disclosure, a fragment refers to the entire amino acid sequence of a domain (e.g., a substrate binding domain or a catalytic domain) of a polypeptide or protein.

As used herein, the term "fusion protein" refers to a protein that is genetically engineered from two or more protein/peptide coding sequences joined together as a single polypeptide. The fusion protein may include a linker (or "spacer") sequence that may facilitate proper folding and activity of each domain of the fusion protein. The fusion protein may also include epitope tags for identification (e.g., in western blots, immunofluorescence, etc.) and/or purification. Non-limiting examples of epitope tags currently in use include: HA. myc, FLAG, and 6-HIS.

The term "identity", as used herein, refers to the sequence identity of subunits between two polymer molecules, particularly between two amino acid molecules (e.g., between two polypeptide molecules). When two amino acid sequences have identical residues at the same position; for example, if a position in each of two polypeptide molecules is occupied by arginine, then they are identical at that position. The identity or degree to which two amino acid sequences have identical residues at the same position in an alignment is typically expressed as a percentage.

As used herein, the terms "improve," "increase," or "enhance" interchangeably refer to a detectable positive change in the amount of a parameter when compared to a standard. Parameters may vary and include, for example, improved production from or expression in a host cell, improved thermostability or an altered temperature-dependent activity profile, improved activity or stability at a desired pH or pH range, improved substrate specificity, improved product specificity, and improved stability with respect to the polypeptide. The degree of improvement may vary. When expressed as a percentage, the improvement can be, for example, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% or more.

As used herein, the term "increased enzymatic activity" or "enhanced catalytic activity" refers to an improved property of an engineered enzyme disclosed herein, which can be expressed in terms of an increase in a particular activity (e.g., product produced/time/weight protein) or an increase in the percent conversion of substrate to product (e.g., percent conversion of an initial amount of substrate to product over a particular time using a particular amount of the engineered enzyme as compared to a reference enzyme).

As used herein, the term "mogroside" refers to a glycoside in which glucose is linked to the aglycon mogrol. Mogrosides are classified into various types according to the attachment position of glucose or the number of glucose units. Mogroside V, mogroside IV, siamenoside I, mogroside IIIE, and 11-oxo-mogrosides such as 11-oxo-mogroside V, 11-siamenoside I are contained in the fruit of Momordica grosvenori. Other mogrosides are also known. The mogrosides are novel in cucurbitane triterpenes in that they are oxygenated regiospecifically at four of C3, C11, C24 and C25 to form tetrahydroxylated cucurbitane mogrol.

As used herein, the term "Luo Han Guo" or "Luo Han Guo" (luohanguo) refers to the fruit of Luo Han Guo, a member of the cucurbitaceae family. The main bioactive component in the fruit extract is cucurbitane-type triterpene saponin, which is called mogroside. It is estimated that the sweetness of the mixed mogrosides is about 200-fold 300-fold that of sucrose.

As used herein, the term "mutant" or "variant" or "derivative" with respect to a protein refers to a protein having one or more residue differences, wherein the activity is preferably increased compared to the wild type due to the mutation. Mutations include substitutions, additions, insertions, deletions, truncations, transversions and/or inversions at one or more positions relative to a reference sequence. The sequence of the mutant protein may comprise a sequence of a protein having at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% homology to the sequence of the wild type.

The terms "polypeptide", "protein" and "amino acid sequence" are used interchangeably herein and include a molecular chain of amino acids linked by peptide bonds. These terms do not refer to a specific length of the product. Thus, "peptide", "oligopeptide" and "protein" are included within the definition of polypeptide.

As used herein, the term "spectrum" or "distribution" refers to the chemical composition of the reaction products produced by enzymatic hydrolysis.

As used herein, the term "purified" with respect to siamenoside I means that the purity of the compound has been increased such that it is present in a form that is more pure than it is present in its natural environment and/or in the extract. Purity is a relative term and does not necessarily imply absolute purity.

As used herein, the term "reaction conditions" refers to the environmental conditions under which the reaction is carried out, such as temperature, pressure, catalyst, and solvent.

As used herein, the term "substrate" refers to a substance (e.g., a chemical compound) on which an enzyme performs an enzymatic activity to produce a product.

As used herein, the term "substrate specificity" refers to an enzyme that exhibits a higher specificity for one substrate than for a competing substrate. Substrate specificity may be the specificity constant (k)_cat/K_m) Is measured. Such ratios can be used to (i) compare specificity or two or more enzymes (e.g., wild-type enzyme versus mutant enzyme) against the same substrate, or (ii) compare a given enzyme against two or more substrates.

The term "suitable" as used herein with respect to a reaction refers to those conditions under which the engineered enzymes disclosed herein are capable of converting a substrate to a desired product compound.

As used herein, "temperature" refers to a physical property that represents heat or cold, typically by means of a thermometer calibrated at one or more temperature scales. The most common scales are the celsius scale (formerly known as celsius) (expressed as ° c), the fahrenheit scale (expressed as ° F), and the kelvin scale (expressed as K).

As used herein, the terms "wild-type" and "naturally occurring" refer to the form found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence that is present in an organism, which can be isolated from a source in nature and has not been intentionally modified by man.

As used herein, the term "yield" refers to the final product or the amount of desired final product obtained using the methods disclosed herein. In some embodiments, the yield is greater than that obtained using methods known in the art. In some embodiments, the term refers to the volume of the final product, while in other embodiments, the term refers to the concentration of the final product.

I. Improved production method of siamenoside I

Disclosed herein are improved methods (e.g., biocatalytic methods) for the production of siamenoside I.

In certain embodiments, the disclosed methods involve modifying one or more reaction conditions, including, for example, reaction temperature, reaction pH, and/or reaction duration, within ranges known in the art.

Siamenoside I is an intermediate in the mogroside V biotransformation pathway. Mogroside V biotransformation can occur by this enzyme via two pathways (Mog V to Sia I to MogIIIE) or (Mog V to Mog IVa to Mog IIIE). In certain embodiments, the methods disclosed herein alter the distribution of siamenoside I and the selectivity of the reaction to increase the yield of siamenoside I.

Increasing siamenoside I can be achieved by: 1) altering the specificity of the enzyme to preferentially switch mogroside V to the pathway with siamenoside I intermediate compared to the pathway with mogroside IV intermediate, and 2) decreasing the conversion of siamenoside I to mogroside IIIE in the second half of the overall bioconversion pathway.

Most of the mutations identified in the G204, C205, V208 libraries showed a weak ability to convert siamenoside I to mogroside IIIE.

Conventionally, the reaction of the enzyme with the fruit extract is carried out at a temperature of from about 20 ℃ to about 80 ℃, at a pH of from about 3 to about 10, and for from about 1 hour to about 96 hours.

In one embodiment, the process of the present invention involves a bioconversion reaction that includes (i) a temperature between about 40 ℃ and 70 ℃, a pH between about 6.0 and about 7.0. When the process is carried out under these reaction conditions, it alters the conversion of mogroside V to siamenoside I to provide a yield of siamenoside I of between about 50% and about 99%, thereby reducing additional contaminating mogroside products in the final reaction mixture.

As will be explained in more detail in the examples, contrary to the previous teachings in the art, the thus obtained siamenoside I profile or distribution can be increased by selecting specific reaction conditions within the ranges described herein.

In one aspect, the present invention provides methods for increasing the conversion and production profile of siamenoside I, comprising:

a) combining the solution of mogroside V with an effective amount of beta-galactosidase in a reaction mixture under suitable pH and temperature conditions to provide a beta-galactosidase/mogroside V solution,

b) incubating the beta-galactosidase/mogroside V solution for a suitable time to provide a solution comprising siamenoside I, an

c) Purifying siamenoside I from a solution comprising siamenoside I,

wherein the purity of the siamenoside I is greater than 90%.

In one embodiment, the yield of siamenoside I from step b) is greater than 60%.

In one embodiment, siamenoside I is greater than 95% pure.

The effective amount of beta-galactosidase can vary. Generally, enzyme concentrations from about 1 to about 100mg/mL will be suitable. In one embodiment, the enzyme concentration is from about 10 to about 90, about 20 to about 80, about 30 to about 70, about 40 to about 60, or about 50 mg/mL. In another embodiment, the enzyme concentration is about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100mg/mL or more.

The total concentration of solids (e.g., mogrol glycosides) in the liquid medium contacted with the one or more enzymes can be, for example, from about 10% by weight to about 50% by weight.

The duration of the reaction may vary. In one embodiment, the reaction run duration is about 6 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, 12 days, 13 days, 14 days, 15 days, 20 days, 25 days, 30 days, or up to 60 days.

Suitable temperatures may vary. In one embodiment, suitable temperatures are about 50 ℃, about 51 ℃, about 52 ℃, about 53 ℃, about 54 ℃, about 55 ℃, about 56 ℃, about 57 ℃, about 58 ℃, about 59 ℃, or about 60 ℃.

In another embodiment, a suitable temperature is between about 45 ℃ and about 65 ℃, between about 50 ℃ and about 60 ℃, between about 51 ℃ and about 59 ℃, between about 52 ℃ and about 58 ℃, between about 53 ℃ and about 57 ℃, or between about 54 ℃ and about 56 ℃.

Suitable pH may vary. In one embodiment, a suitable pH is about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0 or higher.

In another embodiment, a suitable pH is between about 6.0 and about 6.5, between about 6.2 and about 6.5, between about 6.3 and about 6.5, between about 6.4 and about 6.5, between about 6.5 and about 7.0, between about 6.6 and about 7.0, between about 6.7 and about 7.0, between about 6.8 and about 7.0, between about 6.9 and about 7.0, between about 6.2 and about 6.8, between about 6.3 and about 6.7, or between about 6.4 and about pH 6.6.

In various embodiments, the distribution of siamenoside I in the final reaction mixture is about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%.

In various embodiments, the distribution of siamenoside I in the final reaction mixture is between about 40% and about 99%, between about 45% and about 99%, between about 50% and about 99%, between about 55% and about 99%, between about 60% and about 99%, between about 65% and about 99%, between about 70% and about 99%, between about 75% and about 99%, between about 80% and about 99%, between about 85% and about 99%, between about 90% and about 99%, or between about 95% and about 99%.

In another embodiment, the distribution of siamenoside I in the final reaction mixture is about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%.

In another embodiment, the distribution of siamenoside I in the final reaction mixture is between about 60% to about 90%, about 65% to about 85%, or about 70% to about 80%.

In another embodiment, the distribution of siamenoside I in the final reaction mixture is between about 60% and about 70%.

In another embodiment, the distribution of siamenoside I in the final reaction mixture is between about 40% and about 75%.

In another embodiment, the distribution of siamenoside I in the final reaction mixture is greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, or greater than about 90%.

In one embodiment, a suitable temperature is between about 45 ℃ and about 60 ℃, and a suitable pH is between about 6.3 and about 7.0.

In one embodiment, a suitable temperature is between about 50 ℃ and about 60 ℃, and a suitable pH is between about 6.3 and about 7.0.

In one embodiment, a suitable temperature is between about 50 ℃ and about 55 ℃, and a suitable pH is between about 6.3 and about 7.0.

The reaction mixture may further comprise one or more additional components. In one embodiment, the reaction mixture includes glycerol. In another embodiment, the reaction mixture comprises a monovalent or divalent cation.

In one embodiment, when the components are added to the reaction mixture, they may be in any order.

In one embodiment, the method results in a change in the reaction profile for siamenoside I production, which reduces the amount of contaminating mogroside compounds and increases the purity of siamenoside I produced.

In certain embodiments, the amount of contaminating mogroside component is reduced by an amount greater than about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, or more.

In certain embodiments, the purity of siamenoside I is increased by an amount greater than about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, or more.

In one embodiment, the amount of contaminating mogroside component is reduced by an amount greater than about 10%, and the purity of siamenoside I is increased by an amount greater than about 10%.

In one embodiment, the amount of contaminating mogroside component is reduced by an amount greater than about 20%, and the purity of siamenoside I is increased by an amount greater than about 20%.

In one embodiment, the amount of contaminating mogroside component is reduced by an amount greater than about 40%, and the purity of siamenoside I is increased by an amount greater than about 40%.

In one embodiment, the method produces a reaction product having greater than about 60% siamenoside I.

In one embodiment, the method produces a reaction product having between about 60% and about 99% siamenoside I.

In one embodiment, the method produces a reaction product having between about 70% and about 95% siamenoside I.

In one embodiment, the method produces a reaction product having between about 75% and about 90% siamenoside I.

In another embodiment, the method produces a reaction product having about 60%, about 63%, about 65%, about 68%, about 70%, about 73%, about 75%, about 78%, about 80%, about 83%, about 85%, about 88%, about 90%, about 93%, about 95%, or about 100% siamenoside I.

In one embodiment, the method produces a reaction product containing less than 10% mogroside IV.

In certain embodiments, the percent conversion is increased, for example, by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% or more.

In particular embodiments, the percent conversion is between about 55% and about 99%, more particularly, about 60% to about 99%, about 65% to about 99%, about 70% to about 99%, about 75% to about 99%, about 80% to about 99%, about 85% to about 99%, or about 90% to about 99%.

In another particular embodiment, the percent conversion is about 60%, about 63%, about 65%, about 68%, about 70%, about 73%, about 75%, about 78%, about 80%, about 83%, about 85%, about 88%, about 90%, about 93%, about 95%, or about 100%.

In certain embodiments, the transformation specificity is increased, e.g., by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% or more.

In one embodiment of the present invention, a mogroside composition is disclosed comprising siamenoside I and at least one mogrol glycoside selected from the group consisting of mogroside V, mogroside IV and mogroside IIIE, wherein siamenoside I comprises from about 60% to about 85% by weight of the total amount of (mogroside V + mogroside IV and mogroside IIIE), mogroside V comprises from 0 to about 40% by weight of the total amount of (mogroside V + siamenoside I + mogroside IV and mogroside III), mogroside IV comprises from 0 to about 20% by weight of the total amount of (mogroside V + siamenoside I + mogroside IV and mogroside HIE), and mogroside IIIE comprises from 0 to about 40% by weight of the total amount of (mogroside V + siamenoside I + mogroside IV and mogroside HIE), and wherein mogroside IV (if present) is present in an amount no greater than the total amount of siamenoside I.

The reaction may be stopped when the desired mogrol glycoside profile is obtained (e.g., by isolating or inactivating the enzyme from the fruit extract).

Enzyme engineering

Siamenoside I production is catalyzed by the β -galactosidase hydrolysis of a non-reducing terminal β -D-galactose to catalyze the conversion of non-reducing galactose to other compounds. The production of siamenoside I can be catalyzed by beta-galactosidase hydrolysis. Beta-galactosidase is characterized in that it is capable of hydrolyzing lactose by hydrolyzing a non-reducing terminal beta-D-galactose from glucose. However, these enzymes have been shown to hydrolyze other glycosidic linkages. In this case, siamenoside I production is catalyzed by hydrolysis of the non-reducing terminal β -D-glucose of mogroside V.

Beta-galactosidase and beta-glucosidase belong to the enzyme family EC3.2.1.21 and can be divided into several groups and classes. To date, all the β -galactosidase/β -glucosidase enzymes reported to hydrolyze mogroside V belong to one of two different classes of glycosyl hydrolases, GH-a groups: class 2 (including β -galactosidase enzymes from escherichia coli, arthrobacter spp and those from Kluyveromyces fungal species) or class 35 (including most other eukaryotic β -galactosidase enzymes). Class 2 β -galactosidase enzymes have a tetrameric quaternary structure, whereas class 35 enzymes have a monomeric structure, but the substrate range varies widely among the different classes. For example, even if they all appear in class 2, E.coli β -galactosidase cannot hydrolyze mogroside V, whereas enzymes from Kluyveromyces lactis (Kluyveromyces lactis) can hydrolyze. (Pereira-Rodri i guez)Fern-ndez-Leiro R, Gonz lez-Siso MI, Cerd n ME, Becerra M, et al (2012) Structural basisof specific in molecular Kluyveromyces lactis beta-galactosidase [ tetrameric Kluyveromyces lactis beta-galactosidase-specific structural basis]Journal of Structural Biology]177:392-401；Pereira-RodríguezFern-ndez-Leiro R, Gonz-lez Siso MI, Cerd n ME, Becerra M, et al (2010) Crystallization and preseniliny X-ray Crystallization of beta-galactosidase from Kluyveromyces lactis [ Crystallization of beta-galactosidase from Kluyveromyces lactis ] and preliminary X-ray crystallography]Acta Crystallographica Section F Structural Biology and Crystallization Communications [ Section F: structural biology in communication with crystallization]66:297-300). Beta-galactosidase is widely found in mammalian organs, plant seeds, bacteria, fungi and yeast.

In the food industry, β -galactosidase enzymes from yeasts such as Kluyveromyces lactis and Kluyveromyces fragilis (Kluyveromyces fragilis), fungi such as Aspergillus niger and Aspergillus oryzae, and bacteria such as Bacillus circulans have been used. Among them, β -galactosidase derived from Bacillus circulans ATCC 31382 is commercially available under the trade name Biolacta (Daiwa Kasei, U.S. Pat. No. 4,237,230 (1980)). Wild-type beta-galactosidases include G5160 beta-galactosidases from aspergillus oryzae (sold by Sigma), E0025 beta-glucosidase from Clostridium thermocellum (sold by Prozomix), E0110 beta-glucosidase from rhizobium phaseolorum (sold by Prozomix), and E0105 beta-glucosidase from Bacteroides fragilis (sold by Prozomix).

Cloning, nucleotide sequencing and expression of the b-galactosidase coding gene (lacA) from Aspergillus oryzae has been reported. Ito et al, J.Gen.appl.Microbiol. [ J.Bioengineer and applied microorganisms ],48,135-142 (2002). The total sequence (5,319bp) is available from GenBank (accession number E12173). The crystal structure of beta-galactosides from aspergillus oryzae has been reported. Maksimainen MM et al, Int J Biol Macromol. [ International journal of biomacromolecules ]2013 for 9 months; 60:109-115. The genome of wild type aspergillus oryzae (a. oryzae) has been sequenced. Genbank accession numbers AP007150 and AP 007177.

Beta-galactosidase has also been characterized from: aspergillus niger (Kumar V, et al (1992) Biotechnology [ Biotechnology ] (N Y)10: 82-85); aspergillus niger (Hu X, et al (2010) Appl Microbiol Biotechnol [ applied microbiology Biotechnology ]87: 1773-1782); aspergillus carbonarius (O' Connell S et al (2008) applied Biochem Biotechnol [ applied biochemistry and biotechnology ]149: 129-138); and Aspergillus onii (Aspergillus allieus) (Sen, S. et al (2012) Production, purification, mobilization, and characterization of a thermostable β -galactosidase from Aspergillus allieus) [ Production, purification, immobilization and characterization of thermostable β -galactosidase from Aspergillus onii ] Appl Biochem Biotechnol [ applied biochemistry and biotechnology ]167:1938 + 1953).

In certain embodiments, the use of certain β -galactosidases favors the production of siamenoside I over the production of mogroside IV (WO 2014/150127), while other enzymes favor the production of mogroside IV over the production of siamenoside I (WO 2014/150127, incorporated herein by reference). Still other enzymes produce products containing approximately equal amounts of these mogrol glycosides. If a specific ratio of siamenoside I to mogroside IV is desired in the final product, the enzyme may be selected to produce the desired result. FIG. 1 shows the conversion pathway of mogroside V. It was surprisingly found that certain narrower temperature and pH ranges alter the production profile to favor siamenoside I production.

In various embodiments, starch modifying enzymes having glycoside hydrolase activity can be used for these catalytic reactions. Such enzymes may include, but are not limited to, glucanases, cellulases, glucanases (glucanases), lactases, gyrolases (pustulanases), and many other names.

In one aspect, methods are disclosed wherein a luo han guo extract, mogroside V, is contacted with a β -galactosidase for a time and under conditions effective to achieve the desired redistribution of siamenoside I. That is, the reaction conditions are selected to provide the desired degree of conversion of mogroside V to siamenoside I. In general, it is known that shorter reaction times favor the production of siamenoside I and mogroside IV, but not the production of mogroside IIIE (WO 2014/150127, incorporated herein by reference).

In one embodiment, the beta-galactosidase is an aspergillus species beta-galactosidase, such as aspergillus oryzae, aspergillus niger, aspergillus carbonarius, or aspergillus cepacia, or a specific strain thereof.

In one embodiment, the beta-galactosidase is aspergillus oryzae beta-galactosidase (AoBG). Aspergillus oryzae is generally described as an domesticated Aspergillus species derived from Aspergillus flavus and these two species cannot be distinguished by DNA. Aspergillus oryzae is known for its successful expression host on an industrial scale for enzyme production and as a secondary metabolite.

A representative, non-limiting, wild-type strain of Aspergillus oryzae includes RIB40(ATCC 42149).

Representative, non-limiting, industrial Aspergillus oryzae strains include RIB128, RIB915, RIB326, BP2-1, 3.042, and A1560.

In a particular embodiment, the beta-galactosidase is an aspergillus oryzae RIB strain beta-galactosidase. Over 200 strains of Aspergillus oryzae RIB are known. (Murakami, H.1971.J.Gen.appl.Microbiol. [ J.Bioengineer and applied microorganisms ]17: 281-.

In a particular embodiment, the beta-galactosidase comprises the amino acid sequence of SEQ ID NO 1.

In one embodiment, the beta-galactosidase is a fusion protein.

In a particular embodiment, the beta-galactosidase comprises the amino acid sequence of SEQ ID NO 2.

In many reactions, enzyme efficiency may be a limiting step. Thus, efforts to engineer improved mutant β -galactosidases with enhanced reaction selectivity to produce siamenoside I from mogroside V are described.

In one embodiment, the starting (or parent) enzyme sequence used to engineer the improved β -galactosidase is wild-type aspergillus oryzae β -galactosidase (AoBG). Ao- β -gal is a large (1005 residues) multidomain enzyme with a catalytic (α/β) 8-barrel domain. FIG. 14

In a particular embodiment, the starting enzyme comprises an amino acid sequence comprising SEQ ID NO 1.

In one embodiment, the initiator enzyme sequence is a wild-type AoBG fusion protein, in particular, a wild-type AoBG fusion protein comprising a C-terminal s-myc and a hexa-His tag.

In a particular embodiment, the starting enzyme comprises an amino acid sequence comprising SEQ ID NO 2. There are many enzyme engineering methods available to accomplish the modification of beta-galactosidase. These modifications may be, for example, one or more amino acid differences. These differences may be, for example, one or more substitutions, additions, insertions and deletions. Amino acids can be naturally occurring or synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.

In one embodiment, the one or more amino acid substitutions are conservative amino acid substitutions. Amino acids can be grouped according to the similarity of the properties of their side chains (in a.l. lehninger, Biochemistry, 2 nd edition, pages 73-75, Worth press, new york (1975)): (1) non-polar: ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polarity: gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidity: asp (D), Glu (E); (4) alkalinity: lys (K), Arg (R), His (H). Alternatively, naturally occurring residues may be classified into the following groups based on common side chain properties: (1) hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilicity: cys, Ser, Thr, Asn, Gln; (3) acidity: asp and Glu; (4) alkalinity: his, Lys, Arg; (5) residues that influence chain orientation: gly, Pro; (6) aromatic: trp, Tyr, Phe.

In particular embodiments, the modification comprises one or more amino acid differences in the catalytic domain or loop region. In one embodiment, the modification comprises one or more amino acid modifications of amino acid residues 200-212, more particularly 200 and 2010. In particular embodiments, the modification comprises one or more amino acid modifications in amino acid residues 204, 205, or 208. In certain embodiments, the one or more amino acid modifications are substitutions, more particularly, conservative amino acid substitutions.

In particular embodiments, the modification comprises a difference in two or more amino acids in the catalytic domain or loop region. In one embodiment, the modification comprises two or more amino acid modifications of amino acid residues 200-212, more particularly 200 and 2010. In particular embodiments, the modification comprises two or more amino acid modifications in amino acid residues 204, 205, or 208. In certain embodiments, the two or more amino acid modifications are substitutions, more particularly, conservative amino acid substitutions.

In particular embodiments, the modification comprises three or more amino acid differences in the catalytic domain or loop region. In one embodiment, the modifications comprise three or more amino acid modifications of amino acid residues 200-212, more particularly 200 and 2010. In particular embodiments, the modification comprises three or more amino acid modifications in amino acid residues 204, 205, or 208. In certain embodiments, the three or more amino acid modifications are substitutions, more particularly, conservative amino acid substitutions.

A) Orientation method

Molecular dynamics simulations were performed using the 4IUG structure of β -galactosidase with mogroside V using a directed approach. Mogroside V was docked in different orientations to test for predicted Glu/Asp catalytic residues. The residue was modified to Gln/Ala to generate potentially inactive variants. The specific library of relevant amino acid/targeted aa changes in the loop region appears to stabilize the "siamenoside I formed conformation". Inserts were then constructed for recombinant expression and analyzed by liquid chromatography-mass spectrometry (LCMS) for moderate throughput.

B) The semi-inference method comprises the following steps:

the half-inference method, FuncLib/other computational methods, was used to reduce the complexity of the library, predicting the best specificity of loop replacement. Inserts were then constructed for recombinant expression and analyzed by liquid chromatography-mass spectrometry (LCMS) for moderate throughput.

C) Random mutagenesis

Selecting pressure:

using a random mutagenesis approach, mogroside V was used as the sole carbon source for the overall rate increase. The derived mog V/triterpene was used for selective cleavage to siamenoside I (fluorescence/color release). Further release of mogroside III can result in the release of toxic or competitive colors.

Exploration of the catalytic mechanism indicates that the production of siamenoside I is due to a catalytic diad that is different from the traditional cognition of the enzyme.

The traditional catalytic doublet pair was E200, E298, while surprisingly, the inventors found that the catalytic doublet pair used for siamenoside I production was E200, D258. These positions are the subject of mutations to increase the activity of the enzyme for siamenoside I production.

The tested variants showed an increase in activity or specificity for siamenoside I.

In one aspect, the invention provides a modified β -galactosidase enzyme comprising one or more mutations in a catalytic site, a loop region, or a combination thereof. In particular embodiments, the modified β -galactosidase comprises two or more mutations in the catalytic site, the loop region, or a combination thereof.

The mutation may be selected from a substitution, deletion, insertion, or a combination thereof. Substitution means the substitution of an amino acid occupying a position with a different amino acid; deletion means the removal of an amino acid occupying a position; and insertion means addition of 1 to 3 amino acids adjacent to the amino acid occupying a certain position. If the mutant involves more than one mutation type, it is referred to as a combinatorial mutant.

In a specific embodiment, the invention provides a modified β -galactosidase comprising an amino acid sequence variant of SEQ ID No. 1, in particular a variant having at least 50% identity, at least 60% identity, at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% or at least 95% identity to SEQ ID No. 1. In certain embodiments, the modified β -galactosidase can be a functional fragment thereof, i.e., retain β -galactosidase activity.

In another specific embodiment, the invention provides a modified β -galactosidase comprising an amino acid sequence variant of SEQ ID No. 2, in particular a variant having at least 50% identity, at least 60% identity, at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% or at least 95% identity to SEQ ID No. 1. In certain embodiments, the modified β -galactosidase can be a functional fragment thereof.

In one embodiment, the mutation is selected from D258E, E804A, E142Q, E142A, E200A, D258A, D258Q, E200A/E298A, E200Q/E298Q, E298A, E298Q, or D258A/E298A in GenBank accession No. CAW30743.1(UniProt B7VU 80).

In one embodiment, the mutation is selected from E803A, E142Q, E142A, E298A, W298Q, or D258A/E298A.

In one embodiment, the one or more mutations are within amino acid residues 200 to 212, more particularly 200 to 210, even more particularly 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, or 2010.

In one embodiment, the mutation is RRK 67. RRK67 has mutations G204G C205R, and V208E.

In one embodiment, the mutation is E142A RRK 67. E142A RRK67 has mutations E142A, G204G, C205R and V208E.

In one embodiment, the mutation increases the conversion rate and/or specificity of the transformation. In particular embodiments, the mutation increases the conversion and/or specificity of the transformation by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%.

In one embodiment, the one or more mutations increase the specificity constant of the mutant β -galactosidase as compared to the specificity constant of the wild-type β -galactosidase. In particular embodiments, the substrate specificity ratio of the mutant β -galactosidase compared to the wild-type β -galactosidase is about 1.2:1, about 1.3:1, about 1.4:1, about 1.5:1, about 1.6:1, about 1.7:1, about 1.8:1, about 1.9:1, about 2.0:1, about 2.1:1, about 2.2:1, about 2.3:1, about 2.4:1, about 2.5:1, about 2.6:1, about 2.7:1, about 2.8:1, about 2.9:1, about 3.0: 1. In another embodiment, the substrate specificity ratio of the mutant β -galactosidase to wild-type β -galactosidase is about 4:1, about 4.5:1, about 5.0:1, about 5.5:1, about 6.0:1, about 6.5:1, about 7.0:1, about 7.5:1, or about 8.0:1 or higher.

In one embodiment, the mutation changes the distribution to an increase in siamenoside I production. In particular embodiments, the mutation alters the profile to increase siamenoside I by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%.

The invention also provides polynucleotides encoding the beta-galactosidase variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the v beta-galactosidase variants. Polynucleotides encoding the β -galactosidase variants disclosed herein can be expressed enzymatically using an expression vector that typically includes regulatory sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and optionally a repressor gene or various activator genes. The host cell used in the recombinant production of the β -galactosidase variants disclosed herein can be a mammalian, insect, bacterial or fungal cell.

Improvement in the purification of siamenoside I from the reaction mixture

The product obtained from the enzymatic treatment (hereinafter sometimes referred to as "modified fruit extract") may be subjected to further treatment and/or purification steps, such as filtration, treatment with an adsorbent, concentration and/or drying. For example, the product may be in the form of an aqueous mixture containing a desired distribution of different mogrol glycoside isomers, which mixture is concentrated by removal of water or film treatment to provide a more concentrated syrup that can be used as a sweetener or flavor enhancer or dried (e.g., by spray drying) to provide a solid composition (e.g., in powder form) that can also be used as a sweetener or flavor enhancer. The modified fruit extract may be combined with one or more additional sweeteners or other food ingredients (such as bulking agents or carriers) prior to such further processing.

By using the enzymatic treatment methods described herein, siamenoside I can be prepared with a distribution that is different from the naturally occurring production. The enzyme is more selective under more neutral reaction conditions.

In one embodiment, the enzyme is acid-tolerant lactase, but its function at neutral pH is significantly better than its function under acidic conditions.

In various embodiments of the present invention, the conditions under which the fruit extract starting material is contacted with the enzyme are selected to provide a modified fruit extract product in which the siamenoside I content is increased by at least two, at least three, at least five, at least ten, at least fifteen, or at least twenty-fold or more compared to the siamenoside I content of the fruit extract starting material.

The biocatalytic reaction conditions may be selected such that the resulting product is enriched for siamenoside I (i.e., the modified fruit extract has a mogrol glycoside content such that at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least 90%, or even 100% by weight of the mogrol glycosides present are siamenoside I). Thus, pure siamenoside I can be obtained using the treatment methods described herein.

Disclosed is a process for the biotransformation and purification of Momordica grosvenori fruit extracts to give high purity siamenoside I.

Also disclosed are processes for bioconverting and purifying a high purity mogroside mixture from a luo han guo fruit extract.

In one embodiment, the process for purifying siamenoside I from a reaction mixture comprises:

a) providing a mixture of low purity mogrosides;

b) dissolving the low purity mogroside mixture in water or an aqueous alcohol solution to form an initial mogroside solution;

c) mixing the initial solution with an affinity adsorbent to bind mogrosides in the low purity mixture of mogrosides;

d) washing the affinity adsorbent with water to remove enzymes and impurities;

e) eluting the affinity adsorbent with a minimum volume of an organic solvent to obtain a mogroside/solvent solution;

f) distilling the mogroside/solvent solution to obtain a concentrated mogroside aqueous solution;

g) loading the concentrated aqueous mogroside solution onto a C18 resin;

h) eluting the C18 resin with a solvent/water mixture of increasing solvent concentration to produce one or more fractions containing siamenoside I;

i) distilling the one or more fractions containing siamenoside I to obtain a concentrated aqueous siamenoside I solution;

f) the concentrated aqueous solution of siamenoside I is dried to obtain high-purity siamenoside I having a siamenoside content of more than about 60% (w/w).

In one embodiment, the affinity adsorbent is HP20 resin, i.e., a rigid polystyrene/divinylbenzene matrix.

In one embodiment, the affinity adsorbent is added at 25x to 30x (w: w) of the mogroside content of the mixture.

In one embodiment, the organic solvent is selected from acetone, acetonitrile, ethanol, or methanol.

In one embodiment, the organic solvent solution is 100% methanol.

In one embodiment, the organic solvent solution is 100% ethanol.

In one embodiment, another cycle of steps c) -e) is performed before step f).

In another embodiment of the present process, the solvent is an aqueous alcohol solution comprising water and an alcohol selected from the group consisting of: methanol, ethanol, n-propanol, 2-propanol, 1-butanol, and 2-butanol.

In one embodiment, the minimum volume of organic solvent is about 2 vol/wt of resin.

In one embodiment, the affinity adsorbent is a C18 resin, i.e., an octadecyl carbon chain (C18) bonded silica resin.

In one embodiment, the affinity adsorbent/solvent mixture is combined and the solvent is removed by distillation.

In one embodiment, the distillation occurs at a temperature between about 45 ℃ and about 50 ℃.

In one embodiment, the affinity resin column is a C18 resin (Chromatorex SMB 150, 20-45 μm) column.

In one embodiment, siamenoside I is purified with between about 30% and 40% methanol solution.

In one embodiment, siamenoside I is purified with between about 30% -40% methanol solution, yielding siamenoside I with a purity > 95%.

In one embodiment, mogroside IIIE is purified with between about 50% and 100% methanol solution.

In one embodiment, mogroside IIIE is purified with between about 30% -40% methanol solution, yielding mogroside IIIE with purity > 95%.

In another embodiment of the process, the adsorbent is a macroporous polymeric adsorbent resin capable of adsorbing mogroside.

In another embodiment, a method for purifying siamenoside I from a reaction mixture comprises:

a) providing a mixture of low purity mogroside and a reactive mixing reagent;

b) separating mogroside from the reaction mix by: (i) adjusting the pH of the mixture of a) to about 10 or higher, (ii) adding an alcohol to provide an alcohol solution and (iii) filtering the alcohol solution through a first ultrafiltration membrane to provide a first filtered solution;

c) adjusting the pH of the first filtered solution to between about 5 and about 7 and filtering through a second ultrafiltration membrane to provide a second filtered solution;

d) diafiltering the second filtered solution to concentrate the mogroside to provide a mogroside mixture, and then mixing the mogroside mixture with water/ammonia acetate to provide a mogroside/ammonia acetate solution;

e) contacting the mogroside/ammonia acetate solution with a fractionating column;

f) eluting and collecting fractions containing siamenoside I; and

g) the fractions containing siamenoside I are dried to obtain high purity siamenoside I with a siamenoside I content higher than about 60% (w/w).

In one embodiment, the reactive mixing reagent is an enzyme and a salt.

Adjusting the pH of the mixture in b) to about 10 or higher, for example about 10.5 or higher, about 11 or higher, about 11.5 or higher, about 12 or higher or about 12.5 or higher. In a particular embodiment, the pH is adjusted to about 12.4. Any base can be used for conditioning, for example, NaOH.

b) The alcohol added in (a) is any simple alcohol, such as methanol, ethanol, propanol, isobutanol or tert-butanol. In a particular embodiment, the alcohol is ethanol.

b) The alcohol solution of (a) contains from about 5% to about 30% alcohol, e.g., from about 5% to about 25%, from about 5% to about 20%, from about 5% to about 15%, from about 5% to about 10%, from about 10% to about 30%, from about 10% to about 25%, from about 10% to about 20%, from about 10% to about 15%, from about 15% to about 30%, from about 15% to about 25%, from about 15% to about 20%, from about 20% to about 30%, from about 20% to about 25%, and from about 25% to about 30%. In a particular embodiment, the alcohol solution contains about 20% alcohol. In a more specific embodiment, the alcohol solution contains about 20% ethanol.

In one embodiment, the first and/or second ultrafiltration membrane is a 10kDa nominal filtration membrane. In one embodiment, the first and/or second ultrafiltration membrane is a 10kDa nominal filtration membrane.

Adjusting the pH of the filtered solution in c) to about 5 to about 7, e.g., from about 5 to about 6.5, from about 5 to about 6, from about 5 to about 5.5, from about 5.5 to about 7, from about 5.5 to about 6.5, from about 5.5 to about 6, from about 6 to about 7, from about 6 to about 6.5, and from about 6.5 to about 7.

In one embodiment, the fractionation column is a C18 resin column.

In one embodiment, the low purity mogroside mixture in step a) comprises at least 90% mogroside V.

In one embodiment, the low purity mogroside mixture in step a) comprises at least 95% mogroside V.

The present invention further provides sweetener mixtures comprising high purity siamenoside I; wherein the high purity siamenoside I is blended with another high intensity sweetener.

In one embodiment of the sweetener mixture, the another high intensity sweetener is selected from the group consisting of: steviol glycosides (including purified sweet steviol glycoside mixtures), stevioside, rebaudioside a, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I, rebaudioside J, rebaudioside M, rebaudioside N, rebaudioside O, dulcoside a, dulcoside B, rubusoside, and stevia rebaudiana; (ii) a Mogroside IV; mogroside V; isomogroside V, mogroside IIIE, Luo Han Guo (Lo Han Guo) sweetener; monatin (monatin) and salts thereof (monatin SS, RR, RS, SR); glycyrrhizic acid and its salts; curculin; thaumatin; monellin; caper seed sweet protein; brazzein (brazzein); southern doxine (southern dulcin); phyllodulcin; sarsasaponin; phlorizin; trilobatin; gypenoside (baiyunoside); ossadin (ossadin); polybodoside (polypodoside) a; pterocaryoside (pterocaryoside) a; pterocarcoside B; kurososide (mukurozioside); phlomisoside I; glycyrrhizin (periandrin) I; abrin triterpenoid (abrusoside) a; cyclocarioside I; and combinations thereof.

The invention also provides products containing high purity mogrosides. In one embodiment, the product is selected from the group consisting of: a food, a beverage, a pharmaceutical composition, tobacco, a nutraceutical, an oral hygiene composition, or a cosmetic.

The term "mogrosides" refers to mogrol, dihydroxy-mogrol, and oxo-mogrol glycosides, including mogroside IIE, mogroside IIB, mogroside III, mogroside IV, mogroside V, 11-oxo-mogroside V, mogroside VI, siamenoside I, and momordica grosvenori saponin (grosomoside) I.

The term "TM content" means the total mogroside content, and it is calculated as the sum of 4 mogrosides (including mogroside V, mogroside IV, siamenoside I, and mogroside IIIe).

The term "highly purified" or "high purity" means that the siamenoside I content is at least 90% (w/w) on a dry basis.

The term "impurities" means any compounds other than siamenoside I, which are present in the mixture above 0.0001% (w/w) on a dry weight basis. Non-limiting examples of impurities include other mogrosides, proteins, pigments, polysaccharides, aldehydes, unsaturated aldehydes, methyl ketones, butyl crotonates, phenolic compounds, and other non-mogroside compounds in addition to siamenoside I.

The purification process of siamenoside I according to the invention is applicable to any low-purity mogroside mixture in which the siamenoside content is less than 60% w/w on a dry weight basis.

In one embodiment, the purification process of the present invention further comprises filtration using ultrafiltration and/or nanofiltration membranes. Membranes with molecular weight cut-off (MWCO) sizes of 1000, 1500 and 2000 were used. The resulting solution is passed successively through ultrafiltration and/or nanofiltration membranes of MWCO 1000, 1500, 2000 and 2500. For this purpose, a stirred cell membrane system from Sterlitech company (USA) was used. In any event, any suitable filtration system known in the art may be used for this purpose. Non-limiting examples of Membrane manufacturers are Coriolis filter Systems Inc. (Koch Membrane Systems Inc. (USA), GE Oersmonics (GE-Osmonics) (USA), Afa Laval (Alfa Laval) (Sweden). Flat sheets, hollow fibers, spirals, and other membranes may be used. Diafiltration is used to increase the efficiency of the membrane filtration process. Depending on the size of the membrane, the retentate or permeate contains a major amount of mogrosides. After each membrane treatment, the mogroside containing fraction (retentate or permeate) is again concentrated or diluted until the total solids content is 0.1% -50% (wt/vol), preferably 0.5% -10%, and passed through the next membrane. The solution passes through membranes of increasing size (from MWCO 1000 to 2500).

Utility as sweetener in food and beverage manufacture

The siamenoside I thus obtained may be used as a high intensity sweetener, alone or in combination with one or more other high intensity sweeteners or conventional sweeteners, such as sucrose. Siamenoside I can also be used as a taste enhancer in food and beverage products and the like at sub-sweetness concentrations.

The modified fruit extract and siamenoside I obtained according to the present invention may be incorporated as sweeteners or taste enhancers in any type of food or beverage composition. Non-limiting examples of such food and beverage compositions include baked goods, soups, sauces, processed meat products, canned fruits, canned vegetables, dairy products, frozen desserts, carbonated soft drinks, sports drinks, ready-to-drink teas, milk-containing drinks, alcoholic beverages, energy drinks, flavored waters, vitamin drinks, fruit drinks, juices, powdered soft drinks, candies, confectioneries, chewing gums, nutritional food products, and the like. The modified fruit extract and siamenoside I can also be used in medicine, pharmaceutical product and tobacco product. The modified fruit extract and/or siamenoside I are included in an amount effective to impart the desired sweetness to the sweetened product. The product may contain one or more additional sweeteners, for example, a caloric sweetener such as sugar or another high intensity sweetener (natural or synthetic), or may not contain any sweet component other than the modified fruit extract or siamenoside I of the present invention. The modified fruit extracts and siamenoside I described herein may also be used as taste enhancers, wherein they are included in a food or beverage at a concentration below the threshold at which they impart sweetness to the product, but in an amount sufficient to improve, modify or enhance the taste of the product.

The high purity mogrosides can be used alone or in combination with other high intensity sweeteners in the following: a food, a beverage, a pharmaceutical composition, tobacco, a nutraceutical, an oral hygiene composition, or a cosmetic. Other high intensity sweeteners include steviol glycosides (including purified sweet steviol glycoside mixtures), stevioside, rebaudioside a, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I, rebaudioside J, rebaudioside N, rebaudioside O, rebaudioside M, dulcoside a, dulcoside B, rubusoside, and stevia; (ii) a Mogroside IV; mogroside V; luo Han Guo (Lo Han Guo) sweetener; monatin (monatin) and salts thereof (monatin SS, RR, RS, SR); glycyrrhizic acid and its salts; curculin; thaumatin; monellin; caper seed sweet protein; brazzein (brazzein); southern doxine (southern dulcin); phyllodulcin; sarsasaponin; phlorizin; trilobatin; gypenoside (baiyunoside); ossadin (ossadin); polybodoside (polypodoside) a; pterocaryoside (pterocaryoside) a; pterocarcoside B; kurososide (mukurozioside); phlomisoside I; glycyrrhizin (periandrin) I; abrin triterpenoid (abrusoside) a; cyclocarioside I; and combinations thereof.

In some embodiments, siamenoside I obtained according to the invention is present in the foodstuff or beverage in a concentration of at least 25, at least 50, at least 100, at least 200, at least 500, at least 1000, at least 1500 or at least 2000ppm (on a weight basis, calculated on a dry solids basis). At such concentrations, siamenoside I tends to act as a sweetener, i.e., it imparts sweetness to the foodstuff or beverage, or increases the perceived sweetness of the foodstuff or beverage that already (prior to the incorporation of siamenoside I) has some sweetness. In other embodiments, siamenoside I is present in the foodstuff or beverage at a lower concentration (e.g., lower than the concentration at which siamenoside I imparts any perceived sweetness). The maximum sub-sweetness concentration (sometimes referred to as the "sweetness detection threshold") will vary depending on the mogrol glycoside content of the modified fruit extract or the purity of siamenoside I, but typically, the sub-sweetness concentration of siamenoside I will be greater than about 1ppm, but less than about 60 ppm.

For example, concentrations from about 10 to about 50ppm may be effective in improving the taste or flavor of a foodstuff or beverage without increasing the perceived sweetness of such foodstuff or beverage.

The advantages of the present invention will become more apparent from the detailed description given hereinafter. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Examples of the invention

Example 1: modification of the transition profile of mogroside to selectively increase the production of siamenoside I

The reaction conditions that improved the distribution of siamenoside I production were scanned.

The reaction of mogroside V to mogroside III can proceed via 2 pathway pathways, each involving 2 subreactions in the pathway. Since each substrate or intermediate involves a different chemistry/binding, process conditions are used to alter the rate and specificity of the enzyme to perform each of these sub-reactions. (FIG. 1)

The method comprises the following steps:

concentration and preparation of enzymes

Maxilact A4 was prepared by ultrafiltration.(GODO-YNL2 beta-galactosidase (EC 3.2.1.23)) in 4X concentrated enzyme solution. During production, Maxilact A4 contains more than or equal to 50% (w/w) of glycerin and 60-120g/L of sodium chloride. Microfiltration (MF) (0.2 μm) and/or ultrafiltration (UF, 5kDa, 400cm2 membrane) was used to produce about 150mL of each target enzyme concentration (enzymes were microfiltered prior to UF operation). The flux rate of UF was relatively slow, about 1.5 mL/min. The Maxilact a4 batch used in this work was DSM lot No. 417792301.

Evaluation of pH versus temperature versus time

By controlling the pH, temperature and time of the reaction, the reaction is designed to improve the desired profile. The reaction was carried out in a 50mL conical tube, with a total reaction volume of 28.8mL, containing 8mL of 4 Xenzyme concentration in the original receiving enzyme medium, and the final reaction mixture contained 3mM magnesium chloride and 0.1M sodium acetate. The initial pH conditions were titrated to 5.8, 6.0, 6.3, 6.6, and 7.0. The reaction was sterilized by microfiltration (0.2. mu.M). After initial mixing, the reaction was not stirred and the pH was measured to ± 0.1pH units using a micro pH meter. Reaction samples were taken on days 1, 4, 7 and 11 and then analyzed by HPLC methods.

HPLC analysis

Samples were analyzed on a Phenomenex Synergi-Hydro RP, 250mm x 4.6mm, 4 μm (part number 00G-4375-E0) run at 55 ℃ with a gradient of two solvents at a flow rate of 0.5 mL.min. Solvent A consisted of 0.569g of ammonium acetate and 0.231g of acetic acid in 2L of 18 M.OMEGA.cm water. Solvent B consisted of acetonitrile. The elution of the compound was monitored by a UV detector set to a bandwidth of 215nm, 4nm, referenced 265nm, with a bandwidth of 100 nm. The gradient spectra were 25% solvent B at 0min, 58% solvent B at 25 min, 90% solvent B at 35 min, 90% solvent B at 38 min, 25% solvent B at 38.1 min, and 25% solvent B at 43 min. All major peaks were well resolved at this gradient and compared to mogroside standards run in this way. 11-oxomogroside V elutes at about 8.6 minutes, mogroside V elutes at about 9.4 minutes, 11-oxosiamenoside I elutes at about 10.1 minutes, siamenoside I elutes at about 10.6 minutes, mogroside IV elutes at about 11.3 minutes, and mogroside IIIE elutes at about 12.7 minutes.

Samples were ultrafiltered on a 10kDa rotary filter and diluted 5x with 20% aqueous acetonitrile prior to injection. The injection volume was 50 microliters.

Additive experiments at alternating pH

The reactions were set up to evaluate the potential to improve the desired distribution by adding various supplements. The reaction was carried out in 50mL conical tubes, with a total reaction volume of 28.8mL, containing 8mL of 4 Xenzyme concentration in the original receiving enzyme medium, and a final reaction mixture containing 3mM magnesium chloride, 0.1M sodium acetate, and various supplements. In each reaction, glycerol was supplemented to 25% or 50% of the total reaction volume. Sodium chloride was supplemented to a final concentration of 33 g/L. Calcium chloride was supplemented to a concentration of 0.1/L. Potassium chloride was supplemented to 0.5% (w/v). The initial pH conditions were titrated to 6.0 and 6.6. The reaction was sterilized by microfiltration (0.2. mu.M). After initial mixing, the reaction was not stirred and the pH was measured to ± 0.1pH units using a micro pH meter. Reaction samples were taken on days 1, 4, 7 and 11 and then analyzed by HPLC methods.

As a result:

these methods show that temperatures above 50 ℃ and higher pH provide better distribution for siamenoside I production.

Lower temperature and pH increase the proportion of mogroside IIIE more rapidly with less contamination.

Higher pH and temperature significantly improved the yield of siamenoside I:

at pH 6.6 or about pH 6.6 and above 50 ℃, the conversion of mogroside V is greater than 50%.

At pH 6.3 or about pH 6.3 or above 55 ℃, the conversion of mogroside V is greater than 50%.

The low pH and temperature significantly improved the yield of mogroside IIIE:

at pH 5.8 or about pH 5.8 and below 50 ℃, the conversion of mogroside V is greater than 60%.

At pH 5.8 or about pH 5.8 or below 55 ℃, the conversion of mogroside V is greater than 60%.

The key feature of this biotransformation is that it reduces mogroside V levels and keeps mogroside IV levels below 10%, which feature aids in purification.

These results are graphically summarized in fig. 3.

Table 1: change in pH at 45 ℃

Table 2: change in pH at 50 deg.C

Table 3: pH change at 55 deg.C

Additive:

in addition to varying the temperature at the pH of the reaction mixture, additives that increase the profile of siamenoside I production were identified. The addition of glycerol and monovalent and divalent cations changed the distribution of siamenoside I production. The glycerol concentration had only a small effect on the reaction and the observed distribution. However, the addition of monovalent and divalent ions appears to improve the distribution of formation to siamenoside I. The addition of NaCl, CaCl and KCl improves the siamenoside I by 5-10%. NaCl appeared to slow the reactions slightly, but also reduced the amount of mogroside IIE formed in these reactions.

Table 4: effect of additives on the reaction at 50 ℃ and pH 6.0

Table 5: effect of additives on the reaction at 50 ℃ and pH 6.6

Example 2: overexpression of wild-type Aspergillus oryzae beta-galactosidase in yeast

The method comprises the following steps:

creation of wild-type expression vectors

The full length wild-type coding sequence for beta-glucosidase from aspergillus oryzae (PDB accession No. 4IUG _ a) was synthesized in two parts into a gBlock gene fragment (Integrated DNA Technologies, IDT, usa), codon optimized for expression in Pichia pastoris yeast cells (Geneart, Invitrogen, germany) and designed for cloning into the ppicza vector from EasySelectTM Pichia transformation system (Invitrogen, seimer, usa) by three way ligation using compatible restriction enzyme sites Eco R1, Kpn 1 and Sac II (NEB, usa).

Construction of variants (binary evaluation)

To evaluate the catalytic residues that may be responsible for hydrolysis of glucose units from mogroside substrates undergoing digestion, variant enzyme sequences of β -glucosidase from aspergillus oryzae were created. Various amino acid variant enzymes are described in table 1. To replace the coding region of a previously constructed vector carrying the wild-type enzyme sequence, a new vector was constructed as follows: gBlock was constructed including complementary coding sequences between the restriction enzymes listed in table 1 but incorporating the corresponding variant point mutations. These gblocks and the vector backbone are then digested with these restriction enzymes under the conditions provided by the restriction enzyme manufacturer. The isolated restriction DNA fragments are then ligated, transformed and sequenced to verify the correct construction as is standard in the art. (FIG. 4)

Codon optimization for expression in s.cerevisiae (s. cerevisiae) or pichia pastoris and was designed for fusion with the alpha-pairing factor (N-terminal) and s-myc and 6 × His tag (C-terminal) encoded by the pPICZ α a vector and including the relevant restriction endonuclease sites. The stop codon was not included to allow fusion with vector-derived C-terminal s-myc and 6 × His tag.

Transformation of Pichia pastoris cells

The DNA for transformation was prepared by linearizing the plasmid DNA of the desired pPICZ α A expression construct using Sac I. EasyComp was then performed according to the manufacturer's instructions^TMTransformation (invitrogen, seimer feishale, usa). Briefly, 0.5-1. mu.g of linearized DNA was added to 100-200. mu.L of competent cells, mixed with 1mL of solution II, and incubated with shaking at 30 ℃ for 1 hour, followed by heat shock at 42 ℃ for 10 minutes. The cells were then mixed with YPD and grown at 30 ℃ for 1 hour without shaking. Then harvestingTransformed cells (3000g, 5 min) were washed in 500. mu.L of solution III, then resuspended in 200. mu.L of solution III and resuspended in a solution containing 100. mu.g/mL Zeocin^TMYPD plates (Invitrogen, Sammerlow, USA). Transformants were selected after 48-72 hours of growth at 30 ℃ and plated in a medium containing 100. mu.g/mL Zeocin^TMOn YPD plates for subsequent analysis.

Expression of Aspergillus oryzae beta-glucosidase using Pichia pastoris cells

A single colony was inoculated into 10mL of buffered basic glycerol yeast (BMGY) medium in a 50mL tube and incubated overnight at 30 ℃ with shaking (250rpm) until the culture reached OD600nm 2-6. Cells were then harvested (3000g, 5 min) and resuspended at OD600nm 1.0 in buffered minimal methanol medium (BMM) containing 1% methanol and grown at 30 ℃ under shaking (250rpm) for 72 hours, adding methanol to 1% volume daily to maintain induction of the AOX promoter. Cells were harvested (8000g, 10 min) and the supernatant was concentrated using Amicon to produce active partially purified enzyme. The presence of active AoBG enzyme was confirmed by identification using ONPG and mogV enzymatic assays, SDS-PAGE separation and visualization with a Bulldog protein dye, and proteomic analysis of supernatant and pellet fractions from Pichia pastoris (Pichia) cells, comparing strains containing the negative vector to strains expressing AoBG.

Beta-galactosidase ONPG Activity assay

Standard β -galactosidase assays were performed at room temperature using a 200 μ L volume of o-nitrophenyl- β -D-galactopyranoside (ONPG, sigma) as substrate and detecting the o-nitrophenol release at 420nm (UV-Spectra Max spectrophotometer, Molecular Devices, usa). A typical reaction contains 50mM sodium citrate buffer (pH 5.6), 10mM magnesium chloride and 100mM potassium chloride with 2mM ONPG and the desired amount of enzyme (usually 10. mu.L of concentrated supernatant). Partially purified AoBG (Sigma G5160, usa) was used as a positive control at a final concentration of 1U mL "1. Vmax was calculated using SpectraMax Plus software (molecular devices, USA) and the required kinetics were determined by varying the substrate concentration and kinetic determination factors calculated using hyper (j.s. eastby, University of libertol (University)) when required.

Momordica grosvenori biocatalytic assay

Standard mogroside biocatalytic assays were performed in a volume of 500. mu.L of 50mM sodium citrate buffer (pH 5.6), 10mM magnesium chloride, 10mM substrate (usually mogroside V) and 50. mu.g mL-1 enzyme at 37 ℃ and incubated for 24-96 hours. Mogroside glycosylation activity was detected by direct HPLC detection and quantification of mogroside V substrate and mogroside product from the filtered reaction supernatant and quantified by peak to standard curve comparison.

Analytical method

HPLC separation and quantitation of mogroside compounds was performed using a Synergi Hydro-RP column (250 mm. times.4.6 mm or 150 mm. times.4.6 mm) with an initial flow rate of 1mL min-1 and a water acetonitrile gradient as follows, similar to that described by Zhou et al (WO 2014/150127). Compounds were detected at 210nm using a diode array detector (agilent technologies, usa) and calibration curves were established using standard curves from 0.1 to 10 mM.

As a result:

the reaction profiles of G5160 beta-galactosidase (Sigma), Maxilact A4 beta-galactosidase (DSM) and Pp-Aspergillus oryzae beta-galactosidase were evaluated. The ratios of the products of recombinant expression are shown in Table 6.

TABLE 6

	G5160	ML	PpAoBG
				Mog V	19％	22％	24％
Siam I	41％	49％	43％
				Mog IV	11％	12％	21％
Mog III	11％	17％	12％
				Mog II	14％
Mog I	4％

The reaction was carried out using 12.8g/L of MG-V (10mM) at pH 6.0, 50 ℃ for 96 hours

G5160 is a β -galactosidase enzyme activity mixture isolated from Aspergillus oryzae in culture.

(ML) A4 is an acid lactase/beta-galactosidase preparation from Aspergillus oryzae expressed in Aspergillus niger as described in GRAS publication 00510 (FDA, https:// www.fda.gov/default. htm). It is expressed recombinantly in A.niger by a plasmid carrying the A.oryzae TOL gene.

Pp-Aspergillus oryzae beta-galactosidase (PpAoBG) isA4 same acid lactase/beta-galactosidase. Instead of using the A.niger expression system, the P.pastoris system was used. Pichia pastoris has no background beta-galactosidase. Use of the system provides a cleaner system for various evaluations. In addition, the TOL gene// beta-galactosidase was recombinantly expressed in the Pichia pastoris system using a protein export system. The resulting peptide will be reacted withThe peptides in a4 are the same,a4 is an acid lactase/β -galactosidase preparation from aspergillus oryzae expressed in aspergillus niger as described in GRAS publication 00510.

Single colonies were inoculated into 5ml wells of a 96-well growth block, grown to OD 3.0, then pelleted, resuspended in induction medium, and induced/grown for 72-96 hours to achieve active enzyme expression.

So far, the activity was confirmed after 40 hours at 37 ℃ by ONPG, DNS and mogV assay HPLC analysis. After 48 hours the major production of siamenoside I was about 32%. (FIG. 2)

Example 3: structure-based analysis to engineer Aspergillus oryzae beta-galactosidase to produce improved product properties

The goal of this work was to engineer improved β -galactosidases to selectively alter the distribution of siamenoside I production during mogroside V transformation. (FIG. 5)

Library generation

A custom degenerate library was synthesized containing the Eco RI-Kpn I DNA fragment (to replace the Eco RI-Kpn I region between bp 1209 and 2207 in the pPICZ α A-AoBG master plasmid) (FIG. 5). The fragment was synthesized with degenerate RRK codons replacing the codons of amino acids G165, C166 and V169 and amplified with (gene technology, GmBH, seimer feishel) to generate a library. All other codons correspond to the DNA sequence 1.

250ng of the library DNA was digested with Pst 1 and Kpn I, purified and concentrated (DNA purifier & concentrator, Marshall-Nagel) and ligated with 30ng of gel-purified pPICZ α A-AoBG host plasmid digested with Pst 1 and Kpn I using T4 DNA ligase (NEB). The efficiency of the library was tested by: 4uL was transformed into TOPP 10 chemically competent cells (Invitrogen), then the remainder was transformed into DH 5. alpha. CloneCatcher cells, and the grown cells were inoculated into 100mL Luria broth containing Zeocin (100. mu.g mL-1), grown overnight at 37 ℃ and subjected to large scale plasmid preparation (plasmid DNA Midiprep, Qiagen).

Generation and screening of Aspergillus oryzae beta-glucosidase libraries for functional analysis of active site amino acid residues

Enzyme variants with alanine (a) and glutamine (Q) substitutions were prepared for each of the five charged residues identified in stage 1 as likely candidates for glycoside hydrolysis of mogroside V (E142, E200, D258, E298, E804) and expressed in pichia pastoris.

The method comprises the following steps:

library generation

A custom degenerate library was synthesized containing the Eco RI-Kpn I DNA fragment (to replace the Eco RI-Kpn I region between bp 1209 and 2207 in the pPICZ α A-AoBG master plasmid) (FIG. 5). The fragment was synthesized with degenerate RRK codons replacing the codons of amino acids G165, C166 and V169 and amplified with (gene technology, GmBH, seimer feishel) to generate a library. All other codons correspond to the DNA sequence 1.

250ng of the library DNA was digested with Pst 1 and Kpn I, purified and concentrated (DNA purifier & concentrator, Marshall-Nagel) and ligated with 30ng of gel-purified pPICZ α A-AoBG host plasmid digested with Pst 1 and Kpn I using T4 DNA ligase (NEB). The efficiency of the library was tested by: 4uL was transformed into TOPP 10 chemically competent cells (Invitrogen), then the remainder was transformed into DH 5. alpha. CloneCatcher cells, and the grown cells were inoculated into 100mL Luria broth containing Zeocin (100. mu.g mL-1), grown overnight at 37 ℃ and subjected to large scale plasmid preparation (plasmid DNA Midiprep, Qiagen).

Computational simulations were performed using molecular dynamics to better understand the binding of various mogrosides to the predetermined molecular structure of beta-galactosidase from Aspergillus oryzae (Unit prot: B7VU 80; GenBank CAW 30743.1). These simulations allow structural changes in mogroside and protein active site structures to determine which portions of the active site are most likely to direct observed activity and preference for conversion to siamenoside I. The protein structure used in this simulation was reported as 4IUG in the Protein Database (PDB). In different simulations, mogroside V, mogroside IV or siamenoside I were approximately docked to the proposed active sites of the enzyme. In addition, different starting orientations of these mogroside molecules were also evaluated. Simulations indicate that several residues contribute to mogroside V binding and the discrimination or masking of siamenoside I from the active site. The key finding is that the specific loop region in the peptide sequence of the enzyme (between amino acids 202 and 209(B7VU80 sequence)) corresponding to 163 and 170(4IUG sequence) may be responsible for the difference in activity. Notably, molecular dynamics simulations indicate that stabilizing this loop region may promote siamenoside I formation. Directed mutagenesis and screening protocols were developed to create libraries of enzyme variants for evaluation.

Positions (G204, C205, V208) were mutated in combination at each point by an RRK degenerate codon library protocol. An RRK library is one in which any nucleotide (A or G) may be present at the first two positions of the targeted codon (R), and only G or T may be present at the third position (K). The library will allow each of the 3 wild-type amino acid positions to be mutated to R, N, D, E, G, K or S. There are 343 possible combinations of the total library.

After cloning and evaluating the integrity of the library, the variant library was expressed in the pichia expression system. This system allows the culture, expression and export of single enzyme variants into the culture medium without contaminating the beta-galactosidase activity. In doing so, individual enzyme variants are expressed in a well plate format, processed to collect and normalize solutions with relatively pure enzyme concentrations. The resulting normalized enzyme solutions were evaluated by the medium-throughput LC-MS method. Those variant enzymes that showed better function than the wild-type enzyme in terms of siamenoside I yield were sequenced to determine the specific mutation responsible for the improvement.

Transformed into Pichia pastoris

The resulting plasmid DNA was linearized by digestion with Sac I and transformed into chemically competent pichia pastoris cells in aliquots of 1 μ g linearized DNA per 200 μ L cells (× 15), as described in section 1. Positive transformants were selected on 150mm YPD agar plates containing Zeocin (100. mu.g mL-1). Positive transformants were confirmed by isolation of genomic DNA and amplification of the 3kb PCR product encoding the inserted AoBG gene using primers AoBG-F-Eco (SEQ ID NO:4) and AoBG-R (SEQ ID NO: 5). The PCR product was sequenced using the primers AoBG-inner F (SEQ ID NO:6) and AoBG-Kpn-R (SEQ ID NO:7) (Daji Co., Macrogen, Korea) to confirm the substitution at the RRK position.

DNA sequence 2: Eco-Kpn RRK Loop library DNA (Geneart custom degenerate library Synthesis 18ABRBLC, Gene technology Co., GmBH):

the Eco R1 and Kpn 1 cloning sites are underlined. RRK degenerate codons are highlighted in bold text. Degeneracy: R-A or G; K-G or T.

Expression and screening of library variants

A single transformant colony of 3.2.2 was inoculated into 2mL BMGY in a 48-well growth block, grown overnight at 30 ℃ in a bench-top incubator at 1000rpm, then the cell culture of each well was diluted into 2mL BMM medium in an equivalent well in a fresh growth block to an OD600nm of 1.0, and the induced growth block was grown for 72 hours at 30 ℃ at 1000rpm in a bench-top incubator. Cells were then harvested (5000g, 10 min) and supernatants from each well were concentrated using an Amicon Ultra 0.5mL concentration column (10000g, 5 min) with a cut-off of 30 kDa. The enzymatic assay was set up in fresh growth pieces in a total volume of 500. mu.L, containing 50mM sodium citrate buffer (pH 5.6), 10mM magnesium chloride, 10mM mogroside V and 50. mu.L of concentrated supernatant (approximately 35. mu.g mL-1 protein). The assay was incubated at 37 ℃ for 72 hours without shaking. 190. mu.L of the reaction was combined with 10. mu.L of 10mM internal standard (N-Boc-) and 5. mu.L was analyzed by HPLC.

Analytical method

HPLC separation and quantitation of mogroside compounds was performed using a Synergi Hydro-RP column (250 mm. times.4.6 mm or 150 mm. times.4.6 mm) with an initial flow rate of 1mL min-1 and a water acetonitrile gradient as follows, similar to that described by Zhou et al (WO 2014/150127). Compounds were detected at 210nm using a diode array detector (agilent technologies, usa) and calibration curves were established using standard curves from 0.1 to 10 mM. 150mm column retention time in minutes: mog V9.5, sia 110.6, mog IVa 10.5, mog IV 11.3, mog III 12.6 and mogI I13.2. 250mm column retention time: mog V12.3, sia 113.2, mog IVa 13.5, mog IV 14.1, mog III 15.3 and mog II 16.2.

As a result:

cells expressing the enzyme variants grown in buffered minimal medium were induced with 1% methanol for 72 hours (every 24 hours of replacement), culture supernatants were harvested and assayed for standard ONPG activity over 30 minutes and mogroside V activity at pH 5.6, 37 ℃ for 72 hours. The resulting activity data (table 2) identifies that E200 and D258 may be catalytic pairs of acid/base and nucleophilic attack of aspergillus oryzae β -glucosidase on mogroside V. To some extent, it was unexpected that alanine substitution of E298 (one of the typical residues involved in β -glucosidase [1,2] catalysis of galactose) did not result in loss of mogroside V activity, although it did result in decreased activity when o-nitrophenyl- β -galactoside (oNPG) was used as a substrate.

One enzyme variant D258E (putative replacement of catalytic aspartic acid with a glutamic acid residue) resulted in an increase in the initial rate of mogroside hydrolysis, but did not significantly increase the rate of production of siamenoside i (siaI) or the ratio of siaI: mogIII products. This is consistent with molecular dynamics data that indicate that such substitutions bring the active site residues closer to the catalytic site on the mogroside V substrate molecule.

Generation and screening of Aspergillus oryzae beta-glucosidase libraries for functional analysis of active site amino acid residues

Both enzyme variants (E142A and E804A) resulted in an increased rate of siamenoside I production from mogroside V. There are two possible explanations for this:

1) the expansion and/or increase in flexibility within the mogroside V "binding pocket" reduces the likelihood that siamenoside I binds in a catalytic conformation and progresses to smaller mogrosides.

2) The functionality of the amino acid residues involved in the individual opportunistic catalytic sites for enzymatic hydrolysis of siamenoside I glycosides is removed, i.e. in different situations or with different sized substrates, different residues are likely to act as catalytic dyads.

Chromatographic analysis showed comparison with P5160 β -galactosidase from Sigma chemicals (Sigma Chemical). It was previously reported that this enzyme has a better activity in the production of siamenoside I, whereas it was shown in this experiment to produce mogroside IVa, contrary to the previous report in WO 2014/150127.

Comparison of wild-type and variant enzyme activities with mogroside V, siamenoside I, and an equimolar mixture of the two indicates that variants E142A and E804A act by reducing the effectiveness of siamenoside I as a substrate in the presence of mogroside V, resulting in accumulation of siamenoside I and reduced mog IV and mog III production.

TABLE 7 beta-galactosidase and mogroside V hydrolytic Activity of Aspergillus oryzae beta-glucosidase (UniProtKB: B7VU80) variants

Table 8 comparison of mogroside V and siamenoside I hydrolysis activities of aspergillus oryzae β -glucosidase variants: molar percentages of substrate and product.

The reaction was carried out at pH 5.6 at 37 ℃ for the time indicated in parentheses for each column.

Abbreviations: mogV-mogroside V, sai I-siamenoside I, mog IV-mogroside IV, mog III-mogroside III, and mog II-mogroside II.

Conclusion of catalytic dyad variants:

targeted analysis of potential active site amino acid residues involved in the hydrolysis of mogroside V by aspergillus oryzae β -glucosidase revealed some surprising insights that the identification of E200 and D258 might be a catalytic pair for acid/base and nucleophilic attack on mogroside V and highlight several variants with increased yield of siamenoside I glycosylated from mogroside V (E142A, E804A) and/or increased rate of mogroside V hydrolysis (D258E).

The loop replacement library was designed using molecular dynamics simulations to enhance the binding of the "trisaccharide" component of mogroside V to the enzyme loop region 202-212, thereby enhancing the formation of the desired product (siamenoside I) and reducing the rate of further hydrolysis to the undesired mogroside I, II and III compounds. The side chains of amino acid residues G204, C205, V208 are changed in combination to 7 different amino acid residue variants, substituted with charged residues, facilitating the binding of sugar side chains (e.g., Asn, Ser, Lys, Arg, Gly, Glu, Asp [ in a stable loop ]).

Library size was screened and evaluated for 73(643) variants using the pichia expression system.

Selection of library variants with increased yield of siamenoside I

660 gene-enzyme variants transformed into pichia pastoris were co-expressed and screened for improved siamenoside I production from mogroside V. The wild-type enzyme showed a yield of siamenoside I of about 66% under the assay conditions. Thus, variants showing improved function were selected, provided that in the first pass evolution they exceeded yield values > 70% calculated as peak area.

For the second over-evaluation, 48 variants were selected based on a > 70% yield of siamenoside I calculated as peak area. The cell lines were re-grown and assayed independently using mogroside V and siamenoside I as substrates. The PCR amplicons of the AoBG gene inserted in the genomic DNA of the selected strains were sequenced to identify the responsible RRK substitutions and a particularly high yield pattern of siamenoside I was found to be generally associated with the Glu/Asn at position 204 and the motif of Arg or Glu at position (208 UniProtKB: B7VU 80/GenBank: CAW30743.1 numbering).

Additional evidence suggests that screening provides improved function for candidates, and that a set of randomly picked colonies was also evaluated by two-fold assessments. Interestingly, all variants with Gly substitutions showed a decrease in siamenoside I yield from mog V compared to the wild type AoBG enzyme activity.

Table 9: identified candidates with improved function

By combining mutations identified in the RRK variants and/or catalytic dyad assessment results, enzyme variants with increased activity and preferred profiles are created. Thus, beneficial E142A and E804A amino acid substitutions were added to the combinations identified at positions corresponding to C206, G204, C205, and V208 in the RRK variant studies described above.

Example 4: producing siamenoside I; purification of siamenoside I from the crude material by biotransformation of mogroside V: the scale process of biotransformation comprises:

since the stability of the enzyme is affected by higher temperature and pH, conditions for concentrating the enzyme, limiting the reaction time and improving the purification process are sought. Furthermore, this has the added benefit of limiting the possibility of bacterial contamination growth. FIG. 6 shows schematically the general scheme of a scale-up bioconversion process.

A) General Process flow and reactor preparation

Pre-reactor

The Maxilact enzyme was concentrated using a hollow fiber membrane (Koch 6043-PM5 Romicon 5 kDa). The enzyme solution was treated in a batch configuration. The shear stress on the enzyme is reduced using a flexible vane pump. The pressure at the inlet was kept below 2.5 bar. The permeate containing glycerol, salt and water was discarded. (FIG. 6)

Mogroside V90 powder was mixed with UV treated RO water using a stirrer. After the powder was dissolved in the solution, acetic acid, sodium acetate, magnesium chloride hexahydrate, and sodium dihydrogen phosphate were dissolved in the solution. The final reaction concentrations of sodium acetate, sodium phosphate and magnesium chloride were 35mmol, 35mmol and 2.14mmol, respectively. The pH was adjusted to 6.3 using 79% acetic acid.

The mogroside solution was filtered using a 0.2mm hollow fiber filter. The concentrated enzyme solution was then filtered through the same 0.2mm hollow fiber filter. The purpose is to reduce particulate matter to make sterile filtration easier.

Reactor with a reactor shell

The reactor was sterilized with 1 bar steam for 30 minutes. Sterile connections were made using a saoortobran 0.22 filter, and were made in a laminar flow cabinet.

The mogroside solution was pumped through a Sartobran 0.22 filter followed by the enzyme solution. This is to avoid binding of mogroside to the enzyme prior to filtration and possible removal by the filter.

The stirred reactor (30rpm, Rushton stirrer) was maintained sterile and under a positive pressure of 2-4psig with sterile air. The reaction conditions were about 54 ℃. The reaction was stopped on day 12 with 8.9% mogroside V and 63% siamenoside I.

B) Specific example Process

Preparation of mogroside V + buffer solution

Approximately 140L of concentrated, sterile-filtered Maxilact A4 dissolved in approximately 45L of 50mM phosphate buffer (pH 6.5) and 5kg of mogroside V (batch PRF8113001) were transferred aseptically into the fermentor in succession. After heating the contents to 50 ℃, the pH was adjusted to 6.2. The pH was maintained at 6.2 by periodic addition of sterile 1N NaOH solution.

Preparation and operation of the bioreactor:

A650L fermentation vessel was pre-sterilized. Sterile filters (Sartobran 0.22 filters) were attached to the fermentor using aseptic technique. The mogroside V + buffer solution was first added to the vessel followed by 5kg of UV RO water as rinse. Next, maxicat solution was added to the vessel followed by 3kg of UV RO water as rinse (note about 2.5L of this rinse was left in the line and not let into the vessel).

The vessel was set to a bioreactor temperature of about 54 ℃, the operating pressure of the reactor was set to +5psig, and the rotational speed of the agitator was set to 30 rpm. The reaction is monitored approximately every 24-72 hours until the reaction is deemed complete, at which point the mogroside V concentration is about 10% or less.

The reaction progress was monitored by analyzing the reaction mixture at several time points, see table 10 and fig. 7.

After 7 days, the reaction was cooled to ambient temperature and purified

The incubation conditions are within a narrow/optimal pH and temperature window above 40 ℃ to 60 ℃ and pH above 6 to 7 or 7.5. In one example, the mogroside conversion product has a mogrol glycoside profile as follows: mogroside V0 to about 40% by weight, mogroside IV 0 to about 15% by weight, mogroside IIIE 0 to 30% by weight, and siamenoside I60% to about 99% by weight, wherein weight percentages are based on the total mogrol glycoside content of the modified fruit extract, wherein the amount of at least one of mogroside V, mogroside IV, mogroside IIIE or siamenoside I is greater than 0% by weight.

(defined as (([ total mogroside V weight added ] - [ final mogroside V weight ])/100) vs other mogrosides with a siamenoside weight content between 60% and 99% of total mogrosides) (defined as: total mogroside ═ g V + Mog IV + Sia I + Mog IIIE with a mogroside V weight content between)

Example 5: small scale purification

Small scale purification of the crude reaction mixture was accomplished using a Waters XBridge Phenyl column system with HPLC analysis at various stages during the purification process.

1. Materials and methods

The material used to isolate siamenoside I [ batch KTC-B-123(1) ] was a mogroside sample of a mixture of mogroside V (40mg) and Maxiclast A4(0.8 mL; DSM, batch 615495651) in sodium acetate buffer (pH 6; 2mL) stirred at 50 degrees for 7 days and heated at 80 degrees for 20min and then cooled.

Reference standards siamenoside I (batch PRF 8050402) and Mog IVa (batch MS16021403) were also used.

HPLC analysis.

HPLC analysis was performed on a Waters 2695Alliance system coupled to a Waters 996 photodiode array (PDA) detector. In addition, ESA corona + Charged Aerosol Detector (CAD) was used to assess sample purity. Sample analysis was performed using the method conditions described in table 11.

Table 11: analytical HPLC conditions for fraction analysis and final purity analysis in primary and secondary processes.

3. Primary preparative HPLC method

The samples were subjected to primary treatment using a Waters XBridge Phenyl column (19x250mm, 5 μm). The purification process was performed using a Waters Delta prep 2000/4000 system coupled to a Waters 2487UV-Vis detector. Table 12 summarizes details of the preparative method.

Table 12: conditions for the Primary preparative HPLC method

4. Two-stage preparative HPLC method

Secondary treatment was performed using Waters Xbridge Phenyl (19X250mm, 5 μm). The purification process was performed using a Waters 2545 quaternary gradient module system coupled to a Waters 2489UV-Vis detector. Table 13 summarizes the details of the preparative method.

Table 13: conditions for the two-stage preparative HPLC method.

Primary purification approximately 1mL of sample was treated using the primary preparative HPLC method described in table 12. The fractions were analyzed using the analytical methods summarized in table 11. The sample was received in approximately 0.3mL of glycerol. To minimize glycerol contamination, the sample was filtered through a syringe, then mixed with an organic solvent (acetonitrile) and diluted to 10mL with water. Glycerol contamination is considered a problem in primary treatment, in which case the fraction of interest elutes in the wash liquid. The collected fraction (batch No. KTC-B-115 (Wash)) was selected for reprocessing.

Secondary purification: fractions (batch No. KTC-B-115 (Wash)) were reprocessed using the conditions described in Table 12. The fractions were analyzed using the analytical methods summarized in table 10. The collected fraction KTC-B-123(1) (retention time on corresponding preparative HPLC trace of about 13.947min) was considered sufficiently pure for structural resolution by NMR.

And (3) final batch preparation: the fraction (batch No. KTC-B-123(1)) was concentrated by rotary evaporation and further dried by lyophilization for 24 h. The final yield of batch KTC-B-123(1) was 1.4 mg. The final purity was determined using the analytical method summarized in table 10 and found to be 97.09% (AUC, CAD) with a retention time of 13.632 min; fig. 6 provides an analysis. The reference siamenoside I and KTC-B-123(1) are sequentially run, and the retention time is 13.561 min.

Process conditions

Column: phenyl Xbridge (4.6x150mm, 3.5um)

Temperature: ambient temperature

The method comprises the following steps: 80/20 Water/MeCN, 20min isocratic hold.

And (3) detection: CAD, UV @210nm

Table 14: purity analysis of small-Scale Secondary purification

Saimenoside I batch number	RT(min)	Purity (% by area) CAD	Purity (% by area) UV
				KRI-AG-121-8	11.368	93.2	94.6
KRI-AG-122-8	11.832	95.1	98
				KRI-AG-123-5	11.631	96.3	98
KRI-AG-124-8	11.509	97.0	99.1
				KRI-AG-125-8	11.338	97.6	99.1

Example 6: purification for large scale preparation

Figure 8 shows a general purification scheme.

The method comprises the following steps:

as shown in fig. 8, the crude reaction mixture was mixed as a batch with HP20 resin Ca at room temperature. The reaction mixture was mixed with the resin for 1 hour (overhead stirring). HP20 resin was added in an amount of 25X to 30X (w: w) of the mogroside content of the reaction mixture. Mogroside bound to HP20 was loaded into polypropylene SPE cartridges and washed with water to remove enzymes and related impurities. The water wash contained a small amount of sia.i as shown by HPLC analysis. The elution of mogrosides from HP20 resin was tested using 100% organic solvents (methanol, acetone, acetonitrile, and ethanol). The goal of this study was to elute all mogrosides with the smallest volume of solvent. Elution with higher amounts of organic solvent will also facilitate downstream concentration and purification.

As a result:

elution of one or more bound products from HP20 resin was tested using 100% organic solvents (acetone, acetonitrile and methanol; reverse elution order). The objective of the experiment was to elute the product at a lower volume and higher organic solvent concentration.

Table 15: elution of bound mogrosides from HP20 resin (10g) Using 100% organic solvent

^tStandard curves based on the use of the Sia.I reference standard (95% purity; batch No. CDXP-17-0042)

100% methanol (Table 1) or 100% ethanol was found to be suitable for complete elution of the product from HP20 resin in a minimum volume (approximately 2 volumes: weight resin).

Table 16: elution of bound mogrosides from HP20 resin (100g) Using 100% ethanol

^tStandard curves based on the use of the Sia.I reference standard (95% purity; batch No. CDXP-17-0042)

The method can be used for large-scale biotransformation of mogroside V. 340g of Mog.V were dissolved in 1L of potassium phosphate buffer (100mM, pH 6.5) and diluted with 1L of water (2L in total, final buffer concentration 50 mM). It was then filtered through a 0.2 μm sterile filter set.

6L of the most recently concentrated Maxilact A4(3.5 Xconcentrated) was diluted with 6L of buffer (potassium phosphate 100mM, pH 6.5) and the pH of the diluted enzyme was adjusted to about 6.0. 1% filter aid (diatomaceous earth; w/v) was added and the suspension was mixed for about 15 minutes. The suspension was then filtered through a bucket fitted with a sand core (coarse grit). The filtrate was passed through a series of capsule filters (5 μm and 0.2 μm) and then finally filtered into a sterile filtration unit. The final volume of the filtered enzyme was about 11L.

11L of sterile filtered enzyme and 2L of sterile Mog.V solution were transferred separately to the fermentor. The pH was adjusted to 6.2 and the reaction temperature was adjusted to 50 ℃ and the reaction was started.

No significant pH change or microbial contamination was observed during the reaction. However, a higher amount of turbidity/turbidity was observed compared to the previous reaction, probably due to the increased enzyme denaturation due to the higher enzyme concentration.

Samples were withdrawn at regular intervals and analyzed for conversion to siamenoside I.

Table 17 shows the detailed information of the reaction process.

Table 17: results of a third engineering reaction run in a sterile 20L fermentor (run No. KRI-AG-103)

The amount of siamenoside I in the reaction was estimated based on a standard curve using a reference standard (95% purity).

Large-Scale elution of mogrosides from HP20 resin Using methanol

The reaction mixture was worked up batchwise. The reaction mixture was mixed with the resin for 1 hour (overhead stirring). HP20 resin was added in an amount of 25X to 30X (w: w) of the mogroside content of the reaction mixture. The bound resin was washed with water to remove enzymes and related impurities. Elution with 100% methanol was expanded with 2.5Kg of bound HP20

The results are shown in Table 18. As can be seen from the table, mogrosides was completely eluted from the resin using about 2 volumes of 100% methanol.

Table 18: elution of bound mogrosides from 2.5kg of HP20 resin

The method comprises the following steps:

the eluate from the HP20 purification step was prepared for further chromatography. The ethanol containing fractions were combined and the ethanol was completely removed by distillation at a batch temperature of 45 ℃ to 50 ℃. The remaining aqueous solution was further concentrated and the unsaturated portion of the concentrated solution was loaded onto a column of C18 resin (Chromatorex SMB 150, 20-45 μm). The elution of the purified siamenoside I and mogroside IIIE peaks was assessed by an elution step with increasing methanol or ethanol concentration, respectively.

The results for methanol are shown in table 19 (for siamenoside I elution) and in fig. 9.

Table 19: elution profile of siamenoside I from the secondary purification (C18 chromatography) of siamenoside I (using methanol-water mobile phase).

The results of methanol elution for siamenoside I are shown in Table 20

Siamenoside I can be separated from other mogrosides in the mixture with a purity greater than 95% with methanol concentrations between 30% and 40%.

Mogroside IIIE can be separated from other mogrosides in the mixture with a purity greater than 95% with methanol concentrations between 50% and 100% methanol.

Table 20: elution profile of sia.i from the secondary purification of sia.i (C18 chromatography) (using methanol-water mobile phase).

^tEstimating a standard curve based on using a sia.i reference standard; due to 200nThe baselines at m are uneven, and the HPLC peak area integral may be inaccurate; ND is not detected

Total recovery of sia.i 28.9g (about 90%); the recovery of sia.i with a purity of greater than 96% was 22.8g (79.2%); total volume of fractions containing pure sia.i 10L (20RV)

Example 7: purification of mogrosides from the reaction mixture with C18 resin

FIGS. 11-13 show schematically the purification scheme.

After the reactor

After the conversion reaction, mogrosides must be separated from the enzymes and salts. To separate the protein from the mogroside, the reaction mixture was mixed with sodium hydroxide and the pH was raised to 12.4. Adding ethanol to obtain 20% ethanol solution. The mixture was filtered through a 10kDa Koch Romicon membrane with an inlet pressure of 1.7 bar and an outlet at atmospheric pressure. The pH of the permeate was lowered to 5.5 using acetic acid and cooled overnight. The next day, the solution was re-filtered through a 10kDa Koch Romicon membrane.

The water, ethanol and salts were removed using a nanofiltration membrane Koch SR3D with a cut-off of 200 Da. The inlet pressure was 12 bar and the operation was carried out with a pressure drop of 0.6 bar. The solution was diafiltered until the ethanol concentration was less than 3% and concentrated to 20-30L. The concentrated mogroside was mixed with a water/ammonia acetate solution to bring the solution to about 110L.

Preparation of

The mixture was passed through a Biotage SNAP KP-C18-HS 400g guard cartridge. The resulting solution was mixed and aliquoted into 5L HDPE jerry cans (jerry cans) with 1 column load in each jerry can. Each load contained about 69g of siamenoside I. The load was used fresh or frozen at-15 ℃.

Chromatography method

The chromatographic separation has six stages:

1) balance post, ready to load.

2) The column is loaded. The load was followed by a small amount of equilibration solution to distribute the mogrosides throughout the bed.

3) Mogroside V and other early eluting compounds were removed. The first about 120L was sampled and sent to waste. The next approximately 27kg was collected and the target purity appeared in the final fraction.

4) Siamenoside I was removed and two large 18kg fractions followed by 4 × 4.5kg fractions. The final fraction usually does not meet the purity specification.

5) Direct XNS grade ethanol (95%). The first 18kg was used for collection of the Mog IIIe fractions.

6) Direct XNS grade ethanol to clean the column.

The composition of the eluent is shown in table 21.

Table 21: column eluent mixture

Table 22: column eluent mixture

The total mass is 71.2g, the mass average purity is 96.9 percent, and the recovery rate is 92.6 percent

Purity reached greater than 96% as assessed by peak area.

Between 40 and 70g of siamenoside I with a purity of 96% were collected per column run (each column run was loaded with about 5L of material). As shown in fig. 8, mogroside V was hydrolyzed to siamenoside I:

secondary purification-Chromatorex SMB C18.

Diafiltration (Perfect Inflation)

The fractions containing siamenoside I of suitable purity were further processed. These fractions must be freed from ethanol and ammonia acetate. The combined fractions were diafiltered and RO water was added to the solution to maintain the ethanol concentration below 15%. Ammonia, acetate, ethanol and water were removed. The operating pressure may be 12 bar and the pressure drop 0.6 bar. The solution was concentrated to about 50L. After every 10L of permeate collected, RO water was added in a split of 10L. The addition of RO water was continued until the salt and ethanol concentration of the solution decreased 1000 x-fold. About 350-L of diafiltration water was required after concentration. The ammonia salt concentration is expected to be 0.2ppm in solution, or 10ppm in the product.

Freeze drying

The siamenoside I concentrate was filtered through a 0.22mm filter into a lyophilization tray. After freeze-drying, the trays were cut open and ground to a fine powder. Before use, the powder was packaged in a clean-resistant (Nalgene) bottle.

Example 8: comparison of selected engineered enzymes for improved catalytic activity and specificity

Standard mogroside biocatalytic assays were performed in a volume of 500. mu.L of 50mM sodium citrate buffer (pH 5.6), 10mM magnesium chloride, 10mM substrate (usually mogroside V or siamenoside I) and 50. mu.g mL-1 enzyme at 37 ℃ and incubated for 24-96 hours. Mogroside glycosylation activity was detected by direct HPLC detection and quantification of mogroside V substrate and mogroside product from the filtered reaction supernatant and quantified by peak area comparison to a standard curve (see analytical methods).

The analysis method comprises the following steps: HPLC separation and quantitation of mogroside compounds was performed using a Synergi Hydro-RP column (250 mm. times.4.6 mm or 150 mm. times.4.6 mm) with an initial flow rate of 1mL min-1 and a water acetonitrile gradient as follows, similar to that described by Zhou et al (WO 2014/150127). Compounds were detected at 210nm using a diode array detector (agilent technologies, usa) and calibration curves were established using standard curves from 0.1 to 10 mM. 150mm column retention time in minutes: mog V9.5, sia 110.6, mog IVa 10.5, mog IV 11.3, mog III 12.6 and mogI I13.2. 250mm column retention time: mog V12.3, sia 113.2, mog IVa 13.5, mog IV 14.1, mog III 15.3 and mog II 16.2. Table 1 shows the chromatographic gradients.

Table 1:

the biochemical characteristics of the selected variants are reported in table 2:

table 2:

as a general trend, when the results in table 2 (above) and table 3 (below) are compared, an increase in catalytic efficiency (kcat/Km) for mogroside V and a decrease in catalytic efficiency for siamenoside I respectively result in an increase in yield of siamenoside I.

Table 3:

sequence listing

Coca Cola Company (The Coca Cola Company)

Biological catalysis method of mogroside

12600.105185

US 62/810,553

2019-02-26

SEQ ID NO: 1

Amino acids

Aspergillus oryzae

MKLLSVAAVALLAAQAAGASIKHRLNGFTILEHPDPAKRDLLQDIVTWDDKSLFINGERIMLFSGE

VHPFRLPVPSLWLDIFHKIRALGFNCVSFYIDWALLEGKPGDYRAEGIFALEPFFDAAKEAGIYLI

ARPGSYINAEVSGGGFPGWLQRVNGTLRSSDEPFLKATDNYIANAAAAVAKAQITNGGPVILYQPE

NEYSGGCCGVKYPDADYMQYVMDQARKADIVVPFISNDASPSGHNAPGSGTGAVDIYGHDSYPLGF

DCANPSVWPEGKLPDNFRTLHLEQSPSTPYSLLEFQAGAFDPWGGPGFEKCYALVNHEFSRVFYRN

DLSFGVSTFNLYMTFGGTNWGNLGHPGGYTSYDYGSPITETRNVTREKYSDIKLLANFVKASPSYL

TATPRNLTTGVYTDTSDLAVTPLIGDSPGSFFVVRHTDYSSQESTSYKLKLPTSAGNLTIPQLEGT

LSLNGRDSKIHVVDYNVSGTNIIYSTAEVFTWKKFDGNKVLVLYGGPKEHHELAIASKSNVTIIEG

SDSGIVSTRKGSSVIIGWDVSSTRRIVQVGDLRVFLLDRNSAYNYWVPELPTEGTSPGFSTSKTTA

SSIIVKAGYLLRGAHLDGADLHLTADFNATTPIEVIGAPTGAKNLFVNGEKASHTVDKNGIWSSEV

KYAAPEIKLPGLKDLDWKYLDTLPEIKSSYDDSAWVSADLPKTKNTHRPLDTPTSLYSSDYGFHTG

YLIYRGHFVANGKESEFFIRTQGGSAFGSSVWLNETYLGSWTGADYAMDGNSTYKLSQLESGKNYV

ITVVIDNLGLDENWTVGEETMKNPRGILSYKLSGQDASAITWKLTGNLGGEDYQDKVRGPLNEGGL

YAERQGFHQPQPPSESWESGSPLEGLSKPGIGFYTAQFDLDLPKGWDVPLYFNFGNNTQAARAQLY

VNGYQYGKFTGNVGPQTSFPVPEGILNYRGTNYVALSLWALESDGAKLGSFELSYTTPVLTGYGNV

ESPEQPKYEQRKGAY*

SEQ ID NO: 2

Amino acids

Aspergillus oryzae

MKLLSVAAVALLAAQAAGASIKHRLNGFTILEHPDPAKRDLLQDIVTWDDKSLFINGERIMLFSGE

VHPFRLPVPSLWLDIFHKIRALGFNCVSFYIDWALLEGKPGDYRAEGIFALEPFFDAAKEAGIYLI

ARPGSYINAEVSGGGFPGWLQRVNGTLRSSDEPFLKATDNYIANAAAAVAKAQITNGGPVILYQPE

NEYSGGCCGVKYPDADYMQYVMDQARKADIVVPFISNDASPSGHNAPGSGTGAVDIYGHDSYPLGF

DCANPSVWPEGKLPDNFRTLHLEQSPSTPYSLLEFQAGAFDPWGGPGFEKCYALVNHEFSRVFYRN

DLSFGVSTFNLYMTFGGTNWGNLGHPGGYTSYDYGSPITETRNVTREKYSDIKLLANFVKASPSYL

TATPRNLTTGVYTDTSDLAVTPLIGDSPGSFFVVRHTDYSSQESTSYKLKLPTSAGNLTIPQLEGT

LSLNGRDSKIHVVDYNVSGTNIIYSTAEVFTWKKFDGNKVLVLYGGPKEHHELAIASKSNVTIIEG

SDSGIVSTRKGSSVIIGWDVSSTRRIVQVGDLRVFLLDRNSAYNYWVPELPTEGTSPGFSTSKTTA

SSIIVKAGYLLRGAHLDGADLHLTADFNATTPIEVIGAPTGAKNLFVNGEKASHTVDKNGIWSSEV

KYAAPEIKLPGLKDLDWKYLDTLPEIKSSYDDSAWVSADLPKTKNTHRPLDTPTSLYSSDYGFHTG

YLIYRGHFVANGKESEFFIRTQGGSAFGSSVWLNETYLGSWTGADYAMDGNSTYKLSQLESGKNYV

ITVVIDNLGLDENWTVGEETMKNPRGILSYKLSGQDASAITWKLTGNLGGEDYQDKVRGPLNEGGL

YAERQGFHQPQPPSESWESGSPLEGLSKPGIGFYTAQFDLDLPKGWDVPLYFNFGNNTQAARAQLY

VNGYQYGKFTGNVGPQTSFPVPEGILNYRGTNYVALSLWALESDGAKLGSFELSYTTPVLTGYGNV

ESPEQPKYEQRKGAYLEAAAAASFLEQKLISEEDLNSAVDHHHHHH*

SEQ ID NO: 3

DNA

Aspergillus oryzae

atgaagttgttgtctgttgctgccgttgctttgttggctgctcaagctgctggtgcttctatcaaacatagattgaacggtttcaccatcttggaacatccagatccagctaaaagagatttgttgcaagatatcgttacctgggatgacaagtccttgtttattaacggtgaaaggatcatgttgttctccggtgaagttcatccttttagattgccagttccatctttgtggttggacattttccacaaaattagagccttgggtttcaactgcgtttccttttacattgattgggccttgttggaaggtaaaccaggtgattatagagccgaaggtatttttgctttggaaccatttttcgatgctgctaaagaagctggtatctacttgattgctagaccaggttcttacattaacgctcaggtttctggtggtggttttccaggttggttgcaaagagttaacggtactttgagatcttccgatgaaccattcttgaaggctaccgataattacattgctaatgctgctgctgcagttgctaaagctcaaattactaatggtggtccagtcatcttgtaccaaccagaaaatcagtactctggtggttgttgtggtgttaagtatccagatgctgattacatgcaatacgttatggatcaagctagaaaggccgatatcgttgttccattcatttctaatgatgcctctccatctggtcataatgctccaggttctggtactggtgctgttgatatctatggtcatcagtcttacccattgggtttcgattgtgctaatccatctgtttggccagaaggtaaattgccagataatttcagaaccttgcacttggaacaatctccatctactccatactcgttgttgcagtttcaagctggtgcatttgatccatggggtggtcctggttttgaaaaatgttatgccttggtcaaccacgagttctctagagttttttacagaaacgacttgtccttcggtgtttctactttcaacttgtacatgactttcggtggtaccaattggggtaatttgggtcatccaggtggttacacatcttatgattatggttctccaatcaccgaaaccagaaatgttactagggaaaagtactccgacattaagttgttggctaacttcgttaaggcttccccatcttatttgactgctactccaagaaatttgaccactggtgtctatactgatacctctgatttggctgttactccattgataggtgattcaccaggttcattcttcgttgttagacataccgattactcctctcaagaatctacctcctacaaattgaagttgcctacttctgctggtaacttgactattccacaactagaaggtacgctgtctttgaatggtagagattccaaaatccacgttgtcgactataacgtttctggcactaacattatctactctactgccgaagttttcacctggaagaaattcgatggtaacaaggttttggtcttgtacggtggtccaaaagaacatcatgaattggctattgcctccaagtctaacgttactattatcgaaggttccgactctggtatcgtttctactagaaaaggttcctccgttattatcggttgggatgtttcttctaccagaagaatcgttcaagttggtgacttgagagttttcttgttggatagaaactccgcttacaattactgggttccagaattgcctactgaaggtacttctccaggtttttctacttctaagactaccgcctcttccattattgtcaaagctggttatttgttgagaggtgctcatttggatggtgctgacttgcatttgacagctgattttaatgctactaccccaatcgaagttattggtgctccaactggtgctaagaatttgttcgttaatggtgaaaaggcctctcacactgttgataagaatggtatttggtcctccgaagttaagtatgctgctccagaaatcaaattgcctggtttgaaagatttggactggaagtacttggataccctgcctgaaatcaaaagctcttatgatgattctgcatgggtttctgctgatttgccaaagactaagaatacccatagacctttggatactccaacctccttgtattcttctgattacggttttcataccggctacttgatctacagaggtcattttgttgctaacggtaaagagtccgagttcttcattagaactcaaggtggttctgctttcggttcttctgtttggttgaacgaaacttacttaggttcttggacaggtgctgattatgctatggatggtaattctacctacaagttgtcccaattggaatccggtaagaactacgttattaccgttgtcatcgacaacttgggtttagacgaaaattggactgttggtgaagaaaccatgaagaacccaagaggtatcttgtcctataagttgtctggtcaagatgcttctgctattacttggaagttgacaggtaacttaggtggtgaagattaccaagataaggttagaggtccattgaatgaaggtggtctatatgctgaaagacaaggtttccatcaaccacaacctccatctgaatcttgggaatctggttcaccattggaaggtttgtctaaacctggtattggtttctacactgcccaattcgatttggatttgcctaaaggttgggacgttccattatacttcaactttggtaacaatacccaagctgctagagcccaattatatgttaatggttatcagtacggcaagttcactggtaatgttggtccacaaacatcttttccagtacctgagggtattttgaattacagaggtacaaattacgtcgccttgtcattgtgggctttagaatctgatggtgctaaattgggttccttcgaattgtcttataccactccagttttgactggttacggtaacgttgaatctccagaacaacctaaatacgaacaaagaaagggtgcctacctcgaggccgcggcggccgccagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattga

SEQ ID No: 4

DNA

5-GCTCGAATTCATGAAGTTGTTGTCTGTTGCTGCCG-3'

SEQ ID No: 5

DNA

5'-AAGCTTGGATCCTTAATAGGCACCTTTACG-3'

SEQ ID NO:6

DNA

5'-TAGATTGAACGGTTTCACCA-3'

SEQ ID NO:7

DNA

5'- TCAATGGAGTAACAGCCAAA-3'

SEQ ID NO 8

DNA

Aspergillus oryzae

Gtgctcgaattcatgaagttgttgtctgttgctgccgttgctttgttggctgctcaagctgctggtgcttctatcaaacatagattgaacggtttcaccatcttggaacatccagatccagctaaaagagatttgttgcaagatatcgttacctgggatgacaagtccttgtttattaacggtgaaaggatcatgttgttctccggtgaagttcatccttttagattgccagttccatctttgtggttggacattttccacaaaattagagccttgggtttcaactgcgtttccttttacattgattgggccttgttggaaggtaaaccaggtgattatagagccgaaggtatttttgctttggaaccatttttcgatgctgctaaagaagctggtatctacttgattgctagaccaggttcttacattaacgctgaagtttctggtggtggttttccaggttggttgcaaagagttaacggtactttgagatcttccgatgaaccattcttgaaggctaccgataattacattgctaatgctgctgctgcagttgctaaagctcaaattactaatggtggtccagtcatcttgtaccaaccagaaaatgaatactctggtrrkrrktgtggtrrkaagtatccagatgctgattacatgcaatacgttatggatcaagctagaaaggccgatatcgttgttccattcatttctaatgatgcctctccatctggtcataatgctccaggttctggtactggtgctgttgatatctatggtcatgattcttacccattgggtttcgattgtgctaatccatctgtttggccagaaggtaaattgccagataatttcagaaccttgcacttggaacaatctccatctactccatactcgttgttggaatttcaagctggtgcatttgatccatggggtggtcctggttttgaaaaatgttatgccttggtcaaccacgagttctctagagttttttacagaaacgacttgtccttcggtgtttctactttcaacttgtacatgactttcggtggtaccaattggggtaatttgggtcatccaggtggttacacatcttatgat