阅读说明：本技术 L-半乳糖的合成方法 (Method for synthesizing L-galactose ) 是由 黄华 潘思婷 刘励文 于 2021-09-17 设计创作，主要内容包括：本发明公开了L-半乳糖的合成方法,包括以下步骤：取半乳糖醇和氧化型烟酰胺腺嘌呤二核苷酸二钠盐混合,加入甘露醇脱氢酶和NADH氧化酶反应,生成L-半乳糖；或者取D-半乳糖和还原型烟酰胺腺嘌呤二核苷酸二钠盐混合,加入甘露醇脱氢酶和D-半乳糖还原酶反应,生成L-半乳糖。本发明采用市面上廉价、大宗的D-半乳糖或半乳糖醇作为原料,利用酶催化剂一步或者两步直接转化得到昂贵的L-半乳糖。该制备方案具有多方面的工业应用优势,使用原料便宜、制备路线短、反应条件简单且废物排放低；以上这些特点都很好的符合了当今绿色工业生产的基本要求。(The invention discloses a synthetic method of L-galactose, which comprises the following steps: mixing galactitol and oxidized nicotinamide adenine dinucleotide disodium salt, adding mannitol dehydrogenase and NADH oxidase for reaction to generate L-galactose; or mixing D-galactose and reduced nicotinamide adenine dinucleotide disodium salt, adding mannitol dehydrogenase and D-galactose reductase, and reacting to obtain L-galactose. The invention adopts cheap and large amount of D-galactose or galactitol in the market as raw material, and utilizes enzyme catalyst to directly convert in one step or two steps to obtain expensive L-galactose. The preparation scheme has the advantages of various industrial applications, cheap used raw materials, short preparation route, simple reaction conditions and low waste discharge; the characteristics well meet the basic requirements of the current green industrial production.)

A method for synthesizing L-galactose, which is characterized by comprising the following steps:

mixing galactitol and oxidized nicotinamide adenine dinucleotide disodium salt, adjusting the pH value of a system to 7.5-8.0, adding mannitol dehydrogenase (agmDH) and NADH oxidase (NoxE), and reacting at 25-37 ℃ for 6-12 hours to generate L-galactose;

the amino acid sequence of the mannitol dehydrogenase is shown in SEQ ID No. 1.

2. The method of synthesis according to claim 1, characterized in that: the enzyme activity ratio of the mannitol dehydrogenase to the NADH oxidase is 1 (2-3).

3. The method of synthesis according to claim 1, characterized in that: the NADH oxidase is NADH oxidase (ScNoxE) derived from Clostridium scintillans (Clostridium scindens) or NADH oxidase (TtNoxE) derived from Thermus thermophilus (Thermus thermophilus).

4. The method of synthesis according to claim 3, characterized in that: TtNoxE is used in combination with catalase.

A method for synthesizing L-galactose, which is characterized by comprising the following steps:

mixing D-galactose and reduced nicotinamide adenine dinucleotide disodium salt, adjusting the pH value of a system to 7.0-8.5, adding mannitol dehydrogenase (agmDH) and D-galactose reductase (aldR), and reacting at 20-30 ℃ for 5-12 hours to generate L-galactose;

the amino acid sequence of the mannitol dehydrogenase is shown in SEQ.ID.NO. 1;

the amino acid sequence of the D-galactose reductase is shown in SEQ ID No. 4.

6. The method of synthesis according to claim 5, characterized in that: the enzyme activity ratio of the mannitol dehydrogenase to the D-galactose reductase is (2-3) to 1.

7. The synthesis method according to claim 1 or 5, characterized in that: and after the reaction is finished, removing or recovering the enzyme, adjusting the pH value of the reaction system to be neutral, removing impurities by using anion exchange resin, desalting by using an osmotic membrane, concentrating and crystallizing to obtain the L-galactose.

8. The method of synthesis according to claim 7, characterized in that:

the enzyme is liquid enzyme or immobilized enzyme;

if liquid enzyme is adopted, the pH value of a reaction system is adjusted to be strong acid after reaction, protein is precipitated, and the protein is removed by centrifugation;

if immobilized enzyme is adopted, the reaction is filtered and recycled.

9. The synthesis method according to claim 1 or 5, characterized in that: the reactant is added into trihydroxymethyl aminomethane hydrochloric acid, hydroxyethyl piperazine ethanethiosulfonic acid buffer solution or 3-morpholine propanesulfonic acid buffer solution for reaction.

10. The method of synthesis according to claim 8, characterized in that: the immobilized enzyme is prepared by the following steps:

mixing and dissolving the required enzyme according to the activity ratio in 50mM potassium phosphate solution with pH 8.0, then adding 40mM phenoxyacetic acid and LX-1000EP epoxy resin to the buffer solution, stirring for 5 hours at room temperature, filtering out the immobilized enzyme, finally washing three times by using clear water and 25mM phosphate buffer solution with pH 8.0 respectively, and drying at low temperature for standby.

Technical Field

The invention belongs to the field of biological enzyme synthesis, and particularly relates to a synthetic method of L-galactose.

Background

L-galactose (L-galactose) is a rare sugar occurring in nature and is the L-configuration of the common D-galactose. L-galactose is present in milk and sugar beet, and can also be synthesized by the human body. L-galactose is a potential oral treatment for focal and segmental glomerulosclerosis in nephrotic syndrome, and is a structural component of a variety of biologically active saponins (saponins) that are widely used in the fields of food, cosmetics and medicine. Meanwhile, the L-galactose is also a basic raw material for synthesizing various L-nucleoside antiviral drugs and vitamin C biosynthesis. However, the high price greatly limits the popularization and application (the price of L-galactose is 2,300/g on Carbosynth, a sugar selling company known in the world).

At present, L-galactose has a plurality of preparation methods, but all have certain defects, so that the L-galactose cannot be produced and applied on a large scale at low cost.

In the chemical preparation route, the synthesis of the sugar needs to undergo multi-step hydroxyl protection and deprotection, which not only leads to the overlong preparation route and the low overall yield, but also greatly troubles the purification of the product at the later stage due to the chiral racemization problem in the preparation process.

The L-galactose produced by fermentation has low product concentration and purity, so that the purification cost of the final product is too high to realize effective large-scale production.

Disclosure of Invention

The invention aims to provide a synthetic method of L-galactose, which adopts an enzyme catalysis strategy to effectively realize the direct conversion from cheap D-galactose/galactitol to L-galactose, has short preparation route (one-step or two-step conversion), simple reaction conditions and high conversion rate, and therefore has remarkable advantages in production cost and environmental protection.

The purpose of the invention is realized by the following technical scheme:

the synthesis method of the L-galactose comprises the following steps:

collecting galactitol and oxidized nicotinamide adenine dinucleotide disodium salt (NAD)⁺) Mixing, adjusting pH to 7.5-8.0, adding mannitol dehydrogenase (agmDH) and NADH oxidase (NoxE), and reacting at 20-37 deg.C for 6-12 hr to obtain L-galactose;

the enzyme activity ratio of the mannitol dehydrogenase to the NADH oxidase is 1 (2-3);

said NADH oxidase can be derived fromNADH oxidase (ScNoxE for short) of Clostridium scintillans (Clostridium scindens), the oxidation product of which is H₂O, Uniprot ID: B0NJW3, EC 1.6.3.1) (also SEQ. ID. NO.2), or NADH oxidase derived from Thermus thermophilus (Thermus thermophilus) (TtNoxE, the oxidation product is H₂O₂Uniprot ID: Q60049, EC 1.6.99.3) (i.e., seq. ID No. 3);

if TtNoxE is adopted, Catalase (Catalase) is required to be used together; generally, the enzyme activity ratio of catalase to TtNoxE is (0.25-1.2):1, preferably 1: 3.

Preferably, the reaction can be carried out under pressure, particularly preferably at 1.5 atmospheres;

the mannitol dehydrogenase (agMDH) and a wild type (Unit ID: Q38707, EC 1.1.1.255) are from celery (apex graveolens), the enzyme expression and the stability of the mannitol dehydrogenase in escherichia coli are improved through the genetic modification, and mutation sites are as follows: F14I, S47C, N93Y, T301S and S343G, wherein the modified amino acid sequence is shown as SEQ ID No. 1.

The synthesis method of the L-galactose comprises the following steps:

mixing D-galactose and reduced nicotinamide adenine dinucleotide disodium salt (NADH), adjusting the pH value of the system to 7.0-8.5 (the pH value range is kept in the reaction process), adding mannitol dehydrogenase (agmDH) and D-galactose reductase (aldR), and reacting at 20-37 ℃ for 5-12 hours to generate L-galactose;

the enzyme activity ratio of the mannitol dehydrogenase to the D-galactose reductase is (2-3) to 1, preferably 2.5: 1;

the D-galactose reductase (aldR) used by the invention, the wild type (Unit ID: A0A2S9ZVX5, EC 1.1.1.21) of which is from Rhodotorula toruloides, and the wild type coenzyme is NADPH, can effectively utilize NADH after the site-specific modification of the invention, and the mutation sites are as follows: K247W, S248L and R253M, wherein the modified amino acid sequence is shown in SEQ ID No. 4.

In the two methods, after the reaction is finished, enzyme is removed or recovered, the pH value of a reaction system is adjusted to be neutral, then impurity removal is carried out through anion exchange resin, desalting is carried out through a permeable membrane, and then concentration and crystallization are carried out, so that the L-galactose is obtained.

The enzyme is liquid enzyme or immobilized enzyme;

if liquid enzyme is adopted, the pH value of a reaction system is adjusted to be strong acid after reaction, protein is precipitated, and the protein is removed by centrifugation;

if immobilized enzyme is adopted, the reaction is filtered and recycled.

Preferably, the reactant is added into tris (hydroxymethyl) aminomethane hydrochloride (Tris.HCl), hydroxyethyl piperazine ethanesulfonic acid (Hepes) buffer solution or 3-morpholine propanesulfonic acid (MOPS) buffer solution for reaction; however, phosphate buffer systems are not used here, and high concentrations of phosphoric acid have an inhibitory effect on the reaction enzymes.

The immobilized enzyme is prepared by the following steps:

mixing and dissolving the required enzyme in 50mM potassium phosphate solution with pH 8.0 according to a set activity ratio, adding 40mM phenoxyacetic acid and LX-1000EP epoxy resin to the buffer solution, stirring for 5 hours at room temperature, filtering out the immobilized enzyme, washing with clear water and 25mM phosphate buffer solution with pH 8.0 for three times respectively, and drying at low temperature for later use;

the agmDH/ScNoxE immobilized mix enzyme had 70-91% activity corresponding to the liquid enzyme. The aldR/agMDH immobilized mixed enzyme has 78-89% of the activity of liquid enzyme.

Compared with the prior art, the invention has the following advantages and effects:

1. the invention adopts cheap and large amount of D-galactose or galactitol in the market as raw material, and utilizes enzyme catalyst to directly convert in one step or two steps to obtain expensive L-galactose. The preparation scheme has the advantages of various industrial applications, cheap used raw materials, short preparation route, simple reaction conditions and low waste discharge; the characteristics well meet the basic requirements of the current green industrial production.

2. In the first route of the method, galactitol (1200/kg, aladin reagent net) is used as a raw material, and the hydroxyl at the tail end of the galactitol is oxidized into L-galactose by using mannitol dehydrogenase (agmDH, EC 1.1.1.255); the preparation process requires coenzyme Nicotinamide Adenine Dinucleotide (NAD)⁺) By reacting in the presence ofThe NADH oxidase (NoxE, EC 1.6.99.3) is introduced into the system to effectively and circularly regenerate NAD⁺And simultaneously, the conversion rate of the whole reaction is greatly improved.

3. In another route of the method, cheaper D-galactose (480/kg, aladdin reagent net) is used as a raw material, the continuous conversion from D-galactose to L-galactose can be realized by coupling D-galactose reductase (aldR, EC 1.1.1.21) with mannitol dehydrogenase in the first route, and the first step of reaction adopts D-galactose reductase (aldR, EC 1.1.1.21) which can utilize NADH as coenzyme to generate NAD⁺And the second dehydrogenase can convert NAD⁺Reducing the coenzyme into NADH, thereby effectively realizing the cyclic regeneration of the coenzyme in the two-step reaction without adding an additional coenzyme regeneration system; this not only further simplifies the process, but also effectively increases the conversion in both steps.

Drawings

FIG. 1 is an HPLC chromatogram of L-galactose.

FIG. 2 is a 1H-NMR spectrum of L-galactose.

FIG. 3 shows the results of SDS-PAGE gel detection of the prepared enzyme.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Example 1: preparation of L-galactose by using galactitol as raw material and liquid enzyme (agMDH, ScNoxE)

27.3 g of galactitol (150mM), 1.42 g of oxidized nicotinamide adenine dinucleotide disodium salt (NAD), was added to 1L of 50mM Tris-hydrochloric acid (Tris.HCl) solution at pH 8.0⁺2mM), then the pH of the solution was adjusted to 8.0, and then a crude enzyme solution (1500U of agMDH, 3000U of ScNoxE) was added, followed by transferring the reaction solution to a pressure-resistant reactor and slowly stirring the reaction solution at 30 ℃ for 6 hours while maintaining an oxygen pressure of 1.5 atm. After the reaction is finished, HCl aqueous solution is added to adjust the pH value to 1.0 for denaturation, protein is precipitated, andremoving by high-speed centrifugation, adjusting the pH value of the solution to 7.0, removing impurities by using D201 anion exchange resin (Tianjin Kaishi resin science and technology Co., Ltd.), desalting by using a reverse osmosis membrane, concentrating and crystallizing (ethanol: water, 3:1, v: v) to obtain 22 g of L-galactose (colorless granular solid, yield 82%).

The product obtained was analyzed by HPLC. Phenomenex Rezex RHM Series monosaccharide analysis chromatography column (7.8 mm. times.300 mm,8 μm) using pure water as mobile phase at a flow rate of 0.2ml/min was detected at 35 deg.C using a refractive index detector (Agilent,1100Series, G1362A). The HPLC profile is shown in FIG. 1.

The 1H-NMR spectrum of the obtained product is shown in FIG. 2, Varian 600 million nuclear magnetism, and D2O is solvent.

From the results of FIGS. 1 and 2, it was confirmed that the obtained product was L-galactose.

Example 2: preparation of L-galactose by immobilized enzyme (agMDH, ScNoxE) with galactitol as raw material

Similar to example 1, but mixed immobilized agMDH, ScNoxE enzyme was used for catalysis instead of liquid enzyme, and the immobilized enzyme could be recycled after the reaction was finished.

18.2 g of galactitol (100mM), 1.42 g of oxidized nicotinamide adenine dinucleotide disodium salt (NAD), was added to 1L of 50mM Tris-HCl pH 8.0 solution⁺2mM), then adjusting the pH value of the solution to 8.0, adding immobilized mixed enzyme (5000U, the activity unit ratio of agmDH and ScNoxE is 1:5), and then transferring the reaction liquid into a pressure-resistant reactor to maintain the pressure of oxygen at 2 atm and slowly shaking the reaction liquid at 30 ℃ for 12 hours. After the reaction is finished, filtering and recovering the immobilized enzyme (the immobilized enzyme is washed for three times by 50mM of a Tris buffer solution with the pH value of 8.0 and then stored at 4 ℃ for later use), adjusting the pH value of the filtrate to 7.0, purifying by using D201 anion exchange resin, desalting the collected product solution by using a reverse osmosis membrane, concentrating and crystallizing (ethanol: water, 3:1, v: v) to obtain 14.9 g of L-galactose (the yield is 83%), wherein the filtered and recovered immobilized mixed enzyme has the initial enzyme activity of 74%.

The HPLC spectrum and 1H-NMR spectrum of the obtained product are the same as those of FIG. 1 and FIG. 2, respectively, and it can be confirmed that the obtained product is L-galactose.

Example 3: preparation of L-galactose from galactitol by liquid enzyme (agmDH, TtNoxE, Catalase)

18.2 g of galactitol (100mM), 1.42 g of oxidized nicotinamide adenine dinucleotide disodium salt (NAD), was added to 1L of 100mM Tris-HCl pH 7.5 solution⁺2mM), then the pH of the solution was adjusted to 7.5, and then a crude enzyme solution (1000U agMDH, 3000U TtNoxE, 1000U catalase) was added, followed by transferring the reaction solution into a pressure-resistant reactor and slowly stirring the reaction solution at 30 ℃ for 8 hours while maintaining an oxygen pressure of 2 atm. After the reaction, HCl aqueous solution is added to adjust the pH value to 1.0 for denaturation, protein is precipitated and removed by high-speed centrifugation, then the pH value of the solution is adjusted to 7.0, then the solution is purified by D201 anion exchange resin (Tianjin Kaishi resin science and technology Co., Ltd.), and finally, the solution is desalted by a reverse osmosis membrane, concentrated and crystallized (ethanol: water, 3:1, v: v) to obtain 12.8 g of L-galactose (colorless granular solid, yield 71%).

The HPLC spectrum and 1H-NMR spectrum of the obtained product are the same as those of FIG. 1 and FIG. 2, respectively, and it can be confirmed that the obtained product is L-galactose.

Example 4: preparation of L-galactose by using D-galactose as raw material and liquid enzyme (aldR, agmDH)

18 g of D-galactose (100mM) and 0.71 g of reduced nicotinamide adenine dinucleotide disodium salt (NADH,1mM) were added to 1L of 50mM Tris-hydrochloric acid (Tris.HCl) solution having a pH of 8.0, the pH of the solution was adjusted to 8.0, then a crude enzyme solution (800U aldR, 2000U agMDH) was added to start the reaction, the reaction solution was slowly stirred at 25 ℃ for 5 hours, and the pH of the reaction system was maintained at 7.0 to 8.5 by adding an acid base during the reaction. After the reaction, HCl aqueous solution was added to adjust pH to 1.0 to terminate the reaction, precipitate the protein, then adjust the reaction solution to pH 7.0 and purify it with D201 anion exchange resin, the collected product solution was desalted with reverse osmosis membrane and concentrated and crystallized (ethanol: water, 3:1, v: v) to obtain 15.1 g L-galactose (colorless granular solid, yield 84%).

The HPLC spectrum and 1H-NMR spectrum of the obtained product are the same as those of FIG. 1 and FIG. 2, respectively, and it can be confirmed that the obtained product is L-galactose.

Example 5: preparing L-galactose by using D-galactose as raw material and immobilized enzyme (aldR, agmDH)

Similar to example 4, but mixed immobilized aldR, agmDH enzyme was used for catalysis instead of liquid enzyme, and the immobilized enzyme could be recycled after the reaction.

Similarly, 18 g of D-galactose (100mM) and 0.71 g of reduced nicotinamide adenine dinucleotide disodium salt (NADH,1mM) were added to 1L of 50mM Tris-hydrochloric acid (Tris.HCl) solution having a pH of 8.0, the pH of the solution was adjusted to 8.0, 3000U of an immobilized enzyme (the activity ratio of aldR to agmDH was 1:3) was added to start the reaction, the reaction solution was slowly stirred at 25 ℃ for 12 hours, and the pH of the system was maintained at 7.0 to 9.0 during the reaction. After the reaction is finished, filtering and recovering immobilized enzyme (the immobilized enzyme is washed for three times by 50mM of a Tris buffer solution with the pH value of 8.0 and then stored at 4 ℃ for later use), adjusting the pH value of filtrate to 7.0, purifying by using D201 anion exchange resin, desalting the collected product solution by using a reverse osmosis membrane, concentrating and crystallizing (ethanol: water, 3:1, v: v) to obtain 14.2 g of L-galactose (the yield is 79%), and filtering and recovering the immobilized mixed enzyme has 83% of initial enzyme activity.

The HPLC spectrum and 1H-NMR spectrum of the obtained product are the same as those of FIG. 1 and FIG. 2, respectively, and it can be confirmed that the obtained product is L-galactose.

Example 6: fermentative production of enzymes

The enzymes used in the present invention were all produced by laboratory fermentation (except the Catalase available from novifin), and the following is the basic procedure for preparing the enzyme.

Firstly, synthesizing a gene sequence (SEQ. ID. NO. 5-SEQ. ID. NO.8) corresponding to the enzyme by a gene company (Anhui general organisms), then subcloning the gene sequence to a pET28a plasmid through NdeI/XhoI enzyme cutting sites, transferring the plasmid to E.coli (BL21) (engine organisms) cells for plate culture, and finally selecting single clone for liquid step-by-step amplification culture.

Firstly transferring a single colony on a plate into 5ml of LB culture solution (37 ℃) containing 50 mu M kanamycin for culture, inoculating the single colony into 250ml of LB culture solution containing the same antibiotics after the cells grow to a logarithmic phase, and finally transferring the single colony into a 5L culture fermentation tank for culture; when the OD600 of the cells was about 20, 0.5mM isopropyl-. beta. -D-thiogalactopyranoside (IPTG) was added to induce protein expression for 6 hours at 26 ℃ and then centrifuged (4000rpm,15min) to collect 30-55g of wet cells.

To verify the expression of the enzyme, a small amount of cells are taken and uniformly mixed with tris (hydroxymethyl) aminomethane hydrochloride (Tris.HCl) buffer (50mM, pH 8.0), then the cells are crushed by a freeze-thaw method, and after high-speed centrifugation, the supernatant is taken and SDS-PAGE protein gel (sodium dodecyl sulfate-polyacrylamide gel) is run to determine the soluble expression of the protein (figure 3); and (3) uniformly mixing the residual cells with a buffer solution (10 g of wet cells are mixed with about 200ml of buffer solution on the market), then carrying out high-pressure cell disruption and high-speed centrifugation (16000rpm,45min) to remove cell walls, and finally directly carrying out subsequent use on the obtained enzyme-containing clear liquid (namely the crude enzyme liquid) (the activity of the crude enzyme liquid is 180-400U/ml, U is the enzyme amount required for converting 1 mu mol of substrate at room temperature for one minute) or further purifying and then immobilizing for use (solid enzyme reaction).

The LB medium is composed of: 1% tryptone, 0.5% yeast powder, 1% NaCl, 1% dipotassium hydrogen phosphate and 5% glycerol.

Example 7: mixed immobilization of enzymes

To the crude enzyme solutions of mannitol dehydrogenase (agmDH), NADH oxidase (ScNoxE) and galactose reductase (aldR) collected in example 6, ammonium sulfate solids were gradually added until the enzyme was precipitated (30% -60%, w/v ammonium sulfate/buffer). The enzyme solid was then collected by centrifugation (10000rpm,12min) and slowly dissolved in 25mM Tris buffer pH 8.0, and finally desalted on a G25 size exclusion column (from Sigma) and separated on a DEAE Sephate FF anion exchange column to give the primary purified liquid enzymes agmDH, ScNoxE, aldR, which were used directly for subsequent enzyme immobilization.

In the mixed immobilization of the agmDH/ScNoxE enzyme, the primary purified enzymes aldR and ScNoxE were mixed and immobilized with LX-1000EP epoxy resin (Xian blue Xiao Co.).

The basic method of immobilization is that the enzyme is dissolved in 2L 50mM potassium phosphate solution with pH 8.0 according to the activity unit proportion, then 40mM phenoxyacetic acid and 600 g LX-1000EP epoxy resin are added to the buffer solution, the immobilized enzyme is filtered after stirring for 5 hours at room temperature, and finally the immobilized enzyme is washed by clean water and 25mM phosphate buffer solution with pH 8.0 for three times and dried at low temperature for standby; the agmDH/ScNoxE immobilized mix enzyme had 70-91% activity corresponding to the liquid enzyme.

The same method can mix and fix the aldR/agMDH enzyme, and the fixed enzyme has 78-89% of the activity of liquid enzyme.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

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Val Val Leu Val Leu Tyr Ala Asp Leu Glu Asp Ala Leu Ala His Leu

85 90 95

Asp Glu Val Ile His Pro Gly Val Gln Gly Glu Arg Arg Glu Ala Gln

100 105 110

Lys Gln Ala Ile Gln Arg Ala Phe Ala Ala Met Gly Gln Glu Ala Arg

115 120 125

Lys Ala Trp Ala Ser Gly Gln Ser Tyr Ile Leu Leu Gly Tyr Leu Leu

130 135 140

Leu Leu Leu Glu Ala Tyr Gly Leu Gly Ser Val Pro Met Leu Gly Phe

145 150 155 160

Asp Pro Glu Arg Val Arg Ala Ile Leu Gly Leu Pro Ser His Ala Ala

165 170 175

Ile Pro Ala Leu Val Ala Leu Gly Tyr Pro Ala Glu Glu Gly Tyr Pro

180 185 190

Ser His Arg Leu Pro Leu Glu Arg Val Val Leu Trp Arg

195 200 205

<210> 4

<211> 290

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> aldR

<400> 4

Met Gly Ala Glu His Ala Val Ala Ser Pro Phe Thr Leu Ala Ser Ser

1 5 10 15

Val Lys Leu Arg Asn Gly Ala Gln Met Pro Arg Leu Gly Phe Gly Val

20 25 30

Phe Gln Ser Thr Asn Ala Lys Ala Ser Thr Ala His Ala Leu Thr Met

35 40 45

Gly Tyr Arg His Ile Asp Ser Ala Arg Tyr Tyr His Asn Glu Glu Glu

50 55 60

Val Cys Ala Ala Val Gln Lys Phe Ser Gly Gly Asn Leu Pro Asn Glu

65 70 75 80

Gly Thr Gly Lys Val Trp Leu Thr Thr Lys Val Met Gly Gln Glu His

85 90 95

Gly Thr Asp Gln Thr Asn Lys Ala Val Asp Glu Ser Val Ala Ile Ala

100 105 110

Lys Lys Tyr Gly Leu Thr Trp Asp Leu Phe Leu Leu His Asp Pro Thr

115 120 125

Ala Gly Lys Gln Lys Arg Leu Glu Ala Trp Lys Val Leu Ile Glu Lys

130 135 140

Arg Asp Gln Gly Leu Ile Lys Ser Ile Gly Val Ser Asn Phe Gly Val

145 150 155 160

Lys His Leu Glu Gln Ile Lys Glu Ala Gly Leu Glu Thr Pro Glu Val

165 170 175

Asn Gln Ile Glu Leu His Pro Phe Leu Gln Gln Arg Asp Ile Val Glu

180 185 190

Tyr Cys Glu Lys Glu Gly Ile Val Val Glu Ala Tyr Cys Pro Ile Leu

195 200 205

Arg Gly Lys Arg Phe Asp Asp Pro Thr Leu Val Glu Leu Ser Lys Lys

210 215 220

His Ser Val Thr Val Pro Gln Ile Leu Ile Arg Trp Ser Leu Gln Lys

225 230 235 240

Gly Phe Val Pro Leu Pro Trp Leu Asp Thr Pro Gly Met Ile Gln Ala

245 250 255

Asn Ala Asp Leu Trp Asp Phe Glu Leu Asp Glu Gly Asp Met Gln Gln

260 265 270

Met Glu Lys Leu Asp Glu Gly Tyr Ala Val Ser Trp Asn Pro Val Asn

275 280 285

Val Glu

290

<210> 5

<211> 900

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> agMDH

<400> 5

atggcgaaaa gcagcgaaat tgaacatccg gtgaaagcga ttggctgggc ggcgcgcgat 60

accaccggcc tgctgagccc gtttaaattt agccgccgcg cgaccggcga aaaagatgtg 120

cgcctgaaag tgctgttttg cggcgtgtgc catagcgatc atcatatgat tcataacaac 180

tggggcttta ccacctatcc gattgtgccg ggccatgaaa ttgtgggcgt ggtgaccgaa 240

gtgggcagca aagtggaaaa agtgaaagtg ggcgattatg tgggcattgg ctgcctggtg 300

ggcagctgcc gcagctgcga aagctgctgc gataaccgcg aaagccattg cgaaaacacc 360

attgatacct atggcagcat ttattttgat ggcaccatga cccatggcgg ctatagcgat 420

accatggtgg cggatgaaca ttttattctg cgctggccga aaaacctgcc gctggatagc 480

ggcgcgccgc tgctgtgcgc gggcattacc acctatagcc cgctgaaata ttatggcctg 540

gataaaccgg gcaccaaaat tggcgtggtg ggcctgggcg gcctgggcca tgtggcggtg 600

aaaatggcga aagcgtttgg cgcgcaggtg accgtgattg atattagcga aagcaaacgc 660

aaagaagcgc tggaaaaact gggcgcggat agctttctgc tgaacagcga tcaggaacag 720

atgaaaggcg cgcgcagcag cctggatggc attattgata ccgtgccggt gaaccatccg 780

ctggcgccgc tgtttgatct gctgaaaccg aacggcaaac tggtgatggt gggcgcgccg 840

gaaaaaccgt ttgaactgcc ggtgtttagc ctgctgaaag gccgcaaact gctgggcggc 900

<210> 6

<211> 1380

<212> DNA

<213> Clostridium scintillans (Clostridium scindens)

<220>

<223> ScNoxE

<400> 6

atgggcgcgg cgtattgcgt gcatggcggc cgctatcgca aagtgagcga tcgcattatt 60

gtgattggcg cgaaccatgc gggcaccgcg gcgctgaaca ccattctgga taactatacc 120

gataaagatg tgaccgcgtt tgatgcgaac agcaacatta gctttctggg ctgcggcatg 180

gcgctgtgga ttggccgcca gattagcggc ccggaaggcc tgttttatag cgataaagaa 240

accctggaag gcaaaggcgc gaaagtgttt ctggaaacca aagtgagccg cattgatttt 300

gaaaaaaaaa ccgtgtatgc gatggataaa gaaggcgaag aaattgaagc gaactatgat 360

aaactgattc tggcgaccgg cagcctgccg attcagccga aagtgaaagg catggaactg 420

aaaaacgtgc agtttgtgaa actgtatcag aacgcggcgg atgtgattga aaaactggaa 480

gatccgagca ttcagaaagt gaccattatt ggcgcgggct atattggcgt ggaactggcg 540

gaagcgtttg cgcgcaacgg ccgcaaaacc accctggtgg attgcgcgga tacctgcctg 600

agcgcgtatt atgatccgga attttgcaaa attatggaag aaaacctgcg cgaaaacggc 660

gtgcgcaccg cgtttggcga aatggtgcag gaaattcagg gccaggaact ggtggaacgc 720

gtggtgaccg ataaagatag ctatgatacc gatatggcgg tgttttgcat tggctttcgc 780

ccgaacaccc agtatgcgga tggcagcctg gaactgtttc gcaacggcgc gtttctggtg 840

gatctgcatc agcaggcgag ccgcccggaa gtgtatgcga ttggcgattg cgcgaccgtg 900

tttaacaacg cgacccagcg caaagattat attgcgctgg cgaccaacgc ggtgcgcagc 960

ggcattattg cggcgcataa cgcgtgcggc accccgctgg aaagcgcggg cgtgcagggc 1020

agcaacgcga tttgcatttg gggcctgaac atggtgagca ccggcattag cctgaaaaaa 1080

gcgctggaac tgggctatga agcggcggcg gcggattatg aagattggca gaaagcgggc 1140

tttattgaaa gcggcaacga aaaagtgcgc attcgcattg tgtatgataa aaaaacccgc 1200

attgtgctgg gcgcgcagat gtgcagcaaa tatgatatta gcatgggcat tcatatgttt 1260

agcctggcga ttcaggaaca ggtgaccatt gataaactga aactgctgga tctgtttttt 1320

ctgccgcatt ttaaccagcc gtataactat attaccatgg cggcgctgaa agcggaatag 1380

<210> 7

<211> 618

<212> DNA

<213> Thermus thermophilus (Thermus thermophilus)

<220>

<223> TtNoxE

<400> 7

atggaagcga ccctgccggt gctggatgcg aaaaccgcgg cgctgaaacg ccgcagcatt 60

cgccgctatc gcaaagatcc ggtgccggaa ggcctgctgc gcgaaattct ggaagcggcg 120

ctgcgcgcgc cgagcgcgtg gaacctgcag ccgtggcgca ttgtggtggt gcgcgatccg 180

gcgaccaaac gcgcgctgcg cgaagcggcg tttggccagg cgcatgtgga agaagcgccg 240

gtggtgctgg tgctgtatgc ggatctggaa gatgcgctgg cgcatctgga tgaagtgatt 300

catccgggcg tgcagggcga acgccgcgaa gcgcagaaac aggcgattca gcgcgcgttt 360

gcggcgatgg gccaggaagc gcgcaaagcg tgggcgagcg gccagagcta tattctgctg 420

ggctatctgc tgctgctgct ggaagcgtat ggcctgggca gcgtgccgat gctgggcttt 480

gatccggaac gcgtgcgcgc gattctgggc ctgccgagcc atgcggcgat tccggcgctg 540

gtggcgctgg gctatccggc ggaagaaggc tatccgagcc atcgcctgcc gctggaacgc 600

gtggtgctgt ggcgctaa 618

<210> 8

<211> 873

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> aldR

<400> 8

atgggcgcgg aacatgcggt ggcgagcccg tttaccctgg cgagcagcgt gaaactgcgc 60

aacggcgcgc agatgccgcg cctgggcttt ggcgtgtttc agagcaccaa cgcgaaagcg 120

agcaccgcgc atgcgctgac catgggctat cgccatattg atagcgcgcg ctattatcat 180

aacgaagaag aagtgtgcgc ggcggtgcag aaatttagcg gcggcaacct gccgaacgaa 240

ggcaccggca aagtgtggct gaccaccaaa gtgatgggcc aggaacatgg caccgatcag 300

accaacaaag cggtggatga aagcgtggcg attgcgaaaa aatatggcct gacctgggat 360

ctgtttctgc tgcatgatcc gaccgcgggc aaacagaaac gcctggaagc gtggaaagtg 420

ctgattgaaa aacgcgatca gggcctgatt aaaagcattg gcgtgagcaa ctttggcgtg 480

aaacatctgg aacagattaa agaagcgggc ctggaaaccc cggaagtgaa ccagattgaa 540

ctgcatccgt ttctgcagca gcgcgatatt gtggaatatt gcgaaaaaga aggcattgtg 600

gtggaagcgt attgcccgat tctgcgcggc aaacgctttg atgatccgac cctggtggaa 660

ctgagcaaaa aacatagcgt gaccgtgccg cagattctga ttcgctggag cctgcagaaa 720

ggctttgtgc cgctgccgtg gctggatacc ccgggcatga ttcaggcgaa cgcggatctg 780

tgggattttg aactggatga aggcgatatg cagcagatgg aaaaactgga tgaaggctat 840

gcggtgagct ggaacccggt gaacgtggaa tga 873