Enzymatic production of alpha-1, 3-glucan
阅读说明：本技术 α-1,3-葡聚糖的酶促生产 (Enzymatic production of alpha-1, 3-glucan ) 是由 K.D.纳吉 S.M.亨尼西 Y.布伦 M.莱荷曼 于 2018-05-22 设计创作，主要内容包括：公开了一种用于生产不溶性α-1,3-葡聚糖的方法。所述方法的实施例包括提供(i)包含α-1,3和α-1,6糖苷键的低聚糖,或(ii)衍生自葡糖基转移酶反应的低聚糖；和至少接触水、蔗糖、葡糖基转移酶、和在第一步骤中提供的低聚糖。还公开了体现此类方法的葡糖基转移酶反应组合物、及其不溶性产物。当实践所公开的主题时,可实现产率和其他的产物益处。(A method for producing insoluble alpha-1, 3-glucan is disclosed. Embodiments of the method include providing (i) an oligosaccharide comprising alpha-1, 3 and alpha-1, 6 glycosidic linkages, or (ii) an oligosaccharide derived from a glucosyltransferase reaction; and contacting at least water, sucrose, glucosyltransferase, and oligosaccharide provided in the first step. Glucosyltransferase reaction compositions embodying such methods, and insoluble products thereof, are also disclosed. Yields and other product benefits can be realized when practicing the disclosed subject matter.)
1. A method for producing insoluble alpha-1, 3-glucan, the method comprising:
(a) providing an oligosaccharide which:
(i) contain alpha-1, 3 and alpha-1, 6 glycosidic linkages, and/or
(ii) Results from the glucosyltransferase reaction;
(b) contacting at least water, sucrose, said oligosaccharide, and a glucosyltransferase enzyme that synthesizes insoluble alpha-1, 3-glucan,
wherein insoluble alpha-1, 3-glucan is produced; and
(c) optionally, isolating the insoluble alpha-1, 3-glucan produced in step (b).
2. The method of claim 1, wherein the oligosaccharide comprises from about 60% to about 99% alpha-1, 3 glycosidic linkages and from about 1% to about 40% alpha-1, 6 glycosidic linkages.
3. The method of claim 1, wherein the oligosaccharide has a Degree of Polymerization (DP) of 2 to 10.
4. The method of claim 1, wherein the oligosaccharide is purified or unpurified.
5. The method of claim 4, wherein the oligosaccharide is produced from the glucosyltransferase reaction of (a) (ii).
6. The method of claim 5, wherein said glucosyltransferase enzyme of (a) (ii) reacts to synthesize insoluble alpha-1, 3-glucan.
7. The method of claim 5, wherein the oligosaccharide provides a soluble fraction of the glucosyltransferase reaction of (a) (ii), and wherein the soluble fraction is treated or untreated.
8. The method of claim 7, wherein the soluble fraction is a portion or all of the filtrate of the glucosyltransferase reaction of (a) (ii).
9. The method of claim 1, wherein the oligosaccharide is provided in step (b) at an initial concentration of at least about 1 g/L.
10. The method of claim 1, wherein the yield of insoluble alpha-1, 3-glucan produced is increased compared to the yield of insoluble alpha-1, 3-glucan produced if the oligosaccharide was absent in step (b).
11. The method of claim 1, wherein the viscosity of the insoluble a-1, 3-glucan produced is reduced compared to the viscosity of the insoluble a-1, 3-glucan that would be produced if the oligosaccharide were absent in step (b), wherein viscosity is measured as alpha-1, 3-glucan mixed or dissolved in a liquid.
12. The method of claim 1 wherein the insoluble alpha-1, 3-glucan produced has at least 50% alpha-1, 3 glycosidic linkages and a weight average degree of polymerization (DPw) of at least 100.
13. The method of claim 1, wherein steps (a) and (b) are repeated one or more times, and wherein in each repeated step (a), the oligosaccharide is provided from the product produced from each immediately preceding step (b).
14. A reaction composition for the production of insoluble alpha-1, 3-glucan, the reaction composition comprising at least water, sucrose, glucosyltransferase enzyme that synthesizes insoluble alpha-1, 3-glucan, and oligosaccharide, wherein the oligosaccharide is added during preparation of the reaction composition and:
(i) contain alpha-1, 3 and alpha-1, 6 glycosidic linkages, and/or
(ii) Is generated from the reaction of glucosyltransferase,
wherein insoluble alpha-1, 3-glucan is produced in the reaction composition.
15. A composition comprising an insoluble alpha-1, 3-glucan produced by the method of claim 1.
16. The composition of claim 15 wherein the viscosity of the insoluble alpha-1, 3-glucan is less than the viscosity of insoluble alpha-1, 3-glucan that would result if the oligosaccharide were not provided in the method or reaction composition, wherein viscosity is measured for alpha-1, 3-glucan mixed or dissolved in a liquid.
Technical Field
The present disclosure is in the field of enzymatic processes. For example, the present disclosure relates to glucosyltransferase reactions comprising added oligosaccharides.
Reference to electronically submitted sequence Listing
An official copy of this sequence listing was submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file name 20180522_ CL6007WOPCT _ sequence listing, created on day 5/18 of 2018, and having a size of about 157 kilobytes, and submitted concurrently with this specification. The sequence listing contained in this ASCII formatted file is part of this specification and is incorporated by reference in its entirety.
Background
Driven by the desire to use polysaccharides in a variety of applications, researchers have explored polysaccharides that are biodegradable and can be economically manufactured from renewable sources of raw materials. One such polysaccharide is alpha-1, 3-glucan, an insoluble glucan polymer characterized by having alpha-1, 3-glucosidic linkages. For example, such polymers have been prepared using a glucosyltransferase isolated from Streptococcus salivarius (Simpson et al Microbiology 141: 1451-. Also for example, U.S. patent No. 7000000 discloses the preparation of spun fibers from enzymatically produced poly alpha-1, 3-glucan.
Enzymatic synthesis of various glucan polymers has been performed in reactions in which polysaccharides (e.g., dextran) or oligosaccharides (e.g., polysaccharides from hydrolysis) have been added to affect glucosyltransferase function (e.g., Koga et al, 1983, j.gen. microbiol. [ journal of general microbiology ] 129: 751-754; Komatsu et al, 2011, FEBSJ. [ journal of FEBS ] 278: 531-540; Simpson et al; O' Brien et al, U.S. patent No. 8642757). Despite these disclosures, little is known about the regulation of glucosyltransferase reactions for insoluble α -1, 3-glucan synthesis.
Disclosure of Invention
In one embodiment, the present disclosure relates to a method for producing insoluble alpha-1, 3-glucan, the method comprising:
(a) providing an oligosaccharide which:
(i) contain alpha-1, 3 and alpha-1, 6 glycosidic linkages, and/or
(ii) Results from the glucosyltransferase reaction;
(b) contacting at least water, sucrose, an oligosaccharide, and a glucosyltransferase enzyme that synthesizes insoluble alpha-1, 3-glucan,
wherein insoluble alpha-1, 3-glucan is produced; and
(c) optionally, isolating the insoluble alpha-1, 3-glucan produced in step (b).
In another embodiment, the present disclosure relates to a reaction composition for producing insoluble alpha-1, 3-glucan, the reaction composition comprising at least water, sucrose, a glucosyltransferase enzyme that synthesizes insoluble alpha-1, 3-glucan, and an oligosaccharide, wherein the oligosaccharide is added during preparation of the reaction composition and (i) comprises alpha-1, 3 and alpha-1, 6 glycosidic linkages, and/or (ii) results from a glucosyltransferase reaction, wherein insoluble alpha-1, 3-glucan is produced in the reaction composition.
In another embodiment, the present disclosure relates to a composition comprising insoluble alpha-1, 3-glucan produced according to any of the methods of producing insoluble alpha-1, 3-glucan described herein.
Drawings
FIG. 1: of concentrated oligosaccharide preparations1H-NMR spectrum (see example 2).
FIG. 2: the figure shows the aqueous slurry viscosity of each a-1, 3-glucan product prepared in three consecutive reactions, where the second and third reactions comprise filtrates derived from the first and second reactions, respectively (see example 10). Squares, circles and diamonds illustrate viscosity measurements obtained with aqueous slurries of alpha-1, 3-glucan produced in the first, second and third reactions, respectively. Shear rate is given in units of 1/s (shown as "s-1").
TABLE 1 summary of nucleic acid and protein sequence identification numbers
aThis DNA coding sequence is codon optimized for expression in e.coli (e.coli) and is disclosed only as an example of a suitable coding sequence.
Detailed Description
The disclosures of all cited patent and non-patent documents are incorporated herein by reference in their entirety.
The term "a" as used herein is intended to encompass one or more (i.e. at least one) of the referenced feature(s), unless otherwise disclosed.
All ranges, if any, are inclusive and combinable unless otherwise stated. For example, when a range of "1 to 5" is recited, the recited range should be interpreted to include ranges of "1 to 4", "1 to 3", "1 to 2 and 4 to 5", "1 to 3 and 5", and the like.
The term "saccharide" as used herein, unless otherwise indicated, refers to mono-and/or di/oligosaccharides. "disaccharide" herein refers to a carbohydrate having two monosaccharides linked by a glycosidic bond. An "oligosaccharide" herein may refer to a carbohydrate having, for example, 2 to 15 monosaccharides linked by glycosidic linkages. Oligosaccharides may also be referred to as "oligomers". Monosaccharides (e.g., glucose and/or fructose) contained within a di/oligosaccharide can be referred to as "monomeric units," "monosaccharide units," or other similar terms.
The terms "a-glucan", "a-glucan polymer", and the like are used interchangeably herein. Alpha-glucan is a polymer comprising glucose monomer units linked together by alpha-glycosidic bonds. In typical embodiments, an α -glucan herein comprises at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% α -glycosidic linkages. Alpha-1, 3-glucan is an example of alpha-glucan.
The terms "alpha-1, 3-glucan", "poly alpha-1, 3-glucan", "alpha-1, 3-glucan polymer", and the like are used interchangeably herein. Alpha-1, 3-glucan is a polymer comprising glucose monomeric units linked together by glycosidic bonds (i.e., glucosidic bonds), typically wherein at least about 50% of the glycosidic bonds are alpha-1, 3-glycosidic bonds. In certain embodiments, the α -1, 3-glucan comprises at least about 90% or 95% α -1,3 glycosidic linkages. Most or all of the other linkages in the alpha-1, 3-glucan herein are typically alpha-1, 6, although some linkages may also be alpha-1, 2 and/or alpha-1, 4.
The terms "glycosidic linkage" and "glycosidic bond", "linkage", and the like are used interchangeably herein and refer to the type of covalent bond that links carbohydrate (saccharide) molecules to each other. All glycosidic linkages disclosed herein are alpha-glycosidic linkages unless otherwise indicated. "glucoside linkage" refers to a glycosidic linkage between alpha-D-glucose and another carbohydrate molecule. "alpha-D-glucose" will also be referred to herein as "glucose". The terms "α -1,3 glucosyl-glucose linkage", "α -1,3 glucose-glucose linkage" and "glucose- α 1, 3-glucose" herein refer to the α -1,3 glycosidic linkage. The terms "α -1, 6 glucosyl-glucose linkage", "α -1, 6 glucose-glucose linkage" and "glucose- α 1, 6-glucose" herein refer to the α -1, 6 glycosidic linkage.
The glycosidic bond profile of any polysaccharide herein (e.g., a-1, 3-glucan, oligosaccharide) can be determined using any method known in the art. For example, a method using Nuclear Magnetic Resonance (NMR) spectroscopy (e.g.,13c NMR of1H NMR) to determine a bond spectrum. These and other methods that may be used are disclosed in,Food Carbohydrates： Chemistry，Physical Properties，and Applications[food carbohydrate: chemical, physical characteristics and applications of](S.W.Cui eds., Chapter 3, S.W.Cui, Structural Analysis of Polysaccharides [ Structural Analysis of Polysaccharides ]]，Taylor&Francis Group LLC, Boca Raton, FL [ Taylor and Francis Group, Inc. of Pokaladton, Florida]2005), which is incorporated herein by reference.
The "molecular weight" of the large alpha-glucan polymer herein may be expressed as a weight average molecular weight (Mw) or a number average molecular weight (Mn) in units of daltons or grams/mole. Alternatively, the molecular weight of the large α -glucan polymer may be expressed as DPw (weight average degree of polymerization) or DPn (number average degree of polymerization). If desired, the molecular weight of the smaller alpha-glucan polymer (e.g., oligosaccharide) can typically be provided in terms of "DP" (degree of polymerization), which refers only to the amount of glucose contained within the alpha-glucan. Various means for calculating these different molecular weight measurements are known in the art, for example using High Pressure Liquid Chromatography (HPLC), Size Exclusion Chromatography (SEC) or Gel Permeation Chromatography (GPC).
The terms "glucosyltransferase", "GTF", "glucansucrase" and the like are used interchangeably herein. The activity of glucosyltransferase herein catalyzes the reaction of the substrate sucrose to make the products alpha-glucan and fructose. Other products (by-products) of the glucosyltransferase reaction may include glucose, various soluble gluco-oligosaccharides, and leucrose. The wild-type form of glucosyltransferase typically contains (in the N-terminal to C-terminal direction) a signal peptide (usually removed by the cleavage process), a variable domain, a catalytic domain, and a glucan-binding domain. Glucosyltransferases herein are classified under glycoside hydrolase family 70(GH70) according to the CAZy (carbohydrate active enzymes) database (Cantarel et al, Nucleic Acids Res. [ Nucleic Acids research ] 37: D233-238, 2009).
The term "glucosyltransferase catalytic domain" as used herein refers to the domain of glucosyltransferase that provides alpha-glucan synthesis activity to glucosyltransferase. Typically, the glucosyltransferase catalytic domain does not require the presence of any other domain to have this activity.
The terms "enzymatic reaction," "glucosyltransferase reaction," "glucan synthesis reaction," "reaction composition," "reaction formulation," and the like are used interchangeably herein and generally refer to a reaction that initially comprises water, sucrose, at least one active glucosyltransferase, and optionally other components. Components that may further be present in the glucosyltransferase reaction typically after the start of the reaction include fructose, glucose, leucrose, soluble glucose-oligosaccharides (e.g., DP2-DP7) (such sugars may be considered products or by-products based on the glucosyltransferase enzyme used), and/or one or more insoluble alpha-glucan products of DP8 or higher (e.g., DP100 and higher). It will be appreciated that certain glucan products (e.g. alpha-1, 3-glucan) having a Degree of Polymerization (DP) of at least 8 or 9 are water insoluble ("insoluble alpha-1, 3-glucan") and therefore do not dissolve in the glucan synthesis reaction, but may be present outside the solution (e.g. due to precipitation from the reaction). In the glucan synthesis reaction, a step of contacting water, sucrose and glucosyltransferase is performed. The term "under suitable reaction conditions" as used herein refers to reaction conditions that support the conversion of sucrose to one or more alpha-glucan products by glucosyltransferase activity.
As used herein, a "control" reaction may refer to a glucosyltransferase reaction in which no oligosaccharide comprising (collectively) alpha-1, 3 and alpha-1, 6 glycosidic linkages is added directly to the reaction. All other characteristics of the control reaction solution (e.g., sucrose concentration, temperature, pH, GTF type) may be the same as those of the reaction composition to which it is compared.
The "percent dry solids" (percent DS) of a solution (e.g., soluble fraction, aqueous composition) herein refers to the wt% of all materials (i.e., solids) dissolved in the solution. For example, a 100g solution with a DS of 10 wt% contains 10g of dissolved material.
In certain embodiments, the "yield" of α -1, 3-glucan produced by the glucosyltransferase reaction represents the weight of α -1, 3-glucan product converted in the reaction expressed as a weight percentage of sucrose substrate. For example, if 100g of sucrose in the reaction solution is converted into a product and 10g of the product is α -1, 3-glucan, the yield of α -1, 3-glucan is 10%. In some aspects, the "yield" of α -1, 3-glucan produced by the glucosyltransferase reaction represents the molar yield based on sucrose converted. The molar yield of the a-glucan product can be calculated based on the moles of the a-glucan product divided by the moles of sucrose converted. The moles of sucrose converted can be calculated as follows: (initial sucrose mass-final sucrose mass)/sucrose molecular weight [342g/mol ]. These yield calculations (either weight or molar) can be considered as a measure of the selectivity of the reaction to alpha-1, 3-glucan. In some aspects, the "yield" of the a-glucan product in the glucosyltransferase reaction can be based on the glucosyl component of the reaction. Such a yield (glucosyl group-based yield) can be measured using the following formula:
α -glucan yield ═ 100% (IS/2- (FS/2+ LE/2+ GL + SO))/(IS/2-FS/2)).
The fructose balance of the glucosyltransferase reaction can be measured to ensure that the HPLC data (if applicable) is not out of range (90% -110% is considered acceptable). The fructose balance can be measured using the following formula:
fructose equilibrium ═ 100 ((180/342x (FS + LE) + FR)/(180/342x IS)).
In both equations above, IS [ initial sucrose ], FS [ final sucrose ], LE [ leucrose ], GL [ glucose ], SO [ soluble oligomer ] (glucose-oligosaccharide), and FR [ fructose ] (all concentrations are in grams/L and as measured, for example, by HPLC).
The term "relative reaction rate" as used herein refers to the rate of a particular glucan synthesis reaction compared to another glucan synthesis reaction. For example, if reaction A has a rate of x and reaction B has a rate of y, then the relative reaction rate of reaction A relative to the reaction rate of reaction B may be expressed as x/y (x divided by y). The terms "reaction rate" and "rate of reaction" are used interchangeably herein to refer to the change in the concentration/amount of one or more reactants or the concentration/amount of one or more products per unit time per unit enzyme. Given that GTF enzymes are known to follow the kinetics of Michaelis-manten, these rates are typically measured at the beginning of the polymerization, at which time the amount of sucrose is much higher than the Km of the enzyme. In this case, the rate is typically measured when the amount of sucrose in the reaction is higher than at least about 50g/L sucrose. Preferred reactants and products herein for the glucan synthesis reaction are sucrose and alpha-1, 3-glucan, respectively.
As used herein, the "soluble fraction" or "soluble portion" of the glucosyltransferase reaction refers to the portion of the liquid solution of the glucosyltransferase reaction. The soluble fraction may be part or all of the liquid solution from the glucosyltransferase reaction and typically has been separated from the insoluble glucan product synthesized in the reaction. The soluble fraction may alternatively be referred to as "mother liquor". An example of a soluble fraction is the filtrate of the glucosyltransferase reaction. Since the soluble fraction may contain dissolved sugars, such as sucrose, fructose, glucose, leuconostoc, soluble gluco-oligosaccharides, the fraction may also be referred to as a "mixed sugar solution" derived from the glucosyltransferase reaction. The soluble fraction herein may remain untreated after it is obtained, or alternatively, it may be subjected to one or more treatment steps as disclosed herein.
The terms "filtrate," "glucan reaction filtrate," and the like are used interchangeably herein and refer to the soluble fraction filtered from the insoluble glucan product synthesized in the glucosyltransferase reaction.
The terms "percent by volume", "volume percent", "volume (%)", "volume/volume (% vol%)", and the like are used interchangeably herein. The percentage by volume of solute in the solution can be determined using the following formula: [ (solute volume)/(solution volume) ] × 100%.
The terms "percent by weight (percent by weight)", "weight percent (wt%)", "weight-weight percent (wt/w)", and the like are used interchangeably herein. By weight ratio is meant the percentage of a material on a mass basis when the material is contained in a composition, mixture, or solution.
The term "aqueous conditions" and like terms herein refer to a solution or mixture wherein the solvent is, for example, at least about 60 wt% water. The glucosyltransferase reaction herein is carried out under aqueous conditions.
"insoluble", "aqueous insoluble", "water-insoluble" glucan (and similar terms) (e.g., insoluble alpha-1, 3-glucan) is insoluble (or not significantly soluble) in water or other aqueous conditions, optionally wherein the aqueous conditions are further characterized as having a pH of 4-9 (e.g., 6-8) and/or a temperature of about 1 ℃ to 85 ℃ (e.g., 20 ℃ to 25 ℃). In contrast, glucans, such as certain "soluble", "aqueous soluble", "water-soluble" oligosaccharides and the like herein, are significantly solubilized under these conditions.
An "aqueous composition" herein has a liquid component that contains, for example, at least about 10 wt% water (e.g., the liquid component can be at least about 70%, 80%, 90%, 95% water, or 100% water). Examples of aqueous compositions include, for example, mixtures, solutions, dispersions (e.g., colloidal dispersions), suspensions, and emulsions. In certain embodiments, the aqueous composition comprises alpha-1, 3-glucan as produced herein, which is mixed (e.g., by homogenization) or dissolved (e.g., by dissolution under caustic aqueous conditions, e.g., at a pH of at least 11.0 [ as provided using an alkaline solute, such as, for example, NaOH or KOH) in the aqueous composition. The "non-aqueous composition" herein may be "dry" (e.g., containing no more than 2.0, 1.5, 1.0, 0.5, 0.25, 0.10, 0.05, or 0.01 wt% water) and/or contain non-aqueous liquid components (e.g., an organic liquid that can dissolve alpha-1, 3-glucan, such as N, N-dimethylacetamide (DMAc)/0.5% -5% LiCl).
The term "purified" in this context characterizes an oligosaccharide preparation which comprises not more than 25% (by dry weight) of saccharides and/or other salt-free/buffer-free materials which are not comprised by the above definition of oligosaccharides. As implied by the definition, the purified oligosaccharide preparation may optionally comprise salts and/or buffers, the level of either of which does not determine the oligosaccharide purity. The term "unpurified" herein may characterize an oligosaccharide preparation comprising more than 25% (by dry weight) of saccharides and/or other salt-free/buffer-free materials not comprised by the above definition of oligosaccharides.
As used herein, the term "viscosity" refers to a measure of the degree to which a fluid (aqueous or non-aqueous) resists forces that tend to cause it to flow. Various viscosity units that can be used herein include, for example, centipoise (cP, cps) and pascal seconds (Pa · s). One centipoise is one percent of one poise; one poise is equal to 0.100 kg.m-1·s-1. In some aspects, viscosity may be reported as "intrinsic viscosity" (units of IV, η, mL/g); the term refers to a measure of the viscosity contribution of the dextran polymer to a liquid (e.g., a solution) containing the dextran polymer.
The term "increased" as used herein may refer to an amount or activity that is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 50%, 100%, or 200% more than the amount or activity to which the increased amount or activity is compared. The terms "increased", "enhanced", "greater than", "improved", and the like are used interchangeably herein. For example, these terms may be used to characterize "overexpression" or "upregulation" of a polynucleotide encoding a protein.
As used herein, the terms "sequence identity", "identity", and the like with respect to a polynucleotide or polypeptide sequence herein, refer to nucleic acid residues or amino acid residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. Thus, "percent sequence identity," "percent identity," and the like, refer to the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) when the optimal alignment of the two sequences is compared to a reference sequence (which does not comprise additions or deletions). The percentage is calculated by: determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and then multiplying the result by 100 to yield the percentage of sequence identity. It will be understood that when calculating sequence identity between a DNA sequence and an RNA sequence, the T residues of the DNA sequence are aligned with the U residues of the RNA sequence and may be considered "identical" thereto. For the purpose of determining the "percent complementarity" of the first and second polynucleotides, it may be obtained by determining (i) the percent identity between the complement sequences of the first and second polynucleotides (or vice versa), for example and/or (ii) the percentage of bases between the first and second polynucleotides that will result in canonical Watson and Crick base pairs.
Percent identity can be readily determined by any known method, including but not limited to those described in the following documents: 1)computational Molecular Biology [ computational Molecular Biology ]](Lesk, a.m. ed) oxford university: NY (1988); 2)biocomputing: information and Genome Projects [ biological: informatics And genomic items](Smith, d.w. editors) Academic press (Academic): NY (1993); 3)Computer ahaliysis of Sequence Data, Part I [ computer scoring of Sequence DataAnalysis of the first part](Griffin, a.m. and Griffin, h.g. editions) limania press (Humana): NJ (1994); 4)Sequence Analysis in molecular Biology [ sequence analysis in Molecular Biology ]](von Heinje, g. editors) Academic (1987); and 5)Sequence Analysis Primer [ Sequence Analysis Primer ]](Gribskov, m. and Devereux, j., ed.) stokes press (Stockton): NY (1991), incorporated herein by reference in its entirety.
The preferred method for determining percent identity is designed to give the best match between test sequences. For example, methods of determining identity and similarity are programmed into publicly available computer programs. For example, sequence alignments and percent identity calculations can be performed using the MEGALIGN program of LASERGENE bioinformatics computing suite (DNASTAR corporation, Madison, wisconsin). For example, multiple alignments of sequences can be performed using the Clustal alignment method, which covers several algorithms, including the Clustal V alignment method (described in Higgins and Sharp, CABIOS. [ computer applications in biology ] 5: 151-. For multiple alignments, the default values may correspond to a gap penalty of 10 (GAP PENALTY) and a gap length penalty of 10 (GAP LENGTH PENALTY). Default parameters for the calculation of percent identity for alignment-by-alignment pairs and protein sequences using the Clustal method may be KTUPLE-1, gap penalty-3, WINDOW-5, and stored diagonal (DIAGONALS SAVED) -5. For nucleic acids, these parameters may be KTUPLE 2, gap penalty 5, window 4, and stored diagonal 4. Furthermore, the Clustal W alignment method (described in Higgins and Sharp, CABIOS. [ in silico applications ] 5: 151-. Default parameters for multiple alignments (protein/nucleic acid) can be: gap penalty 10/15, gap length penalty 0.2/6.66, delayed divergence sequence (Delav Divergen seq) (%) 30/30, DNA transition weight 0.5, protein weight matrix Gonnet series, DNA weight matrix IUB.
As a feature of certain embodiments, various polypeptide amino acid sequences and polynucleotide sequences are disclosed herein. Variants of these sequences having at least about 70% -85%, 85% -90%, or 90% -95% identity to the sequences disclosed herein may be used or referenced. Alternatively, a variant amino acid sequence or polynucleotide sequence may be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence disclosed herein. Variant amino acid sequences or polynucleotide sequences have the same function/activity of the disclosed sequences, or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the function/activity of the disclosed sequences. Typically, any polypeptide amino acid sequence disclosed herein that does not start with a methionine may further comprise at least one initiating methionine at the N-terminus of the amino acid sequence. In contrast, any polypeptide amino acid sequence disclosed herein that begins with methionine may optionally lack such a methionine residue.
The compositions (e.g., in certain embodiments, insoluble alpha-1, 3-glucan) and glucosyltransferase reactions/methods disclosed herein are believed to be synthetic and non-naturally occurring. Thus, such aspects herein may optionally be characterized as "isolated," meaning that they may, for example, be performed in a manner that would not otherwise exist in nature. It is further believed that the nature/effect of the above-described subject matter is not naturally occurring.
It is now disclosed that yield and other product benefits can be achieved when certain oligosaccharides are used in the glucosyltransferase reaction to produce insoluble alpha-1, 3-glucan.
Embodiments of the present disclosure relate to methods for producing insoluble alpha-1, 3-glucan. The method comprises the following steps:
(a) providing an oligosaccharide which:
(i) contain alpha-1, 3 and alpha-1, 6 glycosidic linkages, and/or
(ii) Results from the glucosyltransferase reaction; and
(b) contacting at least water, sucrose, glucosyltransferase, and oligosaccharide provided in step (a). Insoluble alpha-1, 3-glucan is produced in this process. In certain embodiments, the yield of insoluble alpha-1, 3-glucan product is increased compared to the yield of insoluble alpha-1, 3-glucan that would have been produced if step (b) was performed without the oligosaccharides provided in step (a). The insoluble alpha-1, 3-glucan produced in step (b) of the present method may optionally be isolated.
Notably, disclosed herein are oligosaccharides comprising alpha-1, 3 and alpha-1, 6 glycosidic linkages to modulate the activity of glucosyltransferase enzymes that produce insoluble alpha-1, 3-glucan. Such oligosaccharides may optionally be derived as a by-product of the glucosyltransferase reaction as disclosed herein. Thus, in certain embodiments, the disclosed methods represent advantageous methods for recycling the oligosaccharide by-product of the glucosyltransferase reaction. It is also significant herein that even if provided in an unpurified state, e.g., in a filtrate obtained from a glucosyltransferase reaction, the oligosaccharide by-product can be used to modulate glucosyltransferase activity.
In certain embodiments, the oligosaccharides of the present disclosure comprise alpha-1, 3 glycosidic and alpha-1, 6 glycosidic linkages. For example, an oligosaccharide herein can comprise from about 60% to 99%, 60% to 95%, 70% to 90%, or 80% to 90% alpha-1, 3 glycosidic linkages and from about 1% to 40%, 5% to 40%, 10% to 30%, or 1% to 10% alpha-1, 6 glycosidic linkages. Nonetheless, in some aspects, the oligosaccharide can comprise about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (or ranges between any two of these values) alpha-1, 3 glycosidic linkages and about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, (all inclusive), 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% (or a range between any two of these values). Such a bond profile may characterize an oligosaccharide of any molecular weight herein (e.g., DP2-7, DP2-8, DP2-9, or DP 2-10). The aforementioned spectrogram can optionally characterize the gluco-oligosaccharides.
The oligosaccharides herein may for example "collectively comprise" any of the aforementioned linkage profiles. By "collectively comprising" is meant that the bond profile of the mixture of various oligosaccharides is based on the combination of all bonds present in the composition. Oligosaccharides as used herein can thus include specific oligosaccharide species containing only alpha-1, 3 glycosidic linkages, only alpha-1, 6 glycosidic linkages, and/or containing alpha-1, 3 and alpha-1, 6 glycosidic linkages, provided that the total linkage pattern of all oligosaccharide species present falls within any of the aforementioned linkage patterns (e.g., about 78% alpha-1, 3 linkages and about 22% alpha-1, 6 linkages, or about 87-88% alpha-1, 3 linkages and about 7% alpha-1, 6 linkages). In certain aspects, the oligosaccharides do not/collectively comprise 100% alpha-1, 3 glycosidic linkages or 100% alpha-1, 6 glycosidic linkages.
The gluco-oligosaccharides herein preferably contain a majority of alpha-1, 3 and alpha-1, 6 glycosidic linkages. For example, at least about 95%, 96%, 97%, 98%, 99%, or 100% of the total linkages of the oligosaccharide are alpha-1, 3 and alpha-1, 6 glycosidic linkages. Other linkages, if present in the oligosaccharide, may be, for example, alpha-1, 4 (e.g., ≦ 1.5% or 1%) or alpha-1, 2 (e.g., ≦ 1% or 0.7%) glycosidic linkages.
In some aspects, the oligosaccharides herein can have a Degree of Polymerization (DP) of 2 to 15. By way of example, the oligosaccharide may have a DP of 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14, or 2-15. As will be understood in the art, a group of oligosaccharides herein may be referenced with respect to DP number or range, which specifies the number or range of monomer units of a single oligosaccharide species in the group. For example, the DP2-7 oligosaccharides typically comprise a mixture of DP2, DP3, DP4, DP5, DP6 and DP7 oligosaccharides. The aforementioned oligosaccharides may optionally be referred to as gluco-oligosaccharides.
The distribution of oligosaccharides in the compositions used to provide oligosaccharides herein can vary. For example, a composition comprising DP2-7 oligosaccharides may comprise oligosaccharides having a profile that is the same as or similar to that disclosed below in table 5. Thus, a composition comprising DP2-7 oligosaccharides may comprise, based on the saccharide component of the composition or on dry weight, for example, about 5-15 wt% (e.g., about 9-11 wt%) DP2, about 19-29 wt% (e.g., about 23-25 wt%) DP3, about 27-37 wt% (e.g., about 31-33 wt%) DP4, about 15-25 wt% (e.g., about 19-21 wt%) DP5, about 3-13 wt% (e.g., about 7-9 wt%) DP6, and about 1-10 wt% (e.g., about 4-6 wt%) DP7 oligosaccharides. In some aspects, a composition comprising the oligosaccharide of DP2-7 may comprise an oligosaccharide having the same or similar profile as disclosed below in table 16. Thus, a composition comprising DP2-7 oligosaccharides may comprise, based on the saccharide component of the composition or on dry weight, for example, about 6-16 wt% (e.g., about 10-12 wt%) DP2, about 18-28 wt% (e.g., about 22-24 wt%) DP3, about 23-33 wt% (e.g., about 27-29 wt%) DP4, about 16-26 wt% (e.g., about 20-22 wt%) DP5, about 7-17 wt% (e.g., about 11-13 wt%) DP6, and about 1-10 wt% (e.g., about 4-6 wt%) DP7 oligosaccharides. It is believed that the exact DP distribution is not critical to the present disclosure; other distributions should provide the same behavior as described herein.
In certain embodiments of the present disclosure, the oligosaccharide may be purified or unpurified. Purified oligosaccharides may be provided using any suitable method in the art, for example by chromatography as disclosed in the examples below or by following the disclosure of european patent publication No. EP 2292803B1, which is incorporated herein by reference. For example, the purified oligosaccharides may be provided in dry form or in aqueous form (aqueous solution), each of which may optionally further contain one or more salts (e.g., NaCl) and/or buffers. In certain embodiments, the purified oligosaccharide formulation can comprise less than about 25, 20, 15, 10, 5, 2.5, 2, 1.5, 1.0, 0.5, or 0.1 wt% of (i) saccharides that are not encompassed by the definition of oligosaccharides as disclosed herein (e.g., the oligosaccharides herein are not monosaccharides or DP11+ saccharides) and/or (ii) other salt-free/buffer-free materials.
Unpurified oligosaccharides may be used in certain embodiments of the present disclosure. An unpurified oligosaccharide preparation can comprise, for example, more than about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% wt.% of saccharides and/or other salt-free/buffer-free materials that are not included as a definition of oligosaccharide disclosed herein. An example of an unpurified oligosaccharide preparation herein is a soluble fraction (e.g., filtrate) from a glucosyltransferase reaction. Other "salt-free/buffer-free materials" that may be present in the soluble fractions herein include, for example, sucrose, fructose, glucose, leucrose, and glucosyltransferase proteins.
The oligosaccharides provided in step (a) of the disclosed methods may be produced ("derived from", derived from or obtained from) the glucosyltransferase reaction. For example, the oligosaccharide may be a byproduct of the glucosyltransferase reaction. Such byproducts may result from the glucosyltransferase reaction that synthesizes insoluble alpha-1, 3-glucan in certain embodiments.
The glucosyltransferase reaction from which an oligosaccharide can be produced herein generally refers to an aqueous composition comprising at least sucrose, water and one active glucosyltransferase, and optionally other components. Other components that may be present in the glucosyltransferase reaction include at least fructose, glucose, leucrose, and gluco-oligosaccharides. It will be appreciated that certain glucan products, such as alpha-1, 3-glucan having a DP of at least 8 or 9, may be water-insoluble and therefore insoluble in the glucosyltransferase reaction, but rather be present outside of the solution. Thus, the oligosaccharides herein may be derived from a glucosyltransferase reaction that produces an insoluble glucan product (e.g., alpha-1, 3-glucan).
The glucosyltransferase reaction from which the oligosaccharide may be derived may comprise one or more of the following types of glucosyltransferase enzymes: GTFs that produce alpha-1, 3-glucans having at least 50% alpha-1, 3 glycosidic linkages (e.g., GTFs disclosed herein may also be used as GTFs in the disclosed methods themselves), mutansucrases (mutansucrases), dextran sucrases, reuterin sucrases (reuterenucrase), alternansucrase. In certain embodiments, the oligosaccharide is from a reaction comprising only one or two glucosyltransferases that produce insoluble alpha-1, 3-glucan.
The oligosaccharides herein are typically derived from a glucosyltransferase reaction at a stage where by-product oligosaccharides have been formed in the reaction. Oligosaccharides are formed throughout the polymerization reaction. For example, the oligosaccharide may result from a glucosyltransferase reaction that is only partially complete to near completion (e.g., 80% to 90% complete) or complete (e.g., > 95% complete), where completion is defined as the amount of sucrose consumed divided by the total amount of sucrose fed to the polymerization.
In certain embodiments of the present disclosure, the oligosaccharide may be provided as a soluble fraction of the glucosyltransferase reaction. The soluble fraction herein may be treated or untreated. The soluble fraction may be a portion or all of the liquid solution from the glucosyltransferase reaction. Typically, the soluble fraction is isolated from one or more solid glucan products synthesized in the reaction; this applies to dextran products that are insoluble in water, such as alpha-1, 3-dextran, which precipitate out of solution during their synthesis. In certain embodiments of the present disclosure the soluble fraction is from a glucosyltransferase reaction that produces alpha-1, 3-glucan. However, the soluble fraction may optionally be from a glucosyltransferase reaction that does not produce insoluble glucan products (e.g., dextran).
In certain embodiments, the volume of the collected soluble fraction (prior to optionally treating the soluble fraction, see below) may be at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the volume of the glucosyltransferase reaction obtained therefrom. Typically, in a glucosyltransferase reaction that produces insoluble glucan (e.g., alpha-1, 3-glucan), the soluble fraction will be part (not all) of the liquid solution components of the reaction. The soluble fraction may be obtained at a stage of the glucosyltransferase reaction in which by-product oligosaccharides are formed. For example, the soluble fraction may be from a glucosyltransferase reaction that is only partially completed to near completion (e.g., 80% to 90% complete) or completed (e.g., > 95% complete).
In certain embodiments, examples of soluble fractions of the glucosyltransferase reaction include filtrate and supernatant. Thus, the soluble fraction herein can be obtained (separated) from the glucosyltransferase reaction using a funnel, filter (e.g., a surface filter such as a rotary vacuum filter, a cross-flow filter, a screen filter, a belt filter, a screw press, or a filter press with or without membrane pressing capability; or a depth filter such as a sand filter), centrifuge, and/or any other method or apparatus known in the art that allows for the removal of some or all of the liquid from the solids. For example, filtration can be by gravity, vacuum, or pressure filtration. Filtration preferably removes all or most of the insoluble glucan; any filter material (e.g., cloth, metal mesh, or filter paper) having an average pore size sufficient to remove solids from a liquid (e.g., about 10-50 microns) may be used. The soluble fraction typically retains all or most of its dissolved components, such as certain by-products of the glucosyltransferase reaction. The filtrate or supernatant herein may be derived from a glucosyltransferase reaction that synthesizes insoluble alpha-1, 3-glucan in certain embodiments.
The soluble fraction herein may be treated if desired. Examples of treatments herein include dilution, concentration, hydrolysis treatment, pH change, salt change, and/or buffer change. The treatment may also include inactivating (e.g., heat inactivating) one or more glucosyltransferases used in the glucosyltransferase reaction from which the soluble fraction is obtained. Concentration of the soluble fraction may be carried out using any method or apparatus known in the art suitable for concentrating solutions. For example, the soluble fraction can be concentrated by evaporation, for example, with a rotary evaporator (e.g., a temperature of about 40 ℃ to 50 ℃). Other suitable types of evaporation apparatus include forced circulation evaporators or falling film evaporators. The soluble fraction herein can be concentrated to a volume that is, for example, about or less than about 75%, 80%, 85%, 90%, or 95% of the volume of the original soluble fraction. The concentrated soluble fraction (e.g., concentrated filtrate) may optionally be referred to as syrup.
The soluble fraction herein may optionally be treated using a hydrolysis treatment. The hydrolytic treatment may be an enzymatic treatment in which the soluble fraction is treated with, for example, one or more hydrolytic enzymes. The hydrolase may be, for example, a hydrolase that hydrolyzes one or more byproducts of the glucosyltransferase reaction (e.g., leucrose). Examples of useful hydrolases herein include alpha-glucosidases such as transglucosidase (EC2.4.1.24) ("EC" means enzyme Commission number) and glucoamylase (EC 3.2.1.3). Methods of treating the soluble fraction of a glucosyltransferase reaction with any of these enzymes are disclosed in U.S. patent application publication nos. 2015/0240278 and 2015/0240279, which are incorporated herein by reference.
The soluble fraction herein may be untreated, if desired. The untreated soluble fractions were as follows: wherein the fraction (or a portion of a fraction) is isolated from the glucosyltransferase reaction and used in the disclosed method without any type of modification/treatment after isolation of the soluble fraction. Examples of untreated soluble fractions include pure filtrate and pure supernatant.
In certain preferred embodiments of the present disclosure, the soluble fraction is from an alpha-1, 3-glucan synthesis reaction; such soluble fraction is optionally a filtrate. The soluble fraction of the alpha-1, 3-glucan synthesis reaction herein comprises at least water, fructose and one or more types of saccharides (leuconostoc and/or gluco-oligosaccharides such as DP2-DP 7). Soluble fractions of the type which can be used hereOther components present in (a) include, for example, sucrose (i.e., residual sucrose not consumed in the glucosyltransferase reaction), one or more glucosyltransferases, glucose, buffers, salts, glucose,
It will be appreciated that the exact composition of the saccharides and other materials in the soluble fraction of the glucosyltransferase reaction is not considered critical for use as a source of oligosaccharides in the methods herein. It will also be appreciated that the ratio of saccharide to water (i.e. wt% dry solids), which can be calculated by dividing the mass of initial saccharide by the total initial reaction solution weight, can be adjusted by evaporating water, preferably in vacuo or by adding water at temperatures below 50 ℃, without significantly affecting the relative distribution of saccharide in the soluble fraction of the glucosyltransferase reaction. The percentage of sucrose in the soluble fraction can also be increased by lowering the pH below the activity range of glucosyltransferase, or by heat inactivation of glucosyltransferase to stop the glucosyltransferase reaction before complete conversion (to glucan) is achieved.
Step (b) of the methods herein embodies the glucosyltransferase reaction. Step (a) of providing oligosaccharides is performed before step (b). Thus, the oligosaccharides of step (a) are not provided by means of their possible in situ synthesis during step (b). In other words, performing only the glucosyltransferase reaction in which oligosaccharides are produced as by-products does not completely constitute performing steps (a) and (b); the oligosaccharide must be reacted by the glucosyltransferase enzyme added substantially (manually and/or mechanically) to step (b) to perform step (a). Thus, the oligosaccharides produced by the glucosyltransferase reaction embodied in step (b) may be removed from the reaction (purified or unpurified, treated or untreated, as above; e.g.as a filtrate) and provided as oligosaccharides in step (a). In such embodiments, steps (a) and (b) may be repeated one or more times, such that in each repeated step (a), the oligosaccharide is provided from the product produced in each immediately subsequent step (b). For example, steps (a) and (b) may be repeated 1, 2, 3, 4, 5, 6, or more times. Because of such repetition, the methods according to these examples may optionally be referred to as a continuous reaction process and/or an oligosaccharide recycle process. In view of the foregoing, it is clear that in some methods herein, the glucosyltransferase reaction of step (a) (ii) may be the glucosyltransferase reaction embodied in step (b).
Alternatively, in the methods herein, the glucosyltransferase reaction of step (a) (ii) may be different (distinct) from the glucosyltransferase reaction embodied in step (b). For example, the oligosaccharide can be obtained from a first α -1, 3-glucan synthesis reaction (e.g., a collected filtrate), after which the oligosaccharide is added to a second α -1, 3-glucan synthesis reaction distinct from the first reaction.
The glucosyltransferase is contacted with at least water, sucrose and the oligosaccharide added in step (b) of the process herein. Examples of suitable glucosyltransferases are provided in the following examples, and/or disclosed in U.S. patent No. 7000000 and U.S. patent application publication nos. 2013/0244288, 2013/0244287, 2014/0087431, 2017/0002335, and 2018/0072998 (which are all incorporated herein by reference).
In certain embodiments of the present disclosure, the glucosyltransferase comprises or consists of: for example, a peptide similar to SEQ ID NO: 2.4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 (or optionally any of these sequences without an initiating methionine) is an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical, or 100% identical, wherein the glucosyltransferase is active. All of these glucosyltransferases produce alpha-1, 3-glucans with a high percentage of alpha-1, 3 glycosidic linkages (> 95%) (see, e.g., U.S. application publication No. 2014/0087431, which is incorporated herein by reference).
SEQ ID NO: 16(GTF 7527-short), SEQ ID NO: 14(GTF 2678), SEQ ID NO: 9(GTF 6855), SEQ ID NO: 13(GTF 2919), and SEQ ID NO: 11(GTF 2765) each represents a glucosyltransferase that lacks a signal peptide domain and all or most of the variable domains as compared to its respective wild-type counterpart. Thus, each of these glucosyltransferases has a catalytic domain followed by a glucan-binding domain. The approximate positions of the catalytic domain sequences in these enzymes are as follows: 7527-short (residues 54-957 of SEQ ID NO: 16), 2678 (residues 55-960 of SEQ ID NO: 14), 6855 (residues 55-960 of SEQ ID NO: 9), 2919 (residues 55-960 of SEQ ID NO: 13), 2765 (residues 55-960 of SEQ ID NO: 11). The amino acid sequences of the rough catalytic domains of GTFs 2678, 6855, 2919 and 2765 have approximately 94.9%, 99.0%, 95.5% and 96.4% identity with the GTF 7527-short rough catalytic domain sequence (i.e., amino acids 54-957 of SEQ ID NO: 16), respectively. All five of these glucosyltransferases can produce alpha-1, 3-glucans having about 100% alpha-1, 3 linkages and a DPw of at least 400 (data not shown, see table 4 of U.S. patent application publication No. 2017/0002335, which is incorporated herein by reference). Thus, in certain embodiments, the glucosyltransferase enzyme may comprise or consist of: a glucosyltransferase catalytic domain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% identical or 100% identical to an amino acid sequence of a catalytic domain of GTF 7527-short, 2678, 6855, 2919, or 2765 (e.g., as listed above).
Although it is believed that the glucosyltransferase herein need only have a catalytic domain sequence, such as those described above, the glucosyltransferase may be contained within a larger amino acid sequence. For example, the catalytic domain may be linked at its C-terminus to the glucan-binding domain, and/or at its N-terminus to the variable domain and/or the signal peptide.
Still further examples of glucosyltransferases may be any enzyme as disclosed herein and include 1-300 (or any integer between [ e.g., 10, 15, 20, 25, 30, 35, 40, 45, or 50 ]) residues at the N-terminus and/or C-terminus. Such additional residues may be from the corresponding wild-type sequence from which the glucosyltransferase is derived, or may be a heterologous sequence such as, for example, an epitope tag (at the N-or C-terminus) or a heterologous signal peptide (at the N-terminus). Glucosyltransferases herein typically lack an N-terminal signal peptide.
In certain embodiments, the glucosyltransferase does not occur in nature (i.e., is not native). For example, the enzymes herein are not considered to be naturally secreted (i.e., mature forms) from the microorganism from which the glucosyltransferase herein may be derived. In certain aspects, the non-natural enzyme comprises at least one, two, or three modified/substituted amino acids as compared to its natural counterpart. In certain aspects, the amino acid sequence of a glucosyltransferase enzyme has been modified such that the enzyme produces more products (α -1, 3-glucan and fructose), and fewer by-products (e.g., glucose, oligosaccharides such as leucrose) from a given amount of sucrose substrate. For example, one, two, three or more amino acid residues of the catalytic domain of a glucosyltransferase herein may be modified or substituted to obtain an enzyme that produces more products (alpha-1, 3-glucan and fructose). Suitable examples of such modified glucosyltransferases are disclosed in U.S. patent application publication No. 2018/0072998, which is incorporated herein by reference.
The glucosyltransferase herein may be derived from any microbial source, such as a bacterium. Examples of bacterial glucosyltransferases are those derived from streptococcus species, Leuconostoc (Leuconostoc) species or Lactobacillus (Lactobacillus) species. Examples of Streptococcus species include Streptococcus salivarius, Streptococcus sorbinus, Streptococcus mutans, Streptococcus oralis, Streptococcus gallic acid and Streptococcus sanguis. Examples of leuconostoc species include leuconostoc mesenteroides, leuconostoc amosum (l.amelibiosum), leuconostoc argentatum (l.argentinum), leuconostoc carnosus (l.carnosum), leuconostoc citreum (l.citreum), leuconostoc cremoris (l.cremoris), leuconostoc dextrans (l.dextranium), and leuconostoc fructosum (l.fructisum). Examples of lactobacillus species include lactobacillus acidophilus (l.acidophilus), lactobacillus delbrueckii (l.delbrueckii), lactobacillus helveticus (l.helveticus), lactobacillus salivarius (l.salivariaus), lactobacillus casei (l.casei), lactobacillus curvatus (l.curvatus), lactobacillus plantarum (l.plantarum), lactobacillus sake (l.sakei), lactobacillus brevis (l.brevis), lactobacillus buhneri (l.buchneri), lactobacillus fermentum (l.fermentum), and lactobacillus reuteri.
Glucosyltransferases can produce alpha-1, 3-glucan as disclosed herein. For example, glucosyltransferases can produce alpha-1, 3-glucans having at least 50% alpha-1, 3 glycosidic linkages and a DPw of at least 100. In certain embodiments, the glucosyltransferase enzyme has no or very low (e.g., less than 1%) dextran sucrase, reuterinsucrase, or alternansucrase activity.
The glucosyltransferase herein may be prepared by, for example, fermentation of an appropriately engineered microbial strain. Production of recombinases by fermentation is well known in the art, using microbial strains such as E.coli, Bacillus strains (e.g., Bacillus subtilis), Ralstonia eutropha, Pseudomonas fluorescens, Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, and species of Aspergillus (e.g., Aspergillus awamori) and Trichoderma (e.g., Trichoderma reesei) (see, e.g., Adrio and Delain, Biomolecules (biomasses) 4: 117- A nucleotide sequence of a signal peptide designed to direct secretion of glucosyltransferase. At the end of the fermentation, the cells may break accordingly, and the glucosyltransferase may be isolated using methods such as precipitation, filtration and/or concentration. Alternatively, a lysate comprising glucosyltransferase may be used without further isolation. The activity of glucosyltransferase can be confirmed by biochemical assays, e.g., measuring its conversion of sucrose to glucan polymer.
The glucosyltransferase herein may be primer-independent or primer-dependent. Primer-independent glucosyltransferase does not require the presence of a primer for glucan synthesis. Primer-dependent glucosyltransferases require the presence of a starter molecule in the reaction solution to serve as a primer for the enzyme in glucan polymer synthesis. The term "primer" as used herein refers to any molecule that can act as an initiator for glucosyltransferase. Primers useful in certain embodiments (other than added oligosaccharides as described herein, which are considered to act as primers) include dextran. Dextran that can be used as a primer can be, for example, dextran T10 (i.e., dextran with a molecular weight of 10 kD).
The activity of glucosyltransferase herein can be determined using any method known in the art. For example, glucosyltransferase activity can be determined by measuring the production of reducing sugars (fructose and glucose) in a reaction solution containing sucrose (about 50g/L), dextran T10 (about 1mg/mL), and potassium phosphate buffer (about pH 6.5, 50mM), wherein the solution is maintained at about 22 ℃ to 25 ℃ for about 24 to 30 hours. Can be determined by adding 0.01mL of the reaction solution to a mixture containing 1N NaOH and about 0.1% triphenyltetrazolium chloride, and then monitoring at OD480nmReducing sugars were measured for an increase in absorbance for about 5 minutes.
Insoluble alpha-1, 3-glucan is produced in the methods/reactions of the present disclosure. In certain aspects, the α -1, 3-glucan has at least 50% α -1,3 glycosidic linkages and a DPw of at least 100.
Alpha-1, 3-glucan herein typically comprises at least 50% alpha-1, 3-glycosidic linkages. In certain embodiments, at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% (or any integer between 50% and 100%) of the constituent glycosidic linkages of the α -1, 3-glucan are α -1,3 linkages. In some embodiments, therefore, the α -1, 3-glucan has less than about 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0% (or any integer between 0% and 50%) glycosidic linkages that are not α -1, 3. Typically, the bonds other than α -1,3 are mostly or entirely α -1, 6. It will be appreciated that the higher the percentage of α -1,3 linkages present in α -1, 3-glucan, the higher the likelihood that the α -1, 3-glucan is linear because of the lower incidence of certain linkages forming branch points in the polymer. Thus, α -1, 3-glucans having 100% α -1,3 linkages are believed to be completely linear. In certain embodiments, the α -1, 3-glucan has no branch points or less than about 5%, 4%, 3%, 2%, or 1% branch points as a percentage of glycosidic linkages in the polymer. Examples of branch points include alpha-1, 6, -1, 2, and-1, 4 branch points.
In some aspects, the α -1, 3-glucan herein can have a molecular weight of DPw or DPn of at least about 100. In some embodiments, DPw or DPn may be at least about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, or 1200 (or any integer between 100 and 1200).
The α -1, 3-glucan herein is insoluble in non-caustic aqueous systems, such as those conditions in the glucosyltransferase reaction herein (e.g., pH 4-8, see below). Generally, the solubility of a glucan polymer in the aqueous environment herein is related to its bond profile, molecular weight, and/or degree of branching. For example, in DPw8 and above, alpha-1, 3-glucan having 95% or more of 1,3 bonds is generally insoluble under aqueous conditions at 20 ℃. Generally, as molecular weight increases, the percentage of α -1,3 linkages required for α -1, 3-glucan insolubility decreases.
In some other embodiments, the insoluble α -1, 3-glucan may comprise at least about 30% α -1,3 linkages and a percentage of α -1, 6 linkages such that the sum of both α -1,3 linkages and α -1, 6 linkages in the α -1, 3-glucan amounts to 100%. For example, the percentages of α -1,3 bonds and α -1, 6 bonds may be about 30% to 40% and 60% to 70%, respectively. Glucosyltransferases for producing such alpha-1, 3-glucans are disclosed in U.S. patent application publication No. 2015/0232819, which is incorporated herein by reference. The alpha-1, 3-glucan in these examples does not comprise alternating linkages (alternating 1,3 and 1, 6 linkages).
The disclosed method comprises: in step (b), at least water, sucrose, glucosyltransferase, and oligosaccharide (as provided in step [ a ]) are contacted. This contacting step may optionally be characterized as providing a glucosyltransferase reaction composition comprising water, sucrose, glucosyltransferase, and oligosaccharide. The contacting step of the disclosed method can be performed in any number of ways. For example, the desired amount of sucrose and added oligosaccharide may be first dissolved or mixed in water (optionally, other components, such as buffer components, may also be added at this stage of preparation), followed by the addition of glucosyltransferase. The solution may be held stationary or agitated via, for example, stirring or orbital shaking.
The temperature of the reaction composition herein can be controlled, if desired, and can be, for example, from about 5 ℃ to 50 ℃, 20 ℃ to 40 ℃, 20 ℃ to 30 ℃, 20 ℃ to 25 ℃. In some aspects, the temperature can be about 5 ℃ to 15 ℃ (e.g., about 8 ℃ to 12 ℃, about 9 ℃ to 11 ℃, about 10 ℃), 15 ℃ to 25 ℃ (e.g., about 20 ℃), or 25 ℃ to 35 ℃ (e.g., about 30 ℃).
The oligosaccharide herein may be provided such that its initial concentration in the glucosyltransferase reaction set forth in step (b) is, for example, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 5-10, or 5-15 g/L. Purified or unpurified (e.g., filtrate) oligosaccharide compositions can be used to provide the foregoing concentrations. The "initial concentration" of the oligosaccharide in the present context may for example refer to the concentration of the oligosaccharide in the glucosyltransferase reaction after the smallest set of reaction components (at least water, sucrose, glucosyltransferase, optionally oligosaccharide) has just been added or combined. The oligosaccharides may be added to the glucosyltransferase reaction in batch or fed-batch mode. In batch mode, the oligosaccharide is added in its entirety (all present) at the beginning of the reaction, or within about 10-15 minutes of starting the reaction, while in fed-batch mode, the oligosaccharide is added throughout the reaction. For example, fed-batch may include adding the oligosaccharide in a continuous or incremental manner (e.g., every 30 or 60 minutes of feed) throughout the reaction and/or over a period of time of the reaction (e.g., the first 6 hours). The total amount of oligosaccharides provided in the fed-batch mode reaction may be the same as the amount provided by any of the initial concentrations listed above. In some embodiments, the oligosaccharide is added to the glucosyltransferase reaction at or within the first 1-2 hours of the start of the reaction.
The initial concentration of sucrose in the reaction composition herein may be, for example, about 20-400g/L, 75-175g/L, or 50-150 g/L. In some aspects, the initial sucrose concentration is at least about 50, 75, 100, 150, or 200g/L, or about 50-600g/L, 100-500g/L, 50-100g/L, 100-200g/L, 150-450g/L, 200-450g/L, or 250-600 g/L. "initial concentration of sucrose" refers to the concentration of sucrose in the glucosyltransferase reaction composition immediately after all reaction solution components (at least water, sucrose, glucosyltransferase, optionally added oligosaccharide) have been added/combined.
The sucrose used in the glucosyltransferase reaction solution may be of high purity (. gtoreq.99.5%) or of any other purity or grade. For example, the sucrose may have a purity of at least 99.0%, or may be reagent grade sucrose. As another example, incompletely refined sucrose may be used. Incompletely refined sucrose herein refers to sucrose that has not been processed into white refined sucrose. Thus, incompletely refined sucrose may be completely unrefined or partially refined. Examples of unrefined sucrose are "raw sucrose" ("raw sugar") and solutions thereof. Examples of partially refined sucrose have not undergone one, two, three, or more crystallization steps. The sucrose herein may be derived from any renewable sugar source, such as sugarcane, sugar beet, tapioca, sweet sorghum, or corn. Suitable forms of sucrose for use herein are crystalline forms or non-crystalline forms (e.g., syrup, sugarcane juice, beet juice). Additional suitable forms of incompletely refined sucrose are disclosed in U.S. patent application publication No. 2015/0275256, which is incorporated herein by reference. For exampleThe ICUMSA (International Commission on unified Analysis for Sugar) for incompletely refined sucrose herein may be greater than 150. Methods for determining the ICUMSA value of sucrose are disclosed by the international committee on unified analysis of sugar: for example,ICUMSA Methods of Sugar Analysis：Official and Tentative Methods Recommended by the international Commission for Uniform Methods of Sugar Analysis (ICUMSA) [ Sugar content Analytical ICUMSA method: official and provisional methods recommended by the International Committee for the unified analysis of sugar (ICUMSA)](H.C.S.de Whalley, edited by Elsevier pub.Co. [ Eszeiverer publishing Co. ]]1964) which is incorporated herein by reference. In certain aspects, ICUMSA can be measured by ICUMSA method GS1/3-7, as measured by r.j.mccowage, r.m.urquhart, and m.l.burge (r.l.burge)Determination of the Solution Colour of Raw Sugars， Brown sugar and polar Svrurs at pH 7.0-Offficial [ raw sugar, Brown sugar and colored syrup at pH 7.0 determination of the chroma of the solution-official])Albert barteng, phd publishers, 2011 edition), which is incorporated herein by reference.
In certain embodiments, the pH of the reaction composition may be about 4.0-9.0, 4.0-8.5, 4.0-8.0, 5.0-8.0, 5.5-7.5, or 5.5-6.5. In some aspects, the pH may be about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0. The pH may be adjusted or controlled by adding or incorporating suitable buffers including, but not limited to: phosphate, tris, acetate, citrate, or combinations thereof. The buffer concentration in the reaction composition herein may be, for example, about 0.1-300mM, 0.1-100mM, 10mM, 20mM, or 50 mM. A suitable amount of DTT (dithiothreitol, e.g., about 1.0mM) may optionally be added to the reaction solution.
The glucosyltransferase reaction can be contained in any container (e.g., inert container/vessel) suitable for use in applying one or more of the reaction conditions disclosed herein. In some aspects, the inert container may be stainless steel, plastic, or glass (or comprise two or more of these components) and have a size suitable to contain the particular reaction. For example, the volume/capacity of the inert container (and/or the volume of the reaction composition herein) may be about or at least about 1, 10, 50, 100, 500, 1000, 2500, 5000, 10000, 12500, 15000, or 20000 liters. The inert vessel may optionally be equipped with a stirring device.
The reaction compositions herein may contain, for example, one, two, or more glucosyltransferases. In some embodiments, only one or two glucosyltransferases are included in the reaction composition. The glucosyltransferase reaction herein can be, and typically is, cell-free (e.g., no whole cells are present).
In certain embodiments, completion of the reaction can be determined visually (e.g., no more insoluble glucan accumulates), and/or by measuring the amount of sucrose remaining in the solution (residual sucrose), where a percentage of sucrose consumption of at least about 90%, 95%, or 99% can indicate completion of the reaction. In some aspects, the reaction may be considered complete when its sucrose content is at or below about 5 g/L. For example, the reaction of the disclosed methods can be carried out for about 1 hour to about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 36, 48, 60, 72, 96, 120, 144, or 168 hours. The reaction may optionally be terminated and/or otherwise treated to halt glucosyltransferase activity by heating it to at least about 65 ℃ for at least about 30-60 minutes.
The yield of alpha-1, 3-glucan produced in the glucosyltransferase reaction herein can be, for example, about, at least about, or up to about 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% based on the weight or moles of sucrose converted in the reaction, or on the glucosyl components of the reaction. In some aspects, such yields are achieved in reactions performed for about 16-24 hours (e.g., about 20 hours), and/or as measured using HPLC or NIR spectroscopy.
The alpha-1, 3-glucan produced in the methods of certain embodiments may optionally be isolated. In certain embodiments, separating the insoluble alpha-1, 3-glucan may comprise at least the step of performing centrifugation and/or filtration. The isolation may optionally further comprise washing the alpha-1, 3-glucan once, twice or more times with water or other aqueous liquid, and/or drying the alpha-glucan product.
The isolated alpha-1, 3-glucan product herein, as provided in dry form, can comprise, for example, no more than 2.0, 1.5, 1.0, 0.5, 0.25, 0.10, 0.05, or 0.01 wt% water. In some aspects, the α -1, 3-glucan product is provided in an amount of at least 1 gram (e.g., at least about 2.5, 5, 10, 25, 50, 100, 250, 500, 750, 1000, 2500, 5000, 7500, 10000, 25000, 50000, or 100000 g); such amounts may be, for example, dry amounts.
Examples of suitable conditions and/or components for synthesizing insoluble alpha-1, 3-glucan herein are disclosed in U.S. patent No. 7000000, and U.S. patent application publication nos. 2013/0244288, 2013/0244287, 2013/0196384, 2013/0157316, 2015/0275256, 2015/0240278, 2015/0240279, 2014/0087431, 2017/0002335, and 2018/0072998, which are all incorporated herein by reference.
Any of the disclosed conditions for synthesizing insoluble alpha-1, 3-glucan (e.g., those described previously or in the examples below) can be applied to practice the reaction composition as presently disclosed (and vice versa).
The present disclosure also relates to a reactive composition for producing insoluble alpha-1, 3-glucan. This reaction composition comprises at least water, sucrose, glucosyltransferase enzyme which synthesizes insoluble alpha-1, 3-glucan, and oligosaccharide. These oligosaccharides are added during the preparation of the reaction composition and are:
(i) contain alpha-1, 3 and alpha-1, 6 glycosidic linkages, and/or
(ii) Derived from the glucosyltransferase reaction.
Insoluble alpha-1, 3-glucan is produced in the reaction composition.
The reaction composition herein may be practiced, for example, according to any of the examples disclosed herein or the following examples relating to methods of producing insoluble alpha-1, 3-glucan. Thus, any feature of such embodiments may characterize an embodiment of a reaction composition herein.
In certain embodiments, the yield of alpha-1, 3-glucan produced by the glucosyltransferase reaction may be increased compared to the yield of alpha-1, 3-glucan produced if step (b) is performed without the addition of oligosaccharide (i.e., oligosaccharide without step [ a ]). For example, the yield of α -1, 3-glucan produced may be increased by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, or 120% as compared to the yield of α -1, 3-glucan produced if the added oligosaccharide is absent in step (b). It will be appreciated that, if desired, the percentage increase in the yield of alpha-1, 3-glucan product in the methods herein can be measured relative to a suitable control glucosyltransferase reaction (e.g., a reaction having the same parameters as step [ b ], except for the addition of an oligosaccharide). In some aspects, the increase in yield characterizes a reaction comprising a glucosyltransferase without any catalytic domain amino acid substitutions as compared to its corresponding native amino acid sequence.
In certain embodiments, the relative reaction rate of the glucosyltransferase reaction of step (b) may be increased compared to the reaction rate that would be observed if step (b) were performed without the oligosaccharides provided in step (a). For example, the relative reaction rate of the glucosyltransferase reaction of step (b) can be at least about 1.025, 1.05, 1.075, 1.10, 1.15, 1.20, 1.25, 1.30, 1.40, 1.50, 1.60, 1.70, 1.80, 1.90, 2.00, or 2.10 relative to the reaction rate of a suitable control glucosyltransferase reaction. To illustrate, if the relative reaction rate of the reactions herein is at least about 1.25 relative to the control reaction, the reaction rate of this reaction is at least higher than the reaction rate of the control reactionAbout 25%. The reaction rate of the reaction may be expressed in terms of the change in the concentration/amount of one or more reactants (e.g., sucrose) and/or the change in the concentration/amount of one or more products (e.g., alpha-1, 3-glucan) per unit time per unit concentration of active glucosyltransferase enzyme. May be, for example, at every liter per hour (g L)-1h-1) The reaction rate was measured as grams of α -1, 3-glucan produced.
Byproduct formation may optionally be reduced in the glucosyltransferase reaction of step (b) of the method of producing insoluble alpha-1, 3-glucan herein, compared to byproduct formation that would be observed if step (b) were carried out without the oligosaccharide provided in step (a). For example, the amount of glucose, leucrose, and/or gluco-oligosaccharide by-product formed in step (b) can be reduced as compared to a suitable control glucosyltransferase reaction. Such reduction may be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60%.
In certain embodiments, the viscosity of the α -1, 3-glucan produced by the glucosyltransferase reaction may be reduced compared to the viscosity of the α -1, 3-glucan produced if step (b) is performed without the addition of an oligosaccharide (i.e., the oligosaccharide without step [ a ]). This viscosity of the mixed or dissolved alpha-1, 3-glucan in the liquid can be determined. The viscosity of the α -1, 3-glucan product herein can be at least about 5%, 10%, 15%, 20%, or 25% lower than the viscosity of α -1, 3-glucan that would be produced, for example, if the added oligosaccharide were absent in step (b). It will be appreciated that the percent reduction in viscosity of the alpha-1, 3-glucan product in the methods herein can be measured, if desired, relative to a suitable control glucosyltransferase reaction (e.g., a reaction having the same parameters as step [ b ], except for the addition of an oligosaccharide).
The viscosity of the herein alpha-1, 3-glucan mixed or dissolved in a liquid can be determined. In certain aspects, such assays can be used in aqueous liquids (e.g., water or aqueous solutions that are not caustic) (e.g., a non-caustic aqueous liquid can have a pH of about 4-10, 5-9, 6-8, or 7) Mixed alpha-1, 3-glucan; because the α -1, 3-glucan herein is typically insoluble under such aqueous conditions, mixing is recommended. This mixing can be performed using, for example, any suitable method for effectively mixing alpha-1, 3-glucan in a non-caustic aqueous liquid, such as homogenization or microfluidization (e.g., as disclosed in any one of International patent application publication No. WO2016/126685 or U.S. patent application publication Nos. 2015/0167243, 2005/0249853, 2003/0153746, and 2018/0021238, each of which is incorporated herein by reference in its entirety). Mixing of the alpha-1, 3-glucan in the non-caustic aqueous liquid can typically be carried out to prepare an aqueous slurry and/or dispersion (colloidal dispersion) of the glucan. The viscosity of such aqueous compositions may optionally be measured in the range of about 5-250s-1(e.g., 7-200 s)-1) At a shear rate of about 15-25 ℃ (e.g., about 20 ℃), and/or at a slurry viscosity in cP of 2-10 wt% (e.g., about 4-5 wt%) of the aqueous alpha-1, 3-glucan mixture.
In certain aspects, viscosity measurements can be performed with alpha-1, 3-glucan dissolved in a liquid. Such liquids may be, for example, caustic aqueous solutions having a pH of at least about 11. The caustic aqueous solution may comprise at least a hydroxide (e.g., NaOH, KOH, tetraethylammonium hydroxide), and/or may be disclosed, for example, in: international patent application publication No. WO2015/200612 or WO 2015/200590, or U.S. patent application publication No. 2017/0208823 or 2017/0204203 (each of which is incorporated by reference in its entirety). In some aspects, the liquid used to measure viscosity for dissolving the α -1, 3-glucan herein can be non-aqueous, such as a liquid comprising an organic solvent (e.g., an organic ionic liquid). Examples of suitable organic solvents herein may include N, N-dimethylacetamide (DMAc) (optionally with about 0.5% -5% LiCl), Dimethylsulfoxide (DMSO), N-Dimethylformamide (DMF), pyridine, SO2Diethylamine (DEA)/DMSO, LiCl/1, 3-dimethyl-2-imidazolidinone (DMI), DMSO/tetrabutylammonium fluoride Trihydrate (TBAF), N-methylpyrrolidone, and/or N-methylmorpholine-N-oxide (NMMO). In some aspects, the alpha-1, 3-glucan may be in-situ synthesizedDissolved in a suitable organic solvent such as DMAc/0.5% LiCl to a concentration of about 5-15mg/mL (e.g., 10mg/mL) is used to measure viscosity. In some embodiments, the viscosity of solubilized α -1, 3-glucan herein can be measured as intrinsic viscosity (IV, notation "η", provided in units of mL/g). For example, any suitable method is used, such as those disclosed in the following documents: U.S. patent application publication nos. 2017/0002335 and 2017/0002336, Weaver et al (j.appl.polym.sci. [ journal of applied polymer science)]35: 1631-]195: 701-711), the IV measurements herein may be obtained, and these documents are incorporated by reference in their entirety.
The present disclosure also relates to compositions comprising insoluble alpha-1, 3-glucan produced according to any of the methods herein for producing insoluble alpha-1, 3-glucan. In certain embodiments, such compositions may be aqueous compositions or non-aqueous compositions. In some aspects, the viscosity of the insoluble alpha-1, 3-glucan product herein is less than the viscosity of insoluble alpha-1, 3-glucan that would be produced if the oligosaccharide were not provided in the process/reaction (control glucan). The viscosity can be measured by any of the methods described above with respect to alpha-1, 3-glucan mixed or in solution in a liquid. The viscosity of the α -1, 3-glucan product of the invention can be at least about 5%, 10%, 15%, 20%, or 25% less than the viscosity of, for example, a control glucan. The viscosity/DPw relationship of the α -1, 3-glucan product of the invention can be, for example, as disclosed in the examples below, which show that adding a gluco-oligosaccharide to the glucosyltransferase reaction reduces viscosity.
Non-limiting examples of the compositions and methods disclosed herein include:
1. a method for producing insoluble alpha-1, 3-glucan, the method comprising:
(a) providing an oligosaccharide which:
(i) contain alpha-1, 3 and alpha-1, 6 glycosidic linkages, and/or
(ii) Results from the glucosyltransferase reaction;
(b) contacting at least water, sucrose, a glucosyltransferase enzyme that synthesizes insoluble alpha-1, 3-glucan, and an oligosaccharide, wherein insoluble alpha-1, 3-glucan is produced; and
(c) optionally, isolating the insoluble alpha-1, 3-glucan produced in step (b).
2. The method of embodiment 1, wherein the oligosaccharide comprises from about 60% to about 99% alpha-1, 3 glycosidic linkages and from about 1% to about 40% alpha-1, 6 glycosidic linkages.
3. The method of embodiment 1 or 2, wherein the oligosaccharide has a Degree of Polymerization (DP) of 2 to 10.
4. The method of embodiment 1, 2, or 3, wherein the oligosaccharide is purified or unpurified.
5. The method of embodiment 4, wherein the oligosaccharide is produced from the glucosyltransferase reaction of (a) (ii).
6. The method of example 5, wherein the glucosyltransferase of (a) (ii) reacts to synthesize insoluble alpha-1, 3-glucan.
7. The method of example 5 or 6, wherein the oligosaccharide is provided as the soluble fraction of the glucosyltransferase reaction of (a) (ii), and wherein the soluble fraction is treated or untreated.
8. The method of embodiment 7, wherein the soluble fraction is a portion or all of the filtrate of the glucosyltransferase reaction of (a) (ii).
9. The method of embodiment 1, 2, 3, 4, 5, 6, 7, or 8, wherein the oligosaccharide is provided in step (b) at an initial concentration of at least about 1 g/L.
10. The method of example 1, 2, 3, 4, 5, 6, 7, 8 or 9, wherein the yield of insoluble alpha-1, 3-glucan produced is increased compared to the yield of insoluble alpha-1, 3-glucan produced if the oligosaccharide was absent in step (b).
11. The method of example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 wherein the viscosity of the insoluble alpha-1, 3-glucan produced is reduced compared to the viscosity of the insoluble alpha-1, 3-glucan that would be produced if the oligosaccharide were absent in step (b), wherein viscosity is measured with the alpha-1, 3-glucan mixed or dissolved in a liquid.
12. The method of examples 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, wherein the insoluble alpha-1, 3-glucan produced has at least 50% alpha-1, 3 glycosidic linkages and a weight average degree of polymerization (DPw) of at least 100.
13. The method of examples 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 13 wherein steps (a) and (b) are repeated one or more times and wherein in each repeated step (a) the oligosaccharide is provided from the product (by-product) produced from each immediately preceding step (b).
14. A reaction composition for the production of insoluble alpha-1, 3-glucan, the reaction composition comprising at least water, sucrose, glucosyltransferase enzyme that synthesizes insoluble alpha-1, 3-glucan, and oligosaccharide, wherein the oligosaccharide is added during preparation of the reaction composition and:
(i) contain alpha-1, 3 and alpha-1, 6 glycosidic linkages, and/or
(ii) Is generated from the reaction of glucosyltransferase,
wherein insoluble alpha-1, 3-glucan is produced in the reaction composition, optionally wherein the reaction composition is characterized by any of the features as described in any of examples 2-13.
15. A composition comprising insoluble alpha-1, 3-glucan produced as described in any one of examples 1-13, or produced in the reaction composition of example 14.
16. The composition of embodiment 15 wherein the viscosity of the insoluble alpha-1, 3-glucan is less than the viscosity of insoluble alpha-1, 3-glucan that would result if the oligosaccharide were not provided in the process or reaction composition, wherein viscosity is measured with alpha-1, 3-glucan mixed or dissolved in a liquid.
Examples of the invention
The present disclosure is further illustrated in the following examples. It should be understood that these examples, while indicating certain preferred aspects of the disclosure, are given by way of illustration only. From the above discussion and these examples, one skilled in the art can ascertain the essential characteristics of the disclosed embodiments, and without departing from the spirit and scope thereof, can make various changes and modifications to adapt the disclosed embodiments to various uses and conditions.
General procedure
All reagents were obtained from Sigma Aldrich (Sigma-Aldrich) (st louis, missouri), unless otherwise noted. Sucrose was obtained from VWR corporation (radna, pa).
Preparation of crude extract of Glucosyltransferase (GTF)
The Streptococcus salivarius (Streptococcus salivarius) GTFJ enzyme (SEQ ID NO: 2) used in some of the following examples was expressed in E.coli strain DH10B using an isopropyl β -D-1-thiogalactoside (IPTG) -induced expression system. Compared to the streptococcus salivarius GTFJ amino acid sequence in GENBANK identification number 47527, the amino acid sequence of SEQ ID NO: 2 has a deletion of the N-terminal 42 residues. Briefly, E.coli DH10B cells were transformed to express the DNA sequence of SEQ ID NO: 2. this DNA sequence is contained in an expression vector,
By being at
The GTF enzyme used in example 5 was prepared in the following manner. Subjecting Escherichia coli
Analysis of reaction spectra
Taking periodic samples of the reaction and using a detector equipped with a refractive index detector
Analysis of dextran molecular weight
The insoluble glucan polymer isolated from the glucosyltransferase reaction was treated with N, N-dimethylacetamide (DMAc) with 5% lithium chloride (LiCl) at 100 ℃ for 16 hours to form a glucan polymer solution. This solution (100. mu.L) was then injected into an Alliance equipped with a differential refractometer detector operating at 50 ℃TM2695HPLC (Watts Corporation, Milford, Mass.). The mobile phase (DMAc containing 0.11 wt% LiCl) was passed through four styrene-divinylbenzene columns in series at a flow rate of 0.5 mL/min; specifically, one KD-802, one KD-801, and two linear KD-806M columns (Shorex, Japan). The molecular weight distribution of the dextran polymer sample was determined by comparing the retention time to a broad dextran standard.
Example 1
Glucan polymerization Using GTFJ enzyme (SEQ ID NO: 2)
This example discloses information on the conversion of sucrose to insoluble alpha-1, 3-glucan polymers and soluble sugars and details how the raw materials used in example 2 were produced.
Sucrose (3000g) was added to a clean 5 gallon polyethylene bucket. Water (18.1L) and Fermasure were added to the bucketTM(10mL) and by adding 5 vol% NaOH and 5 vol% H2SO4The pH was adjusted to 7.0. The final volume was about 20L and the initial concentration of sucrose as measured by HPLC was 152.5gAnd L. Dextran polymerization was initiated by adding 0.3 vol% crude GTFJ enzyme (SEQ ID NO: 2) extract prepared as described in the general methods section. This extract contained about 2.9mg/mL protein. Agitation of the reaction solution was provided using an overhead mechanical motor equipped with a glass shaft and PTFE blade.
After 48 hours, HPLC analysis showed 96% of the sucrose had been consumed and the reaction was considered complete. The insoluble alpha-1, 3-glucan was removed by filtration, and then the mother liquor (filtrate) was concentrated to a total sugar concentration of 320g/L of saccharides using a rotary evaporator (bath temperature of 40 ℃ -50 ℃). The composition of the concentrated sugar solution is provided in table 2.
TABLE 2
Composition of concentrated filtrate of glucan Synthesis reaction
aThe weight percentages are relative to the measured saccharide component.
Table 2 shows that the concentrated filtrate obtained at the completion of the above-mentioned glucan synthesis reaction contained saccharides, of which about 14-15 wt% was a by-product of oligosaccharides (DP2-DP 7). This concentrated filtrate was used in example 2 for the chromatographic separation of oligosaccharides.
Example 2
Oligosaccharide separation and analysis using ion exchange resins
This example discloses how to separate oligosaccharides from the concentrated filtrate of a glucan synthesis reaction by chromatographic separation and how to analyze their glycosidic bond patterns. These isolated oligosaccharides were used in examples 3, 5 and 7.
Chromatographic separation using a strong acid cation exchange resin was used to isolate the oligosaccharide fraction of the concentrated filtrate prepared in example 1. The physical parameters of the columns used for this separation are shown in table 3.
TABLE 3
Physical parameters of columns for chromatographic separations
The concentrated sugar solution prepared in example 1 (i.e., concentrated filtrate) was filtered and diluted to 25g dry solids per 100g solution using tap water. Prior to adding the sugar solution to the column resin, the resin was washed with six Bed Volumes (BV) of sodium chloride solution (three BV in the case of 10 wt% sodium chloride followed by three BV in the case of 5 wt% sodium chloride) to convert the resin to the sodium form. The sugar solution (0.6L) was then fed to the column, which was then eluted with water at a flow rate of 50 mL/min. The operating conditions for the chromatographic separations are summarized in table 4.
TABLE 4
Chromatographic separation operating conditions
The oligosaccharide solution was eluted between 11 and 21 minutes. A small amount of salt is eluted at the same time-indicated by an increase in conductivity. The oligosaccharide fractions thus prepared were analyzed by HPLC to determine their product distribution. In summary, the fraction contains > 89% of oligosaccharides containing three or more hexose units and less than 1.5% of identifiable mono-and disaccharides. This fraction was concentrated to a total dry weight of 317g/L using a thin film evaporator (LCI Corporation, Charlotte, N.C.) followed by rotary evaporation using a ROTAVAPOR (R-151; Buchi, N.C.). In table 5 is the product distribution of the concentrated fractions as measured by HPLC.
TABLE 5
Product distribution of concentrated oligosaccharide fraction
aThe weight percentages are relative to the measured saccharide component.
Use of the concentrated oligosaccharide solutions of Table 51H NMR. NMR data were obtained using a 5-mm cryo-triple resonance Pulsed Field Gradient (PFG) probe, used at 500MHz1Operated by H
Thus, the concentrated filtrate from the glucan synthesis reaction was isolated and analyzed for oligosaccharides. In examples 3, 5 and 7, the above concentrated oligosaccharide solutions were used.
Example 3
Comparison of glucosyltransferase reactions in absence or with added oligosaccharides
This example discloses that the addition of a purified oligosaccharide containing a significant DP2+ material fraction (example 2) to an alpha-1, 3-glucan synthesis reaction results in an increased yield of insoluble alpha-1, 3-glucan product compared to reactions lacking such added oligosaccharides. This example also shows that this benefit (increased glucan product yield) is present in a variety of reaction conditions and oligosaccharide loadings.
The glucan synthesis reaction was prepared in the following manner. Mixing sucrose (10g) and potassium dihydrogen phosphate (KH) 0.27g2PO4) 94mL of water, and 50 microliters of FermasureTMAdd to 125-mL clean equipped with polypropylene lidIn a glass bottle. No oligosaccharide was added to the formulation of comparative example 3A (table 6). In examples 3.1 and 3.2 (table 6), an amount of oligosaccharide solution prepared in example 2 (table 5) was added to each corresponding formulation; the amount of water added to each respective formulation was reduced by a comparable volume. Each formulation contained trace amounts of glucose, primarily from the sucrose component; no additional glucose was added to either formulation. Each formulation was agitated in an incubator shaker (temperature controlled at 25 ℃) until a solution formed, at which time the pH of each formulation was adjusted to 5.5 using 5 wt% aqueous sodium hydroxide or 5 wt% aqueous sulfuric acid. A sample of each formulation was taken for analysis by HPLC, after which 0.3 vol% crude GTFJ enzyme (SEQ ID NO: 2) extract prepared as described in the general methods section was added to each formulation to initiate polymerization. As the reactions progressed, samples of each reaction were taken periodically and analyzed by HPLC. The initial rate of the reaction was calculated by the amount of sucrose consumed in the first two hours of polymerization. Once each reaction was deemed complete, the insoluble polymer product was isolated from the reaction by filtration, washed with 200mL of water, washed with 100mL of acetone, and then dried.
The results of each reaction are shown in table 6, which shows the improvement in yield of insoluble alpha-1, 3-glucan polymer product when oligosaccharides were added to the reaction (compare examples 3.1 and 3.2 with example 3A). These results also show a further improvement in the yield of insoluble alpha-1, 3-glucan obtained when additional amounts of oligosaccharides were added (compare example 3.2 with 3.1).
TABLE 6
Profile of glucosyltransferase reaction in absence or presence of added oligosaccharide
Examples of the invention
3A
3.1
3.2
Nominal sucrose (g/L)
100
100
100
Actual sucrose (g/L)
97.2
104.6
107.1
Initial oligosaccharide, DP2+ (g/L)
0.0
5.3
22.0
Initial glucose (g/L)
1.3
1.1
1.6
Initial rate (g sucrose consumed/L-hr)
5.3
14.1
12.3
Sucrose% of reaction%
94.3
96.4
98.1
Production of Polymer (g/L)
15.4
24.6
27.5
Production of glucose (g/L)
8.1
5.9
4.9
Production of oligomers (g/L)
8.2
8.9
24.6
Production of polymer (g/g reacted sucrose)
0.17
0.24
0.26
Production of glucose (g/g reacted sucrose)
0.075
0.048
0.031
Production of oligomers (g/g reacted sucrose)
0.090
0.035
0.025
The benefits conferred upon the addition of oligosaccharides to the alpha-1, 3-glucan synthesis reaction are obtained at some temperatures and sucrose loading. The reactions of examples 3.3-3.5 were prepared and performed in the same manner as described above, except that there was a change in the initial sucrose concentration or temperature. The results of these reactions are summarized in table 7, as well as those of the reactions of comparative examples 3B, 3C and 3D (controls of examples 3.3, 3.4 and 3.5, respectively) without any oligosaccharide added.
TABLE 7
Glucosyltransferase reactions with absence or presence of added oligosaccharides under different sucrose or temperature conditions Corresponding spectrogram
Thus, adding the oligosaccharide by-product of the α -1, 3-glucan synthesis reaction to the new α -1, 3-glucan synthesis reaction can increase the yield of α -1, 3-glucan produced by the new reaction, and increase the rate of polymerization while simultaneously reducing the yield of undesirable oligomers and glucose. Reaction regulation occurs under a variety of conditions.
Example 4
Yield of insoluble alpha-1, 3-glucan glucosyltransferase reaction with glucose added thereto instead of oligosaccharide Reduce
This example discloses that the addition of glucose (in an amount equivalent to the amount of oligosaccharide used in example 3) to the glucosyltransferase reaction is detrimental to the yield of insoluble alpha-1, 3-glucan produced by the glucosyltransferase reaction.
The glucan synthesis reaction was prepared in the following manner. Sucrose (75g) was weighed out and diluted to 0.75L with deionized water in a 1-L baffle-less jacketed flask attached to a LAUDA RK20 circulator. Fermasure was then addedTM(0.5mL/L reaction), and the pH was adjusted to 5.5 using 5 wt% aqueous sodium hydroxide or 5 wt% aqueous sulfuric acid. In comparative example 4A (table 8), the trace amount of glucose was mainly from the sucrose component; no additional glucose was added to the formulation. In example 4.1 (table 8), glucose (18.8g) was added to the formulation in addition to the presence of trace amounts of glucose from the sucrose component. Polymerization reactions were initiated in each formulation by adding 0.3 vol% crude GTFJ enzyme (SEQ ID NO: 2) extractShould be used. An overhead mechanical motor attached to a 4-blade PTFE blade was used to provide agitation for each reaction and the temperature was controlled at 25 ℃. After completion of the reaction as determined by HPLC, the insoluble polymer product of each reaction was isolated by filtration. The polymer product was then washed with water (1.5L), then acetone (0.5L), and then dried under a vacuum oven. The mass of the dry alpha-1, 3-glucan product was recorded.
The results of each reaction are shown in table 8, which shows that the yield of insoluble α -1, 3-glucan polymer obtained in the glucosyltransferase reaction decreases when glucose is added to the reaction.
TABLE 8
Spectra of glucosyltransferase reactions containing various amounts of glucose
Thus, the addition of glucose to the glucosyltransferase reaction is detrimental to the yield of insoluble alpha-1, 3-glucan produced by the glucosyltransferase reaction. It is noteworthy that the amount of glucose in the reaction of example 4.1 is equivalent to the amount of oligosaccharide added in some of the reactions of example 3. The negative results in example 4.1 therefore indicate that the oligomeric nature of the oligosaccharide used in example 3 is essential for the observed yield enhancement of the glucan polymer (i.e. the monomeric component of the oligosaccharide, glucose, is likely to require oligomerization to enhance the yield of glucan product in the reaction).
Example 5
Insoluble alpha-1, 3-glucan yield in reactions containing added oligosaccharides and different GTF enzymes
This example discloses that the glucan polymer yield enhancement effect of adding purified oligosaccharide (from example 2) to the glucosyltransferase reaction is generally applicable to reactions containing enzymes other than GTFJ that produce insoluble alpha-1, 3-glucan.
The different types of GTF enzymes used in this example are GTF 0874(SEQ ID NO: 4), GTF 1724-T1(SEQ ID NO: 7) and GTFJ-T1(SEQ ID NO: 8). Each of these glucosyltransferases may be synthesized, or are expected to be capable of synthesizing, insoluble alpha-1, 3-glucan polymers having about 100% alpha-1, 3 glycosidic linkages (see, e.g., U.S. patent application nos. 2014/0087431 and 2016/0002693, which are incorporated herein by reference).
The glucan synthesis reaction was prepared in the following manner. Mixing sucrose (10g) and potassium dihydrogen phosphate (KH) 0.27g2PO4) And 94mL of water were added to a 125-mL clean glass bottle equipped with a polypropylene cap. No oligosaccharide was added to the formulations in comparative examples 5A, 5B, and 5C (table 9). In examples 5.1, 5.2, and 5.3, an amount of the oligosaccharide solution prepared in example 2 (table 5) was added to each corresponding formulation; the amount of water added to each respective formulation was reduced by a comparable volume. Each formulation contained trace amounts of glucose, primarily from the sucrose component; no additional glucose was added to either formulation. Each formulation was agitated in an incubator shaker (temperature controlled) until a solution formed, at which time the pH was adjusted to 5.5 using 5 wt% aqueous sodium hydroxide or 5 wt% aqueous sulfuric acid. A sample of each formulation was taken for analysis by HPLC, after which 0.3 vol% crude GTF enzyme extract prepared as described in the general methods section was added to each formulation to initiate polymerization. As the reactions progressed, samples of each reaction were taken periodically and analyzed by HPLC. Once each reaction was deemed complete, the insoluble polymer product was isolated from the reaction by filtration, washed with 200mL of water, washed with 100mL of acetone, and then dried.
The results of each reaction are shown in table 9, which shows that the yield of insoluble alpha-1, 3-glucan polymer product is increased when oligosaccharides are added to the reaction. This yield increase occurs in reactions using different types of glucosyltransferases. Notably, the respective rough catalytic domains of each of GTF 1724-T1 and GTF 0874 share approximately only 50% amino acid sequence identity with the rough catalytic domain of GTFJ (see U.S. patent application No. 2017/0002335, incorporated herein by reference). Despite this significant difference in sequence identity, each enzyme showed an increase in the yield of insoluble alpha-1, 3-glucan product.
TABLE 9
Spectra of reactions containing different types of GTF enzymes, and lacking or containing added oligosaccharides
aComparative example 5C was performed at pH 7.0 instead of pH 5.5.
Thus, the addition of the oligosaccharide by-product of the alpha-1, 3-glucan synthesis reaction to the new alpha-1, 3-glucan synthesis reaction can increase the yield of alpha-1, 3-glucan produced by the new reaction. This yield increase occurs in reactions using different types of glucosyltransferases.
Example 6
Yield of insoluble alpha-1, 3-glucan in reactions containing other types of added oligosaccharides
This example discloses that the oligosaccharide may have to contain an alpha-1, 3-glucosidic linkage to bring about a change in selectivity of the glucosyltransferase reaction to the insoluble alpha-1, 3-glucan polymer. Oligosaccharides different from those produced in example 2 (table 5) were added to the glucosyltransferase reaction to determine if they affected alpha-1, 3-glucan yield.
Maltodextrin (5.5, 15, or 18 dextrose equivalents; Sigma Aldrich) having 100% alpha-1, 4 linkages and typically containing mostly oligosaccharides (about DP2-DP20) was used in the alpha-1, 3-glucan polymerization without further purification. Dextran (dextran T-10, average molecular weight 10000 dalton, Sigma Aldrich), which is a polysaccharide containing > 95% alpha-1, 6 linkages, and hydrolyzed dextran, were also used in the polymerization reaction. Hydrolyzed dextran was prepared by heating a solution of T-10 containing 15g dextran in 141mL water to 90 ℃ at pH 1.0. The distribution of oligosaccharides in the hydrolyzed dextran formulation is shown in table 10.
Watch 10
Composition of hydrolyzed dextran preparation
Glucose
DP2
DP3
DP4
DP5
DP6
DP7
DP8-10
DP10+
Total of
g/L
3.8
13.2
14.3
11.7
10.2
16.6
7.7
26.2
10.2
113.9
wt％a
3.4
11.6
12.5
10.3
8.9
14.6
6.8
23.0
8.9
100.0
aThe weight percentages are relative to the measured saccharide component.
The GTFJ reaction was performed following the protocol described in example 3, except that maltodextrin, dextran, or hydrolyzed dextran was used instead of the oligosaccharides produced in example 2 (table 5). Table 11 provides the results of these reactions. The yield of insoluble alpha-1, 3-glucan polymers in reactions using hydrolyzed dextran (examples 6.1 and 6.2) and maltodextrins with various dextrose equivalents (examples 6.3-6.5) was not or only slightly affected by the addition of these different types of oligosaccharides (table 11, compare examples 6.1-6.5 with example 6A).
TABLE 11
Profiles of glucosyltransferase reactions with different types of added oligosaccharides or polysaccharides
aTrace amounts of glucose present in the sucrose component of each reaction.
bDE, dextrose equivalent.
The results in table 11 demonstrate that oligosaccharides containing predominantly alpha-1, 6 linkages (hydrolyzed dextran, examples 6.1 and 6.2) or alpha-1, 4 linkages (maltodextrin, examples 6.3-6.5) do not significantly increase the yield of insoluble alpha-1, 3-glucan polymer when added to the glucosyltransferase reaction. In addition, it appears that in order for the α -1, 6-linked saccharide molecules to increase the yield of the glucan product, such saccharide molecules must be in the form of larger polysaccharides (approximately 10000 daltons) because dextran T-10 (examples 6.6-6.7) increases the yield of the insoluble α -1, 3-glucan product, whereas its oligosaccharide counterpart (examples 6.1-6.2) does not.
On the other hand, tables 6, 7 (example 3), and 9 (example 5) illustrate that oligosaccharides comprising α -1,3 linkages and α -1, 6 linkages can significantly improve the yield of insoluble α -1, 3-glucan in the glucosyltransferase reaction. Based on these data, and the fact that oligosaccharides having only α -1, 6 linkages did not significantly affect the yield of α -1, 3-glucan product (table 11), it appears that the α -1,3 linkage component of the oligosaccharides in table 5 is necessary to increase the yield of insoluble α -1, 3-glucan product.
Thus, the oligosaccharide may have to contain at least a portion of the alpha-1, 3 glycosidic linkages to increase the yield of insoluble alpha-1, 3-glucan in the glucosyltransferase reaction.
Example 7
Separation and recycle of oligosaccharides in alpha-1, 3-glucan synthesis reactions
This example discloses that oligosaccharides isolated from glucan polymerization can be used to obtain consistent glucan product yield improvements after running the polymerization for multiple cycles.
The first glucan synthesis reaction was prepared in the following manner. Sucrose (75g) was weighed out and diluted with deionized water in a 1-L baffle-less jacketed flask with a LAUDA RK20 circulator cooler attachedTo 0.75L. Fermasure was then addedTM(0.5mL/L reaction), and the pH was adjusted to 5.5 using 5 wt% aqueous sodium hydroxide or 5 wt% aqueous sulfuric acid. Purified oligosaccharides obtained from glucan polymerization (table 5, example 2) were added to the formulation to a total concentration of DP2+ of about 5 g/L. An overhead mechanical motor attached to a four-bladed PTFE blade was used to provide agitation to the formulation and the temperature was controlled at 25 ℃. The polymerization was initiated by adding 0.3 vol% of crude GTFJ enzyme (SEQ ID NO: 2) extract. After completion of the reaction as determined by HPLC, the insoluble polymer product was isolated by filtration. The polymer product was then washed with water (1.5L), then acetone (0.5L), and then dried under a vacuum oven. The mass of the dry alpha-1, 3-glucan product was recorded.
The filtrate of the reaction was concentrated to about 30 wt% dry solids using a rotary evaporator. 25-mL fractions of this filtrate were used by column chromatography
TABLE 12
Chromatographic purification operating conditions
Type of resin
BioRad BIO-GEL P-2 GEL
Particle size (micron)
45-90
Bed Length (cm)
100
Column diameter (m)
0.026
Raw material amount (mL)
25
Approximate raw materials Dry solids (g/100g)
30
Column temperature (. degree. C.)
50
Flow rate (mL/min)
50
Fractions separated from chromatography were collected in 10-mL fractions and analyzed by HPLC. The oligosaccharide containing fractions were combined and concentrated by rotary evaporation at 40 ℃.
These purified oligosaccharides were then used as oligosaccharide source in a new glucan synthesis reaction (example 7.1, table 13), following the protocol of the first reaction (see above); about 5g/L of oligosaccharide was supplied to the reaction. After completion of this reaction, the oligosaccharide (DP2+) was thus purified by the above protocol and used in the subsequent reactions. This cycle of running the polymerization reaction containing the oligosaccharide (DP2+) glucan just purified from the previous reaction was repeated four additional times: the oligosaccharide from the reaction of example 7.1 was added to the reaction of example 7.2, the oligosaccharide from the reaction of example 7.2 was added to the reaction of example 7.3, the oligosaccharide from the reaction of example 7.3 was added to the reaction of example 7.4, and the oligosaccharide from the reaction of example 7.4 was added to the reaction of example 7.5. The data from these experiments are summarized in table 13, which shows an improved yield of alpha-1, 3-glucan compared to comparative example 7A, in which no oligosaccharide was added to the reaction.
Watch 13
Spectrum of glucosyltransferase reactions Using oligosaccharides recovered from previous reactions
Thus, based on the results shown in table 13, the oligosaccharide (DP2+) produced from glucan polymerization was useful in obtaining a fairly consistent increase in glucan product yield after running the polymerization for multiple cycles.
Example 8
Glucan polymerization Using modified GTF enzymes
This example discloses an oligosaccharide composition of the filtrate produced in an alpha-1, 3-glucan synthesis reaction catalyzed by an improved glucosyltransferase enzyme.
The amino acid sequence of streptococcus salivarius glucosyltransferase, which produces alpha-1, 3-glucans with about 100% alpha-1, 3 linkages, is modified in its catalytic domain such that the enzyme can produce more products (alpha-1, 3-glucan and fructose) and fewer by-products (e.g., glucose, oligosaccharides such as leuconostoc and DP2-7 glucose-oligosaccharide) from a sucrose substrate, as compared to the unmodified counterpart of the enzyme (see U.S. patent application publication No. 2018/0072998, which is incorporated herein by reference).
The alpha-1, 3-glucan synthesis reaction using the modified glucosyltransferase enzyme was carried out in a 5000-gal stainless steel vessel containing 94g/L white crystalline sucrose dissolved in water. The pH of the reaction was maintained using 10mM potassium phosphate as a buffer and 2N H2SO4It was adjusted to 5.5. The addition of the antimicrobial agent at 100ppmv,XL to prevent contamination during the reaction. The reactor contained three inclined blade impellers set at 33rpm andit was controlled at 23 ℃ with cooling water flowing into the reactor jacket. The reaction was initiated by the addition of 30 pounds of improved glucosyltransferase enzyme and was considered complete after 14 hours when the sucrose concentration reached less than 2 g/L. At the end of the reaction, the glucosyltransferase was inactivated by heating the reaction contents to 65 ℃ for 30 minutes using an external heat exchanger.
The insoluble α -1, 3-glucan polymer (i.e., insoluble fraction) produced in the reaction was separated from the soluble fraction using a filter press, thereby providing a filtrate. Table 14 provides the carbohydrate content (dwb) in the filtrate.
TABLE 14
Carbohydrate composition of filtrate (wt% -dry weight basis)
aIn addition to DP2, the gluco-oligosaccharides include at least sucrose.
Chromatographic separation using a strong acid cation exchange resin is used to separate the oligosaccharide fraction of the filtrate. In table 15 are the physical parameters of the columns used for this separation.
Watch 15
Physical parameters of columns for chromatographic separations
Thus, the filtrate was changed to 30g dry solids per 100g solution using ion exchanged tap water. Before adding this modified filtrate to the column resin, the resin was washed with six Bed Volumes (BV) of sodium chloride solution (three BV in the case of 10 wt% sodium chloride followed by three BV in the case of 5 wt% sodium chloride) to convert the resin to the sodium form. The changed filtrate (15L) was then fed onto the column, after which the column was eluted using ion-exchanged water at a flow rate of 30L/h and a column temperature of 70 ℃.
The oligosaccharide solution was eluted between 140 and 185 minutes and recovered. The oligosaccharide fractions thus prepared were analyzed by HPLC to determine their product distribution. Briefly, the composition of the oligosaccharide fraction was measured using an Agilent 1260HPLC equipped with a refractive index detector. The separation of oligosaccharides was achieved using a BioRad AMINEX HPX-42A column using water as eluent at 85 ℃ and a flow rate of 0.6 mL/min. The composition profile of the oligosaccharides is provided in table 16.
TABLE 16
Composition of oligosaccharides recovered by fractional distillation (wt% -on dry weight basis)
DP2
DP3
DP4
DP5
DP6
DP7+
11
23
28
21
12
5
The oligosaccharide fractions described in Table 16 were subjected to a partially methylated sugar alcohol acetate (PMAA) analysis (according to the method in Pettolino et al, Nature Protocols 7: 1590) -1607) and analyzed by GC-MS. Briefly, a sample was treated with DMSO anion and methyl iodide to methylate the hydroxyl group, and then hydrolyzed with trifluoroacetic acid. The hydroxyl groups resulting from the cleaved glycosidic linkages were then acetylated with acetic anhydride and the resulting glucitol analyzed by GC/MS. The oligosaccharides were found to have the distribution described in table 17 (where all linkages are considered as a). The main bond is α -1, 3. No terminal fructose was detected in the oligosaccharide fraction.
TABLE 17
Linkage distribution of oligosaccharides
Key with a key body
Bond%
1→3
87.5
1→6
7.3
1→3，6
2.8
1→4
1.0
1→2，3
0.7
1→2
0.6
1→3，4
0.3
Thus, the DP2+ oligosaccharides present in the filtrate of the glucan synthesis reaction using the improved glucosyltransferase enzyme were characterized. Such oligosaccharides or e.g. a filtrate comprising such oligosaccharides may be used as a source of added oligosaccharides for carrying out the glucosyltransferase reaction as disclosed herein.
Example 9
Synthesis reaction of glucose, leuconostoc disaccharide, fructose or glucose-oligosaccharide additive on alpha-1, 3-glucan Function of
This example discloses comparing the individual effects of various sugars or oligosaccharides on the enzymatic reactions used to synthesize alpha-1, 3-glucan. Consistent with the above data (e.g., examples 3, 5, 7), this example shows that the addition of certain oligosaccharides to an alpha-1, 3-glucan synthesis reaction can improve product yield. In addition, this higher yield reacted α -1, 3-glucan product had significantly reduced intrinsic viscosity.
Mixing 100g/L sucrose/10 mM KH2PO4The solution (500mL, pH adjusted to 5.5 with sodium hydroxide or sulfuric acid) was added to a separate 500-mL resin kettle with overhead agitation. The following materials were then added: glucose to 10g/L, leucrose to 10g/L, purified gluco-oligosaccharide to 10g/L (produced in a similar manner as in example 8), fructose to 5g/L, fructose to 10g/L, fructose to 15g/L, or fructose to 30 g/L. One kettle did not receive any additional material and was set as a control. The temperature of each kettle was adjusted to 25 ℃. Each reaction was then initiated by adding an aliquot (610 μ L) of the glucosyltransferase enzyme used in example 8 and allowed to run at 25 ℃ with moderate agitation for about 16 hours. Samples of each reaction were then taken, centrifuged and subjected to liquid analysis by HPLC to obtain the sugar content. The insoluble alpha-1, 3-glucan produced in each reaction was then filtered, washed with about 1L of water, and then dried in a vacuum oven at 45 ℃ for several days. The reaction filtrate was discarded.
More than 99% of the sucrose was converted in each reaction. Table 18 provides the α -1, 3-glucan yields for each reaction on HPLC (different glucosyl group consumption) and on dry solids weight. These yield measurements are all in good agreement with each other.
Watch 18
Effect of glucosyltransferase reaction additives on alpha-1, 3-glucan yield
Table 18 shows that upon addition of glucose (consistent with example 4) or increased amounts of fructose, the alpha-1, 3-glucan yield decreased, which was useful to increase the yield of the leucrose by-product (data not shown). Although the addition of leuconostoc disaccharide has no effect, the addition of glucose-oligosaccharides purified from the glucosyltransferase reaction alone improves the yield of alpha-1, 3-glucan.
The molecular weight (DPw) and intrinsic viscosity (. eta., provided in mL/g) of α -1, 3-glucan produced in some of the above reactions were measured (abbreviated as "IV") and are shown in table 19. IV measurements for this example were obtained according to U.S. patent application publication No. 2017/0002335, which is incorporated herein by reference.
Watch 19
Effect of glucosyltransferase reaction additives on alpha-1, 3-glucan products IV and DPw
aThe same control reaction as described above (table 18). The IV of the α -1, 3-glucan product of this reaction was not measured.
bA separate control reaction was performed in a similar manner to the control-1 reaction.
Table 19 shows that the IV of alpha-1, 3-glucan was significantly reduced after addition of glucose-oligosaccharides purified from the glucosyltransferase reaction alone. Although the DPw of this a-1, 3-glucan is also reduced, this change is not believed to explain the reduction in IV, as other additives also reduce DPw, but do not significantly reduce IV. This is evident, for example, on fructose addition (30g/L) which produces essentially the same reduction in alpha-1, 3-glucan DPw, but no observable effect on IV.
Thus, adding the gluco-oligosaccharide by-product of the alpha-1, 3-glucan synthesis reaction to a new alpha-1, 3-glucan synthesis reaction can (i) increase the yield of alpha-1, 3-glucan produced by the new reaction, and (ii) decrease the IV of alpha-1, 3-glucan produced by the new reaction.
Example 10
Alpha-1, 3-glucan production in glucosyltransferase reactions in the absence or presence of added gluco-oligosaccharides Comparison of
This example discloses that the addition of a gluco-oligosaccharide produced from the polymerization of alpha-1, 3-glucan can be used to obtain an alpha-1, 3-glucan product having a lower aqueous slurry viscosity and a lower dissolved polymer solution viscosity than alpha-1, 3-glucan produced in a reaction without the added gluco-oligosaccharide.
In each reaction prepared in this example, the glucosyltransferase used in example 8 was used.
The first alpha-1, 3-glucan reaction without addition of gluco-oligosaccharides was prepared in a 4-L jacketed glass reactor with overhead stirring and an external cooler/heater to maintain a constant temperature. The reaction medium is prepared by the following steps: 299g of sucrose was added to 2412g of water, followed by 3.54g of potassium phosphate and 130. mu.L of
The α -1, 3-glucan produced in the first reaction was filtered in a vacuum buchner funnel with filter paper and the filtrate (which contained the glucose-oligosaccharides) was collected for use in the subsequent reaction. The alpha-1, 3-glucan cake was then washed and filtered with more than 8L of water to separate the sugars from the alpha-1, 3-glucan, providing a cake with greater than 10 wt% solids (the percent solids were measured accordingly).
In the same reactor vessel, a second reaction was prepared by adding 299g of sucrose to 780g of filtrate from the first reaction and 1632g of water. Since the filtrate contained 1.03g of potassium phosphate, 2.51g of potassium phosphate was added to the second reaction. The solutions were mixed and the temperature was controlled at 20 ℃ followed by the addition of 130. mu.L
The third reaction was set as a repeat of the second reaction, but the filtrate collected from the second reaction was used. Table 20 summarizes the components of each of the first through third reactions (reactions 1-3, respectively).
Watch 20
Saccharide composition of reactions 1-3
Components
Reaction 1
Reaction 2
Reaction 3
Initial sucrose (g/L)
114
115
116
Initial Leuconostoc disaccharide (g/L)
0.0
4.9
9.0
Initial glucose (g/L)
0.5
2.0
1.4
Initial fructose (g/L)
0.3
16.0
19.6
Initial glucose-oligosaccharides (g/L)
0.0
3.5
3.9
The aqueous slurry viscosity of the alpha-1, 3-glucan product of each of the first, second and third reactions was measured by first adding sufficient water to the glucan cake to make a 4 wt% aqueous mixture and then homogenizing the mixture. The viscosity of each mixture was then measured on a rheometer at 20 ℃ and the shear rate was varied from 7s-1Increasing to 200s-1And the viscosity was measured in centipoise (cP). Fig. 2 shows the measured values showing the reduction of the aqueous slurry viscosity of the α -1, 3-glucan product as continuously prepared in the first to third reactions.
After dissolving in DMAc/0.5% LiCl to a concentration of 10mg/mL, the molecular weight (DPw) and Intrinsic Viscosity (IV) of each of the α -1, 3-glucan products in the first to third reactions were measured (Table 21, reactions 1-3, respectively).
TABLE 21
Molecular weight and IV of the alpha-1, 3-glucan product of reactions 1-3
Reaction 1
Reaction 2
Reaction 3
DPw
773
753
736
IV(mL/g)
292
262
248
Thus, consistent with the results of example 9 above, the addition of the gluco-oligosaccharide by-product of the α -1, 3-glucan synthesis reaction to the new α -1, 3-glucan synthesis reaction can reduce the viscosity of the α -1, 3-glucan produced by the new reaction (as measured both in the aqueous slurry and dissolved polymer form).
Example 11
Alpha- Comparison of 1, 3-Glucan polymers
This example discloses that the addition of gluco-oligosaccharides produced from the polymerization of alpha-1, 3-glucan can be used in batch or fed-batch mode for further reactions. Specifically, this example discloses that the addition of the gluco-oligosaccharides during the polymerization reaction (fed-batch addition) of alpha-1, 3-glucan reduces the viscosity of the glucan polymer produced over the reaction time. However, the final α -1, 3-glucan polymer produced at the end of the reaction has a higher viscosity than the α -1, 3-glucan polymer produced in a reaction in which all of the added glucose-oligosaccharides are provided in portions at the beginning of the reaction (added in portions). The higher final Intrinsic Viscosity (IV) of the polymer product of the fed-batch mode reaction compared to the batch reaction is likely due to the lower initial gluco-oligosaccharide concentration of the reaction.
In each reaction prepared in this example, the glucosyltransferase used in example 8 was used. The gluco-oligosaccharides used in these reactions are provided, for example, in the form of a glucosyltransferase reaction filtrate prepared in example 10.
The fed-batch reaction was prepared in a 4-L jacketed glass reactor with overhead stirring and an external cooler/heater to maintain a constant temperature. The reaction medium is prepared by the following steps: 260g of sucrose was added to 1656g of water, followed by 2.51g of potassium phosphate and 130. mu.L of
In the same reactor vessel, 1656g of water and 780g of a glucose-oligosaccharide containing liquid were mixed with 260g of sucrose to prepare a batch reaction. The solutions were mixed and the temperature was controlled at 20 ℃ followed by the addition of 130. mu.L
Table 22 shows the change in the glucose-oligosaccharide concentration during the fed-batch and batch reactions and confirms that the initial glucose-oligosaccharide concentration is initially higher in the batch reaction than in the fed-batch reaction.
TABLE 22
Glucoso-oligosaccharide concentration during fed-batch and batch reactions
After dissolution in DMAc/0.5% LiCl to a concentration of 10mg/mL, the Molecular Weight (MW) and Intrinsic Viscosity (IV) of the α -1, 3-glucan products of the fed-batch and batch reactions, respectively, were measured (tables 23 and 24).
TABLE 23
Viscosity of alpha-1, 3-glucan products of fed-batch and batch reactions
Table 23 shows that in the fed-batch reaction, the viscosity of the α -1, 3-glucan polymer decreased with time. However, the fed-batch final α -1, 3-glucan viscosity was higher than the batch final α -1, 3-glucan viscosity (both measured at the 22-hour time point, table 23).
Watch 24
Molecular weight of alpha-1, 3-glucan products of fed-batch and batch reactions
Thus, consistent with the results of examples 9-10 above, adding the gluco-oligosaccharide by-product of the α -1, 3-glucan synthesis reaction to a new α -1, 3-glucan synthesis reaction in either a batch or fed-batch mode can reduce the viscosity of the α -1, 3-glucan produced by the new reaction. It is noted, however, that such batch mode addition works much more to reduce polymer viscosity.
Example 12
Alpha-1, 3-glucan polymers produced in glucosyltransferase reactions with the addition of glucose-oligosaccharides at various temperatures Comparison of Compounds
This example discloses that the addition of glucose-oligosaccharides produced from the polymerization of alpha-1, 3-glucan to other alpha-1, 3-glucan polymerizations at different temperatures reduces the viscosity of the latter glucan polymer products. At lower reaction temperatures, this change in viscosity is significantly higher.
In each reaction prepared in this example, the glucosyltransferase used in example 8 was used. The gluco-oligosaccharides used in these reactions are provided, for example, in the form of a glucosyltransferase reaction filtrate prepared in example 10.
The reaction was carried out in a 500-mL jacketed glass reactor with overhead stirring and an external cooler/heater to maintain a constant temperature. The reaction medium is prepared by the following steps: 50g of sucrose was added to 469g of water, followed by 0.68g of potassium phosphate and 25. mu.L of potassium phosphate
Reactions (1-9) were run at three temperatures and three glucose-oligosaccharide concentrations. The gluco-oligosaccharide concentration was varied by adding the appropriate amount of filtrate from the previously described alpha-1, 3-glucan polymerization. The liquid added to each reaction is a suitable mixture of water and filtrate. Table 25 shows the reaction temperature and initial gluco-oligosaccharide concentration for reactions 1-9. After the entire reaction is complete, the α -1, 3-glucan product is filtered and washed with more than 1L of water to produce a glucan wet cake with greater than 10 wt% solids. Each cake was dissolved in DMAc/0.5% LiCl to a concentration of 10mg/mL, and the molecular weight and intrinsic viscosity of the glucan polymer product were then measured (Table 25).
TABLE 25
Fraction of alpha-1, 3-glucan produced in reactions with different temperatures and initial glucose-oligosaccharide concentrations Quantum and viscosity
Table 25 shows that the α -1, 3-glucan product was less viscous at the reaction with higher initial gluco-oligosaccharide concentration (maintained at the same temperature), consistent with the results described above. It is clear that this viscosity change is more pronounced (in terms of percentage) in reactions maintained at lower temperatures.
Example 13
Alpha-1, 3-glucan polymers produced in glucosyltransferase reactions with delayed addition of glucose-oligosaccharides
This example discloses that the addition of a gluco-oligosaccharide generated from the polymerization of alpha-1, 3-glucan to another polymerization of alpha-1, 3-glucan four hours after the start of the reaction (initiated by the addition of glucosyltransferase) produced a glucan polymer with a similar viscosity as compared to the polymerization of alpha-1, 3-glucan without the addition of gluco-oligosaccharide.
The glucosyltransferase used in example 8 was used in the reaction prepared in this example. The gluco-oligosaccharides used in this reaction are provided, for example, in the form of a glucosyltransferase reaction filtrate prepared in example 10.
The reaction was prepared in a 500-mL jacketed glass reactor with overhead stirring and an external cooler/heater to maintain a constant temperature. The reaction medium is prepared by the following steps: 46g of sucrose was added to 364g of water, followed by 0.44g of potassium phosphate and 20. mu.L of potassium phosphate
After the reaction is complete, the α -1, 3-glucan product is filtered and washed with more than 1L of water to make a glucan wet cake with greater than 10 wt% solids. The cake was dissolved in DMAc/0.5% LiCl to a concentration of 10mg/mL and the molecular weight and intrinsic viscosity of the glucan polymer product were then measured (Table 26).
Watch 26
Molecular weight and viscosity of alpha-1, 3-glucan produced in reactions with delayed glucose-oligosaccharide addition
As is apparent from table 26, the addition of the gluco-oligosaccharide resulting from the alpha-1, 3-glucan polymerization reaction to the start of the reaction for a period of time resulted in a glucan polymer having a similar viscosity as compared to the alpha-1, 3-glucan polymerization reaction with only an appropriate amount of added gluco-oligosaccharide.
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