CN113355367A

CN113355367A - Application of ketoacid reductase in synthesis of chiral aromatic 2-hydroxy acid

Info

Publication number: CN113355367A
Application number: CN202110401448.4A
Authority: CN
Inventors: 薛亚平; 郑裕国; 王闯; 柳志强
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2018-03-22
Filing date: 2018-03-22
Publication date: 2021-09-07
Anticipated expiration: 2038-03-22
Also published as: CN113355367B; CN108410831A; CN113355299B; CN113355299A; CN108410831B

Abstract

The invention discloses an application of ketoacid reductase in synthesizing chiral aromatic 2-hydroxy acid, wherein the amino acid sequence of the ketoacid reductase is shown as SEQ ID NO. 10; the present invention provides a highly efficient ketoacid reductase derived from Leuconostoc lactis, which is capable of catalyzing a wide-spectrum aromatic 2-keto acid and whose substrate loading with acetophenone acid as a substrate is increased from 100mM to 400 mM. A single-bacterium two-plasmid three-enzyme tandem redox cascade system established by ketoacid reductase, 2-hydroxy acid dehydrogenase and glucose dehydrogenase can catalyze most of racemic aromatic 2-hydroxy acid to be efficiently racemized into chiral aromatic (R) -2-hydroxy acid, and both the yield and the e.e. value are more than 99%.

Description

Application of ketoacid reductase in synthesis of chiral aromatic 2-hydroxy acid

The application is a divisional application of a patent application, namely' application No. 201810239718.4, application date 2018, 3 and 22 months, namely ketoacid reductase, gene, engineering bacteria and application in synthesizing chiral aromatic 2-hydroxy acid

(I) technical field

The invention relates to a ketoacid reductase gene derived from Leuconostoc lactis (Leuconostoc lactis), a coding enzyme, a vector, an engineering bacterium and application in synthesizing chiral aromatic 2-hydroxy acid.

(II) background of the invention

Chiral 2-hydroxy acid is a compound with hydroxyl (-OH) substitution at C1 position at carboxyl (-COOH) side, has wide distribution and active property, is an important chiral building block for producing medicaments and fine chemicals, and has important application value in the fields of chemical industry, medicaments and the like. Wherein, the representative is a kind of derivatives containing benzene ring in the structure, such as (R) -mandelic acid is a key intermediate for producing semi-synthetic penicillin, cephalosporin, antineoplastic and weight-reducing drug, and is an important chiral resolving agent; the (R) -o-chloromandelic acid is a key chiral building block for synthesizing the antithrombotic drug (clopidogrel), and has high market demand and economic benefit; (R) -4-chloromandelic acid is the synthetic precursor of the first commercial mandelamide fungicide (mandipropamid); the (R) -4-hydroxymandelic acid can be used for producing levo-p-hydroxyphenylglycine, and further synthesizing various broad-spectrum antibiotics, such as amoxicillin, amoxicillin and cefaloxime, and the like.

Due to the important application of chiral 2-hydroxy acid in the fields of medicine and fine chemistry, how to obtain optically pure chiral 2-hydroxy acid becomes a research hotspot. Traditionally, the preparation of optically pure chiral 2-hydroxy acids has relied primarily on chemical kinetic resolution, i.e., the selection of appropriate chiral resolving agents to convert racemic mixtures into diastereomeric salts, change the physical properties of the two configurations, and then perform subsequent separation and extraction. However, this method has many disadvantages, such as a maximum yield of only 50%, low optical purity, expensive resolving agent, severe reaction conditions, and environmental pollution. In recent years, the biocatalytic method has been increasingly researched and developed due to its advantages of high stereoselectivity and catalytic activity, mild reaction conditions, environmental friendliness, etc., and is used for the efficient production of optically pure chiral 2-hydroxy acid. Among them, the comparison typically includes enzymatic resolution, nitrilase method, asymmetric reduction and redox cascade racemization.

Enzymatic resolution, in which optical pure chiral 2-hydroxy acid is prepared by enzymatic resolution, belongs to kinetic resolution, and generally means that one configuration in racemic 2-hydroxy acid is selectively degraded by a biological enzyme method to obtain another target configuration. It retains the advantages of the biological enzyme reaction, but has the greatest disadvantage that the theoretical yield is only 50%. In addition, the method of separating o-chloromandelic acid ester by using lipase to obtain an esterified substance with a single configuration and then hydrolyzing to obtain the optically pure o-chloromandelic acid is also available, but the method has complicated steps and is not suitable for industrial application.

The nitrilase method is an important hydrolase, can catalyze nitrile substrates to be converted into corresponding carboxylic acid in one step, and has good catalytic property, so that the preparation of the optical pure chiral 2-hydroxy acid by using the nitrilase to catalyze the 2-hydroxy nitrile biologically is widely researched. However, the method requires a large amount of highly toxic hydrocyanic acid (HCN) in the catalytic process, thereby causing certain dangerousness and operational difficulty in practical application.

And thirdly, asymmetric reduction, namely, the prochiral 2-keto acid is asymmetrically reduced into the optically pure 2-hydroxy acid by utilizing the reductase with stereoselectivity in the microorganism body. The method has the advantages of high theoretical yield, simple operation and the like; the disadvantages are that the reaction process generally needs to add coenzyme, the coenzyme is expensive, the production cost is greatly increased, and the industrial application is not facilitated. In addition, prochiral 2-keto acid substrates are not readily available or expensive relative to the racemic compound, limiting their utility.

And fourthly, racemization of an oxidation-reduction cascade, and catalyzing the racemic 2-hydroxy acid to be converted into a single configuration product by using 2-hydroxy acid dehydrogenase, ketoacid reductase and glucose dehydrogenase with enantioselectivity. The method saves time, omits the extraction or purification of the product, and also can reduce the reversible reaction to the direction of the substrate, thereby being the most valuable chiral 2-hydroxy acid acquisition method. Meanwhile, by means of a multigene coexpression technology, a three-enzyme coexpression system can be successfully constructed for efficiently descemizing racemic 2-hydroxy acid.

In the preparation of optically pure chiral 2-hydroxy acids using a strategy of redox cascade deracemization, it was found that the activity of the keto acid reductase limits the overall catalytic efficiency, resulting in relatively low substrate loading and yields. Therefore, the ketoacid reductase with high activity and substrate tolerance is searched for being used in a three-enzyme coexpression catalytic system, and has significant significance for realizing the high-efficiency racemization of the racemic 2-hydroxy acid.

Ketoacid reductases are an important class of oxidoreductases capable of catalyzing the asymmetric reduction of prochiral ketoacids to the corresponding hydroxyacids while requiring NADH (nicotinamide adenine dinucleotide) or NADPH (nicotinamide adenine dinucleotide phosphate) as a cofactor for the reaction. In the field of biocatalysis, biological enzymes as catalysts often have specific catalytic substrates, and a single enzyme generally does not have catalytic activity for both aliphatic and aromatic compounds. The ketoacid reductase excavated in the research belongs to 2-dehydropantoate-2-reductase (2-dehydropantoate 2-reductase), reported catalytic substrates are aliphatic compounds, but experiments show that the ketoacid reductase can efficiently catalyze a plurality of aromatic 2-ketoacids to be converted into chiral aromatic 2-hydroxy acid, and the ketoacid reductase has important application value for realizing efficient racemization of racemic aromatic 2-hydroxy acid.

Disclosure of the invention

The invention aims to provide a high-efficiency ketoacid reductase gene derived from Leuconostoc lactis (Leuconostoc lactis), a coding enzyme, a vector, an engineering bacterium and application thereof in synthesizing chiral aromatic 2-hydroxy acid. The excavated ketoacid reductase can catalyze a wide-spectrum aromatic 2-ketoacid, and has higher substrate loading and catalytic efficiency.

The technical scheme adopted by the invention is as follows:

the invention provides a ketoacid reductase, wherein the amino acid sequence of the ketoacid reductase is shown in one of SEQ ID NO.4, SEQ ID NO.8 or SEQ ID NO.10, and more preferably, the amino acid sequence of the ketoacid reductase is shown in SEQ ID NO. 4. The invention selects 5 ketoacid reductases with amino acid sequences of SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10 or SEQ ID NO.12 from NCBI database by using ketoacid reductases LeKAR (with amino acid sequence of SEQ ID NO.2 and nucleotide sequence of SEQ ID NO.1) from Leuconostoc mesenteroides (Leuconostoc mesenteroides) as a template, and the ketoacid reductases are respectively from Leuconostoc lactis (Leuconostoc lactis), Leuconostoc pseudomesenteroides (Leuconostoc pseudomesenteroides), Leuconostoc mesenteroides (Leuconostoc mesenteroides), Klebsiella oxytoca (Klebsiella oxytoca) and Salmonella enterica (Salmonella enterica), and have the amino acid sequence homologies of 84%, 78%, 74%, 49% and 49%, and only have catalytic activity of the ketoacid reductases of SEQ ID NO.4, SEQ ID NO.8 and SEQ ID NO. 10.

Any polypeptide fragment or variant thereof obtained by carrying out deletion, insertion or substitution treatment of one or more amino acids on the amino acid sequences shown in SEQ ID NO.4, SEQ ID NO.8 and SEQ ID NO.10 is within the protection scope of the present invention as long as the polypeptide fragment or variant thereof has homology of more than 95% with the amino acid sequence.

The invention also provides a coding gene of the ketoacid reductase, and the nucleotide sequence of the coding gene is shown in one of SEQ ID NO.3, SEQ ID NO.7 and SEQ ID NO. 9. Any nucleotide sequence obtained by substituting, deleting or inserting one or more nucleotides into the nucleotide sequence shown in SEQ ID NO.3, SEQ ID NO.7 or SEQ ID NO.9 is within the protection scope of the present invention as long as the nucleotide sequence has more than 90% homology with the nucleotide sequence.

The invention also relates to a recombinant vector and a recombinant genetic engineering bacterium constructed by the encoding gene of the ketoacid reductase, wherein the recombinant genetic engineering bacterium is one of the following: (1) the ketonic acid reductase coding gene is introduced into host bacteria; (2) a ketoacid reductase-encoding gene, a 2-hydroxy acid dehydrogenase-encoding gene, and a glucose dehydrogenase-encoding gene are introduced into a host bacterium.

In addition, the invention also provides application of the ketoacid reductase in catalyzing and synthesizing chiral aromatic 2-hydroxy acid.

The application method comprises the following steps: when the recombinant genetic engineering bacteria are obtained by introducing encoding genes of ketoacid reductase into host bacteria, the application method comprises the following steps: taking supernatant of ketonic acid reductase obtained by ultrasonication of wet thallus obtained by fermentation culture of engineering bacteria containing ketonic acid reductase coding gene and supernatant of glucose dehydrogenase obtained by ultrasonication of wet thallus obtained by fermentation culture of engineering bacteria containing glucose dehydrogenase coding gene (nucleotide sequence is shown in SEQ ID NO.15) as catalysts, taking acetophenone acid as substrate, and NAD (nicotinamide adenine dinucleotide) as substrate⁺As coenzyme, glucose is used as auxiliary substrate, and KH of 100mM and pH7.0 is used₂PO₄-K₂HPO₄The buffer solution is used as a reaction medium, the reaction is carried out at 35 ℃ and 700rpm, and after the reaction is completed, a reaction solution containing (R) -mandelic acid is obtained; the dosage of the ketoacid reductase supernatant is 800U/mL buffer solution calculated by ketoacid reductase enzyme activity, the dosage of the glucose dehydrogenase supernatant is 800U/mL buffer solution calculated by glucose dehydrogenase enzyme activity, the dosage of glucose is 200-800 mM calculated by buffer solution volume, the dosage of the substrate is 100-400 mM calculated by buffer solution volume, and NAD⁺The amount used was 0.5mM based on the volume of the buffer.

Further, the catalyst in the first method is prepared according to the following method: inoculating engineering bacteria containing a ketoreductase coding gene into an LB liquid culture medium containing 50 mu g/mL kanamycin, and performing shake culture for 8-10 h at 37 ℃ and 150rpm to obtain a seed solution; the seed liquid was inoculated into LB liquid medium containing 50. mu.g/mL kanamycin in an inoculum size of 2% (volume concentration), and cultured with shaking at 37 ℃ and 150rpm to OD₆₀₀Reaching 0.4-0.8 (preferably 0.6), adding IPTG (isopropyl-beta-D-thiogalactoside) until the final concentration is 0.1mM, carrying out shake culture for 10-12 h under the conditions of 28 ℃ and 150rpm, centrifugally collecting wet thalli, and washing twice with normal saline to obtain resting cells; resuspending resting cells in KH₂PO₄-K₂HPO₄In a buffer (100mM, pH7.0), ultrasonication was performed for 20min (disruption power 40W, working 1s, stop 1s) under ice bath conditions, and the disrupted solution was centrifuged at 12000rpm for 10min at 4 ℃ to collect the supernatant of ketoacid reductase. The method for preparing the supernatant of glucose dehydrogenase isoketoacid reductaseAnd (4) liquid.

The application method of the invention is to utilize the reductase with stereoselectivity in the microorganism body to asymmetrically reduce prochiral 2-keto acid into optically pure 2-hydroxy acid.

The application method II comprises the following steps: when the recombinant genetic engineering bacteria are obtained by introducing a ketoacid reductase encoding gene, a 2-hydroxy acid dehydrogenase encoding gene and a glucose dehydrogenase encoding gene into host bacteria together, the application method comprises the following steps: using wet bacteria obtained by fermentation culture of engineering bacteria containing ketoreductase (preferably LlKAR), 2-Hydroxy Acid Dehydrogenase (HADH) and Glucose Dehydrogenase (GDH) encoding genes as catalysts, racemic aromatic 2-hydroxy acid as substrate, glucose as co-substrate, and buffer solution (preferably KH of pH7.0) of pH 6.0-8.0₂PO₄-K₂HPO₄Buffer solution) as a reaction medium, and reacting completely at 20-45 deg.C (preferably 30 deg.C) and 700rpm to obtain a conversion solution containing optically pure aromatic (R) -2-hydroxy acid.

Further, the racemic aromatic 2-hydroxy acid is one of the following: mandelic acid 1 a; 2-fluoromandelic acid 1 b; 4-fluoromandelic acid 1 c; 2, 4-difluoromethanoic acid 1 d; 3, 5-difluoromethanoic acid 1 e; 2-chloromandelic acid 1 f; 1g of 3-chloromandelic acid; 4-chloromandelic acid for 1 h; 2-bromomandelic acid 1 i; 3-bromomandelic acid 1 j; 4-bromomandelic acid 1 k; 1l of 4-methylmandelic acid; 1m of 4-trifluoromethyl mandelic acid; 3-hydroxymandelic acid 1 n; 4-hydroxymandelic acid 1 o; 4-methoxymandelic acid 1 p; 3-methoxy-4-hydroxymandelic acid 1 q; 3-hydroxy-4-methylmandelic acid 1 r; 3-hydroxy-4-trifluoromethylmandelic acid 1 s; 3-methyl-4-methoxymandelic acid 1t, preferably 1a to 1m, the letters being numbering only and having no meaning.

Further, in the reaction system of the second application method, the final concentration of the substrate is 20-300mM, the concentration of the co-substrate is 10-300mM, and the dosage of the catalyst is 4-20g/L in terms of dry weight of wet thalli.

Further, the second engineering bacterium according to the present invention is constructed by introducing genes encoding a ketoacid reductase (preferably LlKAR), a 2-Hydroxy Acid Dehydrogenase (HADH) and a Glucose Dehydrogenase (GDH) into a host bacterium E.coli BL21(DE 3); the nucleotide sequence of the coding gene of the 2-hydroxy acid dehydrogenase is shown in SEQ ID NO.13, and the nucleotide sequence of the coding gene of the glucose dehydrogenase is shown in SEQ ID NO. 15.

Specifically, the engineering bacteria containing coding genes of ketoacid reductase (LlKAR), 2-Hydroxy Acid Dehydrogenase (HADH) and Glucose Dehydrogenase (GDH) are constructed according to the following steps:

(1) construction of E.coli BL21(DE3)/pET28b-LlKAR Strain

The nucleotide sequence of ketoacid reductase LlKAR is connected into expression plasmid pET28b to obtain recombinant plasmid pET28b-LlKAR, and the recombinant plasmid pET28b-LlKAR is transformed into E.coli BL21(DE3) to construct recombinant bacterium E.coli BL21(DE3)/pET28 b-LlKAR.

(2) Coli BL21(DE3)/pCDFDuet-LlKAR-GDH strain was constructed

Glucose Dehydrogenase (GDH) gene (nucleotide sequence SEQ ID NO.15) derived from Exiguobacterium sibiricum and LlKAR gene are connected with an expression vector pCDFDuet-1 in sequence to obtain a recombinant plasmid pCDFDuet-LlKAR-GDH, and the recombinant plasmid pCDFDuet-LlKAR-GDH is transformed into E.coli BL21(DE3) to construct a recombinant bacterium E.coli BL21(DE 3)/pCDFDuet-LlKAR-GDH.

(3) Construction of E.coli BL21(DE3)/pET28b-HADH Strain

2-Hydroxy Acid Dehydrogenase (HADH) gene (nucleotide sequence SEQ ID NO.13) derived from Pseudomonas aeruginosa is synthesized into a corresponding nucleotide sequence in vitro and is connected into an expression plasmid pET28b to obtain a recombinant plasmid pET28b-HADH, and the recombinant plasmid pET 28-HADH is transformed into E.coli BL21(DE3) to construct a recombinant bacterium E.coli BL21(DE3)/pET28 b-HADH.

(4) Coli BL21(DE3)/pET28b-HADH/pCDFDuet-LlKAR-GDH strain was constructed

Plasmids pET28b-HADH and pCDFDuet-LlKAR-GDH are extracted from recombinant bacteria E.coli BL21(DE3)/pET28b-HADH and E.coli BL21(DE3)/pCDFDuet-LlKAR-GDH respectively, after being mixed uniformly according to the molar concentration ratio of 1:1, the mixture is transformed into E.coli BL21(DE3) together, the E.coli BL21(DE3)/pET28b-HADH/pCDFDuet-LlKAR-GDH is coated on a double-antibody LB plate containing 50 mu g/mL streptomycin, and the recombinant bacteria E.coli BL21(DE3)/pET28b-HADH/pCDFDuet-LlKAR-GDH, abbreviated as E.coli (HADH-LlKAR-GDH) containing HADH, LKAR and GDH genes simultaneously is screened, thereby constructing a single-bacterium double-plasmid tandem redox cascade system.

The reaction mechanism of the single-bacterium double-plasmid three-enzyme tandem redox cascade system is as follows: racemic aromatic 2-hydroxy acid is taken as a substrate, the (S) -2-hydroxy acid in the substrate is oxidized into 2-keto acid by HAHD asymmetry with S-stereoselectivity, and the consumed cofactor FMN can be regenerated by itself; further asymmetrically reducing the produced 2-keto acid to an (R) -2-hydroxy acid by using a keto acid reductase having R-stereoselectivity (preferably LlKAR), and finally obtaining an optically pure (R) -2-hydroxy acid, wherein the GDH can realize efficient cyclic regeneration of the coenzyme NADH without adding an exogenous coenzyme. The specific reaction mechanism is shown in FIG. 1, which lists representative 20 racemic aromatic 2-hydroxy acids, and the substrates catalyzed by the three-enzyme coexpression system of the present invention include, but are not limited to, the 20 racemic aromatic 2-hydroxy acids.

Further, the catalyst in the second method is prepared according to the following method: inoculating engineering bacteria containing coding genes of ketoreductase, 2-hydroxy acid dehydrogenase and glucose dehydrogenase into LB liquid culture medium containing 50 mug/mL kanamycin and 50 mug/mL streptomycin, and performing shake culture at 37 ℃ and 150rpm for 8-10 h to obtain seed liquid; the seed liquid was inoculated into LB liquid medium containing 50. mu.g/mL kanamycin and 50. mu.g/mL streptomycin in an amount of 2% (volume concentration), and cultured with shaking at 37 ℃ and 150rpm to OD₆₀₀And (3) reaching 0.4-0.8 (preferably 0.6), adding IPTG (isopropyl-beta-D-thiogalactoside) until the final concentration is 0.1mM, carrying out shake culture at 28 ℃ and 150rpm for 10-12 h, centrifuging, collecting wet thalli, and washing twice with normal saline to obtain the wet thalli.

Coli (HADH-LlKAR-GDH) is finally further used for catalyzing the racemization of the o-chloromandelic acid with high concentration. The reaction takes resting cells of recombinant bacteria E.coli (HADH-LlKAR-GDH) as a catalyst, racemic o-chloromandelic acid as a substrate, glucose as a co-substrate and KH₂PO₄-K₂HPO₄The buffer is the reaction medium. In the reaction process, the pH value is automatically controlled to be 7.0 by using 3.0M NaOH, and the conversion solution containing (R) -o-chloromandelic acid is obtained after the reaction is completed.

Compared with the prior art, the invention has the following beneficial effects:

(1) the present invention provides a highly efficient ketoacid reductase (preferably LlKAR) derived from Leuconostoc lactis (Leuconostoc lactis) capable of catalyzing a wide-spectrum aromatic 2-keto acid and increasing the substrate loading with acetophenone acid as a substrate from 100mM to 400 mM. A single-bacterium two-plasmid three-enzyme tandem redox cascade system established by ketoacid reductase (preferably LlKAR), 2-Hydroxy Acid Dehydrogenase (HADH) and Glucose Dehydrogenase (GDH) can catalyze most of racemic aromatic 2-hydroxy acid to be efficiently racemized into chiral aromatic (R) -2-hydroxy acid, and both the yield and the e.e. value are more than 99%. Finally applied to racemization removal of 300mM o-chloromandelic acid to prepare optically pure (R) -o-chloromandelic acid, and the yield reaches 83.8 g/(L.d).

(2) A single-bacterium two-plasmid three-enzyme tandem redox cascade system constructed by ketoacid reductase (preferably LlKAR), HADH and GDH can be used for efficiently descemating racemic chiral aromatic 2-hydroxy acid and preparing optically pure aromatic (R) -2-hydroxy acid with high utilization value. The reaction has the advantages of low cost, environmental protection, simple process, high catalytic efficiency, no need of adding exogenous coenzyme and the like, and has wide industrial prospect.

(IV) description of the drawings

FIG. 1 shows the reaction mechanism of a single-strain double-plasmid three-enzyme tandem redox cascade system.

FIG. 2 is a comparative analysis of the substrate loading of ketoacid reductases LeKAR, LlKAR, LmKAR and KoKAR.

FIG. 3 is the construction scheme of recombinant strain E.coli BL21(DE3)/pET28 b-HADH/pCDFDuet-LlKAR-GDH.

FIG. 4 shows the electrophoretic analysis of the co-expression of HADH, LlKAR and GDH, lane M shows standard protein molecular weight marker, lane 1 shows non-induced recombinant bacterium E.coli BL21(DE3)/pCDFDuet-LlKAR-GDH, lane 2 shows IPTG-induced recombinant bacterium E.coli BL21(DE3)/pCDFDuet-LlKAR-GDH, lane 3 shows non-induced recombinant bacterium E.coli BL21(DE3)/pET28b-HADH/pCDFDuet-LlKAR-GDH, and lane 4 shows IPTG-induced recombinant bacterium E.coli BL21(DE3)/pET28 b-HADH/pCDFDuet-LlKAR-GDH.

FIG. 5 shows the course of the racemization reaction of 200mM o-chloromandelic acid catalyzed by E.coli (HADH-LeKAR-GDH).

FIG. 6 shows the course of racemization reaction of 200mM o-chloromandelic acid catalyzed by E.coli (HADH-LlKAR-GDH).

FIG. 7 shows the course of racemization reaction of 300mM o-chloromandelic acid catalyzed by E.coli (HADH-LlKAR-GDH).

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

example 1: screening for highly efficient ketoacid reductase LlKAR

The amino acid sequence (shown in SEQ ID NO.2 and the corresponding nucleotide sequence (shown in SEQ ID NO. 1)) of the known ketoacid reductase LeKAR is taken as a template, and is subjected to NCBI-Blastp online comparison to obtain 5 potential ketoacid reductase sequences, namely LlKAR (shown in the amino acid sequence SEQ ID NO.4 and the nucleotide sequence shown in the SEQ ID NO. 3), LpKAR (shown in the amino acid sequence SEQ ID NO.6 and the nucleotide sequence shown in the SEQ ID NO. 5), LmKAR (shown in the amino acid sequence SEQ ID NO.8 and the nucleotide sequence shown in the SEQ ID NO. 7), KoKAR (shown in the amino acid sequence SEQ ID NO.10 and the nucleotide sequence shown in the SEQ ID NO. 9) and SnKAR (shown in the amino acid sequence SEQ ID NO.12 and the nucleotide sequence shown in the SEQ ID NO. 11), wherein the amino acid sequence homologies are respectively 84%, 78%, 74%, 49% and 49%. Subsequently, the corresponding nucleotide sequence was synthesized in vitro and ligated into expression plasmid pET28b to obtain 6 recombinant plasmids pET28b-LeKAR, pET28b-LlKAR, pET28b-LpKAR, pET28b-LmKAR, pET28b-KoKAR and pET28b-SnKAR, which were transformed into E.coli BL21(DE3), coated on LB plate containing 50. mu.g/mL kanamycin, and 6 recombinant bacteria E.coli BL21(DE3)/pET28b-LeKAR, E.coli BL21(DE3)/pET28b-LlKAR, E.coli BL21(DE 847)/pET 3628-LpKAR, E.coli 21(DE 21)/pET 21-LpKAR, E.coli BL 21/DE 21-LpKAR, E.874 BL 21-Sn72/21/DE 21/21).

Respectively inoculating 6 recombinant strains of ketoacid reductase and recombinant strains of glucose dehydrogenase E.coli BL21(DE3)/pET28b-GDH (the nucleotide sequence of the GDH gene is shown as SEQ ID NO.15, and the amino acid sequence is shown as SEQ ID NO. 16) into an LB liquid culture medium containing 50 mu g/mL kanamycin, and carrying out shake culture at 37 ℃ and 150rpm for 8-10 h to obtain seed liquid; inoculating the seed solution into LB liquid medium containing 50. mu.g/mL kanamycin at an inoculum size of 2% (volume concentration), and shake-culturing at 37 deg.C and 150rpm to OD₆₀₀Up to 0.4 to 0.8: (Preferably 0.6), adding IPTG to the final concentration of 0.1mM, carrying out shake culture for 10-12 h under the conditions of 28 ℃ and 150rpm, centrifuging, collecting wet bacteria, and washing twice with normal saline to obtain resting cells of the 4 ketoacid reductase recombinant bacteria and the glucose dehydrogenase recombinant bacteria respectively. Resuspending resting cells in KH₂PO₄-K₂HPO₄In a buffer solution (100mM, pH7.0), carrying out ultrasonic crushing for 20min (crushing power 40W, working for 1s, stopping for 1s) under ice bath conditions, centrifuging the crushed solution for 10min at 4 ℃ and 12000rpm, and collecting supernatant, namely the corresponding crude enzyme solution.

The enzyme activities of 6 ketoacid reductases were measured in a 1mL reaction system containing KH₂PO₄-K₂HPO₄Buffer (100mM, pH7.0), acetophenone acid (10mM), NADH (5mM) and appropriate amount of crude enzyme (0.2. mu.g). The reaction system and the crude enzyme solution were incubated at 35 ℃ for 5min, reacted at 35 ℃ for 2min at 700rpm, and then quenched with HCl (6.0M). The sample was centrifuged (12000rpm, 2min), diluted 2-fold with ultrapure water, filtered through a 0.22 μm membrane, and subjected to chiral HPLC analysis. Unit enzyme activity definition (1U): under standard conditions, the enzyme amount required for catalyzing the formation of 1 mu mol of product from the phenylacetic acid per min is one unit of enzyme activity. Specific enzyme activity unit: kU/mg crude enzyme. The results showed that the specific enzyme activities of 6 ketoacid reductases LeKAR, LlKAR, LpKAR, LKAR, KoKAR and SnKAR were 1.11kU/mg, 3.71kU/mg, 0kU/mg, 3.37kU/mg, 2.89kU/mg and 0kU/mg, respectively, of which only 4 ketoacid reductases LeKAR, LlKAR, LmKAR and KoKAR had catalytic activities, and LlKAR had the highest specific enzyme activity.

The detection of the enantiomeric excess (e.e.) is carried out in a 1mL reaction system comprising KH₂PO₄-K₂HPO₄Buffer (100mM, pH7.0), acetophenone acid (10mM), NADH (15mM) and crude enzyme at the appropriate concentration. After reacting at 35 ℃ for 10h at 700rpm, the reaction was stopped with HCl (6.0M). The sample was centrifuged (12000rpm, 2min), diluted 2-fold with ultrapure water, filtered through a 0.22 μm membrane, and subjected to chiral HPLC analysis. The results show that the e.e. values of 4 ketoacid reductases LeKAR, LlKAR, LmKAR and KoKAR for catalyzing the formation of (R) -mandelic acid from phenylacetic acid are all more than 99%.

4 ketoacid reductases LeKAR, LlKAR, LmKAR andfurther comparative analysis of KoKAR at high substrate concentrations was performed in a 10mL reaction system comprising KH₂PO₄-K₂HPO₄Buffer (100mM, pH7.0), acetophenone acid (both 100mM and 400mM concentrations are selected), glucose (2-fold higher than acetophenone acid, i.e., 200mM and 800mM, respectively), NAD⁺(0.5mM), KAR (ketoacid reductase, 800U/mL), and GDH (glucose dehydrogenase, 800U/mL). After 3 hours at 35 ℃ and 700rpm, the reaction was stopped with HCl (6.0M). The sample was centrifuged (12000rpm, 2min), diluted 2-fold with ultrapure water, filtered through a 0.22 μm membrane, and subjected to chiral HPLC analysis. The pH was automatically controlled at 7.0 with 3.0M NaOH during the reaction. As shown in FIG. 2, the conversion rates and e.e. values of 4 ketoacid reductases LeKAR, LlKAR, LmKAR and KoKAR were all greater than 99% when catalyzing asymmetric reduction of 100mM acetophenone acid; the e.e. values were all greater than 99% when catalyzing the asymmetric reduction of 400m acetophenone acid, but the conversions were 65.8%, 99.2%, 75.5%, and 86.4%, respectively. Only LlKAR can catalyze 100mM and 400mM of acetophenone acid to be completely converted into (R) -mandelic acid, and the conversion rate and the e.e. value are both more than 99%, so that the LlKAR with the highest specific enzyme activity and substrate loading capacity is selected for constructing the three-enzyme co-expression recombinant bacterium E.coli BL21(DE3)/pET28 b-HADH/pCDFDuet-LlKAR-GDH.

The chiral HPLC analysis method was as follows: reverse phase chiral column (model Chirobiotic R250X 4.6mm, Sigma, USA) with mobile phase of 0.5% ammonia water: CH₃OH (10:90, v/v), detection wavelength of 215nm, and sample injection amount of 3 μ L.

e.e. value calculation method: ee (%) - (R-S)/(R + S) × 100%. Wherein R represents the concentration of (R) -2-hydroxy acid after the completion of the reaction, and S represents the concentration of (S) -2-hydroxy acid after the completion of the reaction.

Example 2: construction of recombinant bacterium E.coli BL21(DE3)/pET28b-HADH/pCDFDuet-LlKAR-GDH

Glucose Dehydrogenase (GDH) gene (nucleotide sequence is shown in SEQ ID NO.15, and amino acid sequence is shown in SEQ ID NO. 16) from Exiguobacterium sibiricum (WP _012369122.1) is synthesized into corresponding nucleotide sequence in vitro and is connected into expression plasmid pET28b to obtain recombinant plasmid pET28b-GDH, the recombinant plasmid pET 28-GDH is transformed into E.coli BL21(DE3), and the recombinant plasmid E.coli BL21(DE3)/pET28b-GDH is screened after being coated on an LB plate containing 50 mu g/mL kanamycin.

With the aid of a seamless cloning kit (

II, Vazyme Biotech co., Ltd), the nucleotide sequences of GDH and preferably LlKAR were ligated with the expression vector pCDFDuet-1 in sequence to obtain recombinant plasmid pCDFDuet-LlKAR-GDH, transformed into e.coli BL21(DE3), spread on LB plates containing 50 μ g/mL streptomycin, and the recombinant bacterium e.coli BL21(DE3)/pCDFDuet-LlKAR-GDH was selected.

2-Hydroxy Acid Dehydrogenase (HADH) gene (nucleotide sequence is shown as SEQ ID NO.13, amino acid sequence is shown as SEQ ID NO. 14) derived from Pseudomonas aeruginosa (AGM49308.1) is synthesized into a corresponding nucleotide sequence in vitro and is connected into an expression plasmid pET28b to obtain a recombinant plasmid pET28b-HADH, the recombinant plasmid pET 28-HADH is transformed into E.coli BL21(DE3), the E.coli BL 3538 (DE3)/pET28b-HADH is coated on an LB plate containing 50 mu g/mL kanamycin, and the recombinant bacterium E.coli BL21(DE3)/pET28b-HADH is screened.

Plasmids pET28b-HADH and pCDFDuet-LlKAR-GDH were extracted from recombinant bacteria E.coli BL21(DE3)/pET28b-HADH and E.coli BL21(DE3)/pCDFDuet-LlKAR-GDH, respectively, mixed uniformly at a molar concentration ratio of 1:1, co-transformed into E.coli BL21(DE3), spread on a double-resistant LB plate containing 50. mu.g/mL kanamycin and 50. mu.g/mL streptomycin, and screened for recombinant bacteria E.coli BL21(DE3)/pET28b-HADH/pCDFDuet-LlKAR-GDH containing genes of HADH, LKAR and GDH simultaneously. Coli (HADH-LeKAR-GDH) was constructed in the same manner.

The construction of recombinant plasmids and recombinant bacteria is shown in FIG. 3.

Example 3: induction of recombinant bacterium E.coli BL21(DE3)/pCDFDuet-LlKAR-GDH double enzyme co-expression

Inoculating recombinant Escherichia coli E.coli BL21(DE3)/pCDFDuet-LlKAR-GDH into LB liquid culture medium containing 50 μ g/mL streptomycin, and performing shake culture at 37 ℃ and 150rpm for 8-10 h to obtain seed solution; inoculating the seed solution into LB liquid culture medium containing 50. mu.g/mL streptomycin in an inoculum size of 2% (volume concentration), and performing shake culture at 37 deg.C and 150rpm to OD₆₀₀Up to 0.6, addAnd (3) carrying out IPTG to a final concentration of 0.1mM, carrying out shake culture for 10-12 h at 28 ℃ and 150rpm, centrifuging, collecting wet thalli, and washing twice with physiological saline to obtain the resting cells of E.coli BL21(DE 3)/pCDFDuet-LlKAR-GDH. SDS-PAGE protein electrophoresis analysis is carried out by using non-induced E.coli BL21(DE3)/pCDFDuet-LlKAR-GDH as a control, and the result is shown in figure 4, and it can be seen that a distinct band appears near 32kDa and 28kDa respectively after IPTG induction, the molecular weight is consistent with the theoretical protein molecular weight of LlKAR and GDH, and the success construction of the recombinant bacterium E.coli BL21(DE3)/pCDFDuet-LlKAR-GDH is proved.

Example 4: coli (HADH-LlKAR-GDH) three-enzyme co-expression

Inoculating recombinant Escherichia coli E.coli (HADH-LlKAR-GDH) into LB liquid culture medium containing 50. mu.g/mL kanamycin and 50. mu.g/mL streptomycin, and performing shake culture at 37 ℃ and 150rpm for 8-10 h to obtain seed solution; the seed liquid was inoculated into LB liquid medium containing 50. mu.g/mL kanamycin and 50. mu.g/mL streptomycin in an inoculum size of 2% (volume concentration), and cultured with shaking at 37 ℃ and 150rpm to OD₆₀₀And (3) reaching 0.6, adding IPTG to a final concentration of 0.1mM, carrying out shake culture at 28 ℃ and 150rpm for 10-12 h, centrifuging, collecting wet thalli, and washing twice with normal saline to obtain the resting cells of E.coli (HADH-LlKAR-GDH). The results of SDS-PAGE protein electrophoresis analysis using uninduced E.coli (HADH-LlKAR-GDH) as a control show in FIG. 4, and it can be seen that a distinct band appears near 41kDa, 32kDa and 28kDa after IPTG induction, and the molecular weight is consistent with the theoretical protein molecular weight of HADH, LlKAR and GDH, thus proving that recombinant strain E.coli (HADH-LlKAR-GDH) is successfully constructed. Coli (HADH-LeKAR-GDH) resting cells were prepared in the same manner.

Example 5: coli (HADH-LlKAR-GDH) catalytic condition optimization

By the method of example 4, resting cells of recombinant bacterium e.coli (HADH-LlKAR-GDH) were obtained, and an optimized reaction system was designed to be 10 mL: selecting racemic o-chloromandelic acid with a final concentration of 20mM as a substrate, and examining the cell concentrations of 4g/L, 8g/L and 12g/L (calculated by dry weight), the cosubstrate glucose concentrations of 10mM, 20mM and 30mM and a reaction medium KH₂PO₄-K₂HPO₄The pH of the buffer solution is 6.0, 7.0 and 8.0, and the temperature is 25 ℃, 30 ℃, 35 ℃ and other factors influence the reaction. After reacting for 2h at 700rpm, the reaction was terminated with HCl (6.0M), and the sample was analyzed by the chiral HPLC method of example 1, using the yield of the target product as the evaluation index, and the results are shown in the following table:

TABLE 1

Coli (HADH-LlKAR-GDH) catalyzes the racemization of 20mM racemic aromatic 2-hydroxy acid, the concentration of the cells is preferably 8g/L based on the dry cell weight, the concentration of the co-substrate is preferably 20mM, and the reaction medium is preferably KH at pH7.0₂PO₄-K₂HPO₄Buffer, temperature preferably 30 ℃.

Example 6: coli (HADH-LlKAR-GDH) catalyzed racemization of racemic aromatic 2-hydroxy acid

The reaction was carried out in 10mL of a conversion system containing KH₂PO₄-K₂HPO₄Buffer (100mM, pH7.0), racemic aromatic 2-hydroxy acid (final concentration 20mM, see Structure in FIG. 1 and Table 2), glucose (final concentration 20mM), the recombinant bacterium co-expressed by the three enzymes obtained in example 4 (amount of 8g/L based on dry weight of the cell body). The reaction was carried out at 30 ℃ and 700rpm for 4 hours, samples were taken at intervals of 1 hour, the reaction was terminated with HCl (6.0M), and the samples were centrifuged (12000rpm, 2min), diluted 4-fold with ultrapure water, filtered through a 0.22 μ M membrane, and then analyzed by chiral HPLC as described in example 1. The results are given in the following table:

TABLE 2

The catalytic results show that most of the racemic aromatic 2-hydroxy acids (1a-1m) are efficiently converted to the corresponding aromatic (R) -2-hydroxy acids over 2h, with yields greater than 98% and e.e. values greater than 99%. Even if the racemic aromatic 2-hydroxy acid (1f-1h,1i-1k) is substituted at ortho, meta or para positions on the benzene ring, the resulting steric effect does not affect the overall catalytic efficiency. The racemic aromatic 2-hydroxy acid (1n-1q) had a relatively low yield, but all of them reached 80% or more, because a small amount of (S) -2-hydroxy acid was not completely converted or a small amount of intermediate keto acid was accumulated after the reaction. Whereas for racemic aromatic 2-hydroxy acids (1r-1t), the three-enzyme co-expression system is essentially catalytically inactive. On the whole, a high-efficiency ketoacid reductase LlKAR is discovered and can effectively catalyze the conversion of aromatic 2-ketoacid into chiral aromatic 2-hydroxy acid, and the chiral aromatic 2-ketoacid is coupled with HADH and GDH to construct a single-bacterium two-plasmid three-enzyme coexpression redox cascade system, so that the high-efficiency racemization of most racemic aromatic 2-hydroxy acid is realized, and the application value in practical production is important.

Example 7: coli (HADH-LlKAR-GDH) catalyzing deracemization of 200mM o-chloromandelic acid

The reaction was carried out in 20mL of a conversion system containing KH₂PO₄-K₂HPO₄Buffer (100mM, pH7.0), racemic o-chloromandelic acid (final concentration 200mM), glucose (final concentration 200mM), and the recombinant bacterium co-expressed with three enzymes obtained in example 4 (the amount used was 20g/L based on the dry weight of the bacterium). The reaction was carried out at 30 ℃ and 700rpm for 20 hours, samples were taken at intervals of 2 hours, and the samples were quenched with HCI (6.0M), centrifuged (12000rpm, 2min), diluted, and subjected to membrane filtration for detection and analysis by the HPLC method in example 1. In the reaction process, the pH value is automatically controlled to be 7.0 by using 3.0M NaOH, and the conversion solution containing (R) -o-chloromandelic acid is obtained after the reaction is completed. The catalytic reaction process is shown in FIG. 6, the racemic o-chloromandelic acid of 200mM is completely converted into optically pure (R) -o-chloromandelic acid at 16h, the yield and the e.e. value are both more than 99%, and the space time yield is as high as 55.9 g/(L.d).

Coli (HADH-LeKAR-GDH) obtained in example 4 was used as a catalyst under the same conditions, and the results were as follows: coli (HADH-LeKAR-GDH) in the racemization of 200mM racemic o-chloromandelic acid, the intermediate keto acid was accumulated in a large amount, and the reaction was continued for 22 hours, so that racemic o-chloromandelic acid could not be completely converted into (R) -o-chloromandelic acid, and the results are shown in FIG. 5.

Example 8: coli (HADH-LlKAR-GDH) catalyzing deracemization of 300mM o-chloromandelic acid

The reaction was carried out in 20mL of a conversion system containing KH₂PO₄-K₂HPO₄Buffer (100mM, pH7.0), racemic o-chloromandelic acid (100mM), glucose (100mM), and the recombinant bacterium co-expressed with the three enzymes obtained in example 4 (the amount of the recombinant bacterium was 20g/L, based on the dry weight of the bacterium). After the reaction was started at 30 ℃ and 700rpm, racemic o-chloromandelic acid (100mM) and glucose (100mM) were added every 4 hours for 2 times. The reaction was sampled every 1h, and the samples were quenched with HCI (6.0M), centrifuged (12000rpm, 2min), diluted, and subjected to membrane filtration for detection and analysis by HPLC as in example 1. In the reaction process, the pH value is automatically controlled to be 7.0 by using 3.0M NaOH, and the conversion solution containing (R) -o-chloromandelic acid is obtained after the reaction is completed. The catalytic reaction progress is shown in FIG. 7, and finally, 300mM of racemic o-chloromandelic acid is completely converted into optically pure (R) -o-chloromandelic acid at 16h, the yield and the e.e. value are both more than 99%, and the space-time yield is as high as 83.8 g/(L.d). The (R) -o-chloromandelic acid is an important chiral building block for synthesizing antithrombotic drug (clopidogrel), so the catalytic reaction has great application value.

Sequence listing

<110> Zhejiang industrial university

Application of <120> ketoacid reductase in synthesis of chiral aromatic 2-hydroxy acid

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 954

<212> DNA

<213> Leuconostoc mesenteroides (Leuconostoc mesenteroides)

<400> 1

atgaaaatcg caattgccgg tttcggtgct ctgggcgcac gtctgggcgt gatgctgcaa 60

gccggtggtc atgaagtcac cggtattgac ggttggccgg cacacatcgc tgctatcaac 120

accaaaggcc tgaccgttgt taaagacaac gatgcaccgc agaaatactt cgtacctgta 180

atgccagctt ctgaggttac tggcactttc gatctgatca tcctgctgac caaaacccct 240

caactggacc gcatgctgac tgatatccag ccaatcatta ctgatactac taaactgctg 300

gttctgtcta acggtctggg taacatcgaa gtgatggcta aacacgtgtc ccgccaccag 360

atcctggccg gcgttacgct gtggacctct tctctgatca aaccgggtga aatccacgta 420

accggttctg gctctatcaa actgcaggct atcggtgacg cagacgtaca gtctattgct 480

gacgccctga accaggcagg tctgaacgct gaaatcactc ctgacgtcat gacggctatc 540

tggcataaag cgggcatcaa cgcggtactg aacccgctgt ccgtactgct gaatgcgaac 600

attgctgaat tcggcaccgc gggtaacgcg atggatctgg ctctgaacat cctggacgaa 660

atgaaacagg taggcgcttc tcagggtatc aaagtcgacg taagcggtat catgacggac 720

ctgtcccagc tgctgaagcc ggaaaatgct ggcaaccact ttccgtctat gtatcaagac 780

attcagaacg gtaaacgtac cgagatcgac ttcctgaacg gctacttcgc gaagattggc 840

cacgaatccg gtatcccgac tccgttcaac gcactggtta cgcgtctgat ccacgctaaa 900

gaggacatcg aacgcgttaa actggcgaaa cagcaagaga acttcgagat ctga 954

<210> 2

<211> 317

<212> PRT

<213> Leuconostoc mesenteroides (Leuconostoc mesenteroides)

<400> 2

Met Lys Ile Ala Ile Ala Gly Phe Gly Ala Leu Gly Ala Arg Leu Gly

1 5 10 15

Val Met Leu Gln Ala Gly Gly His Glu Val Thr Gly Ile Asp Gly Trp

20 25 30

Pro Ala His Ile Ala Ala Ile Asn Thr Lys Gly Leu Thr Val Val Lys

35 40 45

Asp Asn Asp Ala Pro Gln Lys Tyr Phe Val Pro Val Met Pro Ala Ser

50 55 60

Glu Val Thr Gly Thr Phe Asp Leu Ile Ile Leu Leu Thr Lys Thr Pro

65 70 75 80

Gln Leu Asp Arg Met Leu Thr Asp Ile Gln Pro Ile Ile Thr Asp Thr

85 90 95

Thr Lys Leu Leu Val Leu Ser Asn Gly Leu Gly Asn Ile Glu Val Met

100 105 110

Ala Lys His Val Ser Arg His Gln Ile Leu Ala Gly Val Thr Leu Trp

115 120 125

Thr Ser Ser Leu Ile Lys Pro Gly Glu Ile His Val Thr Gly Ser Gly

130 135 140

Ser Ile Lys Leu Gln Ala Ile Gly Asp Ala Asp Val Gln Ser Ile Ala

145 150 155 160

Asp Ala Leu Asn Gln Ala Gly Leu Asn Ala Glu Ile Thr Pro Asp Val

165 170 175

Met Thr Ala Ile Trp His Lys Ala Gly Ile Asn Ala Val Leu Asn Pro

180 185 190

Leu Ser Val Leu Leu Asn Ala Asn Ile Ala Glu Phe Gly Thr Ala Gly

195 200 205

Asn Ala Met Asp Leu Ala Leu Asn Ile Leu Asp Glu Met Lys Gln Val

210 215 220

Gly Ala Ser Gln Gly Ile Lys Val Asp Val Ser Gly Ile Met Thr Asp

225 230 235 240

Leu Ser Gln Leu Leu Lys Pro Glu Asn Ala Gly Asn His Phe Pro Ser

245 250 255

Met Tyr Gln Asp Ile Gln Asn Gly Lys Arg Thr Glu Ile Asp Phe Leu

260 265 270

Asn Gly Tyr Phe Ala Lys Ile Gly His Glu Ser Gly Ile Pro Thr Pro

275 280 285

Phe Asn Ala Leu Val Thr Arg Leu Ile His Ala Lys Glu Asp Ile Glu

290 295 300

Arg Val Lys Leu Ala Lys Gln Gln Glu Asn Phe Glu Ile

305 310 315

<210> 3

<211> 954

<212> DNA

<213> Leuconostoc lactis (Leuconostoc lactis)

<400> 3

atgaaaatcg ctatcgctgg tttcggtgct ctgggtgctc gtctgggtat catgctgcag 60

gctgctggtc acgacgttac cggtatcgac ggttggccgg ctcacatcgc tgctatcaac 120

accaaaggtc tgaccgttgt tcacgacgac caggacccga aagtttacta cctgccggtt 180

atgaccccgc aggaagttac cggtaccttc gacctgatca tcctgctgac caaaaccccg 240

cagctggacc gtatgctgac cgacatcgct ccgatcatca ccgaccagac ccagctgctg 300

atcctgtcta acggtctggg taacatcgaa gttatggcta aacacgttgc taaatctcag 360

atcgttgctg gtgttaccct gtggacctct tctctggtta aaccgggtga aatccacacc 420

accggttctg gttctatcaa actgcaggct ctggctggtg gtgacgctca gccgatcgtt 480

gaagctctga acgaagctgg tctgaacgct gaactggttc cggacgttat gaccgctatc 540

tggcacaaag ctggtatcaa cgctgttctg aacccgctgt ctgttctgct ggacgctaac 600

atcgctgaat tcggtaccgc tggtaacgct atggacatgg ctctgaacat cctggacgaa 660

atgaaacagg ttggtgcttc tcagggtatc aaagttgacg ttgctggtat catcgctgac 720

ctgtctcgtc tgctgaaacc ggaaaacgct ggtaaccact acccgtctat gtaccaggac 780

atccagaacg gtaaacgtac cgaaatcgac ttcctgaacg gttacttcgc tcgtctgggt 840

cagcaggaag gtatcccgac cccgttcaac gctctggtta cccgtctgat ccacgctaaa 900

gaagacatcg ttcgtaccaa actggctaaa gaaaaagaaa acttcgaaat ctaa 954

<210> 4

<211> 317

<212> PRT

<213> Leuconostoc lactis (Leuconostoc lactis)

<400> 4

Met Lys Ile Ala Ile Ala Gly Phe Gly Ala Leu Gly Ala Arg Leu Gly

1 5 10 15

Ile Met Leu Gln Ala Ala Gly His Asp Val Thr Gly Ile Asp Gly Trp

20 25 30

Pro Ala His Ile Ala Ala Ile Asn Thr Lys Gly Leu Thr Val Val His

35 40 45

Asp Asp Gln Asp Pro Lys Val Tyr Tyr Leu Pro Val Met Thr Pro Gln

50 55 60

Glu Val Thr Gly Thr Phe Asp Leu Ile Ile Leu Leu Thr Lys Thr Pro

65 70 75 80

Gln Leu Asp Arg Met Leu Thr Asp Ile Ala Pro Ile Ile Thr Asp Gln

85 90 95

Thr Gln Leu Leu Ile Leu Ser Asn Gly Leu Gly Asn Ile Glu Val Met

100 105 110

Ala Lys His Val Ala Lys Ser Gln Ile Val Ala Gly Val Thr Leu Trp

115 120 125

Thr Ser Ser Leu Val Lys Pro Gly Glu Ile His Thr Thr Gly Ser Gly

130 135 140

Ser Ile Lys Leu Gln Ala Leu Ala Gly Gly Asp Ala Gln Pro Ile Val

145 150 155 160

Glu Ala Leu Asn Glu Ala Gly Leu Asn Ala Glu Leu Val Pro Asp Val

165 170 175

Met Thr Ala Ile Trp His Lys Ala Gly Ile Asn Ala Val Leu Asn Pro

180 185 190

Leu Ser Val Leu Leu Asp Ala Asn Ile Ala Glu Phe Gly Thr Ala Gly

195 200 205

Asn Ala Met Asp Met Ala Leu Asn Ile Leu Asp Glu Met Lys Gln Val

210 215 220

Gly Ala Ser Gln Gly Ile Lys Val Asp Val Ala Gly Ile Ile Ala Asp

225 230 235 240

Leu Ser Arg Leu Leu Lys Pro Glu Asn Ala Gly Asn His Tyr Pro Ser

245 250 255

Met Tyr Gln Asp Ile Gln Asn Gly Lys Arg Thr Glu Ile Asp Phe Leu

260 265 270

Asn Gly Tyr Phe Ala Arg Leu Gly Gln Gln Glu Gly Ile Pro Thr Pro

275 280 285

Phe Asn Ala Leu Val Thr Arg Leu Ile His Ala Lys Glu Asp Ile Val

290 295 300

Arg Thr Lys Leu Ala Lys Glu Lys Glu Asn Phe Glu Ile

305 310 315

<210> 5

<211> 954

<212> DNA

<213> Leuconostoc pseudomesenteroides (Leuconostoc pseudosensoides)

<400> 5

atgaaaattg caattgcggg ttttggtgca ttgggtgcgc gagttggtgt aatgttgcaa 60

cgcgctggtc atgatgtaac tggtatagat ggctgggcag aacacattgc agcaatcaac 120

actaagggat tgactgtgac agaggatgac gggtcatcta aaaaatattt tattccagtc 180

atgacatcca aagaagttac tggtgaattt gatttggtga ttttattgac gaaaacgcca 240

caattggatc gtatgttaac tgatattcaa ccgcttatca caaaacaaac gcagttatta 300

gtcttgtcaa atggactagg taatgttgaa gtaatggcaa agcatgtgtc atcacaacaa 360

attattgctg gggtaacttt gtggacatct gatttggttc aacctggtga aattcatgtg 420

accggtacag gctccatcaa gttgcaagcg attgatcacg cagatattac cgctgttgtc 480

actgctttga atgaagcagg gcttaatgct gaggtatccg ataatgttgt agaagctatt 540

tggcataaag cgggcattaa ttctgtcctt aacccattga cagtcttact tgatgctaat 600

attgctgaat ttggcacagc agggaatggt atggatcttg cattgaacat tttagatgag 660

attaagcaag ttggtgacgt ggctggcgtt aatgtcgatg tgaatagtat tttaagcgat 720

ttgtcgaatt tgctaaaacc agaaaacgcg ggtaatcact acccatcaat gtaccaagat 780

attcaagctg gtaaacgaac tgaaatcgac tttttgaatg gctattttgc taaattggga 840

cgtgaaaatc atatcgccac accttttaat gcacttgtaa cacgattaat tcatgcaaaa 900

gaagatattg aacgtgttaa attggccaaa caacaagaaa cctttgaaat ttga 954

<210> 6

<211> 317

<212> PRT

<213> Leuconostoc pseudomesenteroides (Leuconostoc pseudosensoides)

<400> 6

Met Lys Ile Ala Ile Ala Gly Phe Gly Ala Leu Gly Ala Arg Val Gly

1 5 10 15

Val Met Leu Gln Arg Ala Gly His Asp Val Thr Gly Ile Asp Gly Trp

20 25 30

Ala Glu His Ile Ala Ala Ile Asn Thr Lys Gly Leu Thr Val Thr Glu

35 40 45

Asp Asp Gly Ser Ser Lys Lys Tyr Phe Ile Pro Val Met Thr Ser Lys

50 55 60

Glu Val Thr Gly Glu Phe Asp Leu Val Ile Leu Leu Thr Lys Thr Pro

65 70 75 80

Gln Leu Asp Arg Met Leu Thr Asp Ile Gln Pro Leu Ile Thr Lys Gln

85 90 95

Thr Gln Leu Leu Val Leu Ser Asn Gly Leu Gly Asn Val Glu Val Met

100 105 110

Ala Lys His Val Ser Ser Gln Gln Ile Ile Ala Gly Val Thr Leu Trp

115 120 125

Thr Ser Asp Leu Val Gln Pro Gly Glu Ile His Val Thr Gly Thr Gly

130 135 140

Ser Ile Lys Leu Gln Ala Ile Asp His Ala Asp Ile Thr Ala Val Val

145 150 155 160

Thr Ala Leu Asn Glu Ala Gly Leu Asn Ala Glu Val Ser Asp Asn Val

165 170 175

Val Glu Ala Ile Trp His Lys Ala Gly Ile Asn Ser Val Leu Asn Pro

180 185 190

Leu Thr Val Leu Leu Asp Ala Asn Ile Ala Glu Phe Gly Thr Ala Gly

195 200 205

Asn Gly Met Asp Leu Ala Leu Asn Ile Leu Asp Glu Ile Lys Gln Val

210 215 220

Gly Asp Val Ala Gly Val Asn Val Asp Val Asn Ser Ile Leu Ser Asp

225 230 235 240

Leu Ser Asn Leu Leu Lys Pro Glu Asn Ala Gly Asn His Tyr Pro Ser

245 250 255

Met Tyr Gln Asp Ile Gln Ala Gly Lys Arg Thr Glu Ile Asp Phe Leu

260 265 270

Asn Gly Tyr Phe Ala Lys Leu Gly Arg Glu Asn His Ile Ala Thr Pro

275 280 285

Phe Asn Ala Leu Val Thr Arg Leu Ile His Ala Lys Glu Asp Ile Glu

290 295 300

Arg Val Lys Leu Ala Lys Gln Gln Glu Thr Phe Glu Ile

305 310 315

<210> 7

<211> 954

<212> DNA

<213> Leuconostoc mesenteroides (Leuconostoc mesenteroides)

<400> 7

atgaaaatcg ctatcgctgg tttcggtgct ctgggtgctc gtgttggtgt tatgctgcag 60

caggctggtc acgaagttac cggtatcgac ggttgggctg ctcacatcgc tgctatctct 120

accgacggtc tgaccgttca ccaggacgac ggtgctacca aaaaatacta catcccggtt 180

atgaccgcta aagaaatcga cggtaaattc gacctgatca tcctgctgac caaaaccccg 240

cagctggaca tgatgctgac cgacatcaaa cacatcatca ccaaaaacac caaactgctg 300

gttctgtcta acggtctggg taacatcgaa gttatggaaa aacacgttaa ccgtaaccag 360

atcctggctg gtgttaccct gtggacctct gaactgatca acccgggtga aatccgtgtt 420

accggtaccg gttctatcaa actgcaggct atcggtgaag ctaacgctaa gccaatcgta 480

agcgctctga acaaagctgg tctgaacgtt accctgtctc agaacgttat cgaagctatc 540

tggcacaaag ctggtatcaa ctctgttctg aacccgctga ccgttctgct ggacgctaac 600

atcgctgaat tcggtatggc tggtaacggt atggacctgt ctctgaacat cctggacgaa 660

atcaaaaaaa tcggtgaact ggaaggtatc aacgttgacg ttaacgctat catgaaagac 720

ctggctctgc tgatccgtcc ggaaaacgct ggtaaccact acccgtctat gtaccaggac 780

atcaaagctg gtaaacacac cgaaatcgac ttcctgaacg gttacttcgc taaactgggt 840

tctgaacacg acgttgctat gccgttcaac gctctggtta cccgtctgat ccacgctaaa 900

gaagacatcg aacgtaccaa actggctaaa aaacaggaaa ccttcgaaat ctaa 954

<210> 8

<211> 317

<212> PRT

<213> Leuconostoc mesenteroides (Leuconostoc mesenteroides)

<400> 8

Met Lys Ile Ala Ile Ala Gly Phe Gly Ala Leu Gly Ala Arg Val Gly

1 5 10 15

Val Met Leu Gln Gln Ala Gly His Glu Val Thr Gly Ile Asp Gly Trp

20 25 30

Ala Ala His Ile Ala Ala Ile Ser Thr Asp Gly Leu Thr Val His Gln

35 40 45

Asp Asp Gly Ala Thr Lys Lys Tyr Tyr Ile Pro Val Met Thr Ala Lys

50 55 60

Glu Ile Asp Gly Lys Phe Asp Leu Ile Ile Leu Leu Thr Lys Thr Pro

65 70 75 80

Gln Leu Asp Met Met Leu Thr Asp Ile Lys His Ile Ile Thr Lys Asn

85 90 95

Thr Lys Leu Leu Val Leu Ser Asn Gly Leu Gly Asn Ile Glu Val Met

100 105 110

Glu Lys His Val Asn Arg Asn Gln Ile Leu Ala Gly Val Thr Leu Trp

115 120 125

Thr Ser Glu Leu Ile Asn Pro Gly Glu Ile Arg Val Thr Gly Thr Gly

130 135 140

Ser Ile Lys Leu Gln Ala Ile Gly Glu Ala Asn Ala Lys Pro Ile Val

145 150 155 160

Ser Ala Leu Asn Lys Ala Gly Leu Asn Val Thr Leu Ser Gln Asn Val

165 170 175

Ile Glu Ala Ile Trp His Lys Ala Gly Ile Asn Ser Val Leu Asn Pro

180 185 190

Leu Thr Val Leu Leu Asp Ala Asn Ile Ala Glu Phe Gly Met Ala Gly

195 200 205

Asn Gly Met Asp Leu Ser Leu Asn Ile Leu Asp Glu Ile Lys Lys Ile

210 215 220

Gly Glu Leu Glu Gly Ile Asn Val Asp Val Asn Ala Ile Met Lys Asp

225 230 235 240

Leu Ala Leu Leu Ile Arg Pro Glu Asn Ala Gly Asn His Tyr Pro Ser

245 250 255

Met Tyr Gln Asp Ile Lys Ala Gly Lys His Thr Glu Ile Asp Phe Leu

260 265 270

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

275 280 285

Phe Asn Ala Leu Val Thr Arg Leu Ile His Ala Lys Glu Asp Ile Glu

290 295 300

Arg Thr Lys Leu Ala Lys Lys Gln Glu Thr Phe Glu Ile

305 310 315

<210> 9

<211> 918

<212> DNA

<213> Klebsiella oxytoca (Klebsiella oxytoca)

<400> 9

atgaaaatcg ctatcgctgg tgctggtgct atgggttgcc gtttcggtta catgctgctg 60

ggtgctggtc acgacgttac cctgatcgac ggttggcacg aacacgttaa cgctatctgc 120

tctaacggtc tgttcgttga aaccgaagtt tctcagcagt actacccgat cccggctatg 180

ctggctgacg aatctcaggg tgaattcgaa ctgatcatcc tgttcaccaa agctatgcag 240

ctggaccgta tgctgcagca catcaaaccg ctgctgccgg ctgctaaagt tgttatgatc 300

ctgtctaacg gtctgggtaa catcgaaacc ctggaaaaat acgttgaccg tcagaaaatc 360

tacgctggtg ttaccctgtg gtcttctgaa ctggaaggtc cgggtcacat catggctacc 420

ggtaccggta ccatcgaact gcagccggtt gcttctcagg acgctgctct ggaagaaaac 480

atcgttgctg ttctgaactc tgctggtctg aacgctgaaa tctctccgga cgttctgctg 540

tctatctgga aaaaagctgc tttcaactct gttatgaaca cctactgcgc tctgctggac 600

tgcaacgttg gtggtttcgg tcagctgccg ggtgctctgg acctggctca ggctgttgtt 660

gacgaattcg ttctggttgc tgcttctcag aacatcccgc tgtctggtga acgtgttatg 720

aacaccgtta aaaaagtttt cgacccgcgt gaatctggtc accactaccc gtctatgtac 780

caggacctgc agaaaggtcg tctgaccgaa atcgactacc tgaacggtgc tatcgctcgt 840

atcggtatgc agaacaacat cccggttccg gttaacaccc tgctgaccca gctgatccac 900

gctaaagaag ctcagtaa 918

<210> 10

<211> 305

<212> PRT

<213> Klebsiella oxytoca (Klebsiella oxytoca)

<400> 10

Met Lys Ile Ala Ile Ala Gly Ala Gly Ala Met Gly Cys Arg Phe Gly

1 5 10 15

Tyr Met Leu Leu Gly Ala Gly His Asp Val Thr Leu Ile Asp Gly Trp

20 25 30

His Glu His Val Asn Ala Ile Cys Ser Asn Gly Leu Phe Val Glu Thr

35 40 45

Glu Val Ser Gln Gln Tyr Tyr Pro Ile Pro Ala Met Leu Ala Asp Glu

50 55 60

Ser Gln Gly Glu Phe Glu Leu Ile Ile Leu Phe Thr Lys Ala Met Gln

65 70 75 80

Leu Asp Arg Met Leu Gln His Ile Lys Pro Leu Leu Pro Ala Ala Lys

85 90 95

Val Val Met Ile Leu Ser Asn Gly Leu Gly Asn Ile Glu Thr Leu Glu

100 105 110

Lys Tyr Val Asp Arg Gln Lys Ile Tyr Ala Gly Val Thr Leu Trp Ser

115 120 125

Ser Glu Leu Glu Gly Pro Gly His Ile Met Ala Thr Gly Thr Gly Thr

130 135 140

Ile Glu Leu Gln Pro Val Ala Ser Gln Asp Ala Ala Leu Glu Glu Asn

145 150 155 160

Ile Val Ala Val Leu Asn Ser Ala Gly Leu Asn Ala Glu Ile Ser Pro

165 170 175

Asp Val Leu Leu Ser Ile Trp Lys Lys Ala Ala Phe Asn Ser Val Met

180 185 190

Asn Thr Tyr Cys Ala Leu Leu Asp Cys Asn Val Gly Gly Phe Gly Gln

195 200 205

Leu Pro Gly Ala Leu Asp Leu Ala Gln Ala Val Val Asp Glu Phe Val

210 215 220

Leu Val Ala Ala Ser Gln Asn Ile Pro Leu Ser Gly Glu Arg Val Met

225 230 235 240

Asn Thr Val Lys Lys Val Phe Asp Pro Arg Glu Ser Gly His His Tyr

245 250 255

Pro Ser Met Tyr Gln Asp Leu Gln Lys Gly Arg Leu Thr Glu Ile Asp

260 265 270

Tyr Leu Asn Gly Ala Ile Ala Arg Ile Gly Met Gln Asn Asn Ile Pro

275 280 285

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

290 295 300

Gln

305

<210> 11

<211> 918

<212> DNA

<213> Salmonella enterica

<400> 11

atgaaaattg caatcgcagg tgcaggcgct atggggtgtc gttttggcta tatgctgctg 60

gaggccgggc acgacgtgac gcttatcgat agctggcagg agcatgtcga cgctattcgt 120

agcaaggggt tgtttgtcga aacggaaacg acgcagaagt attaccccat ccctgctatg 180

ttggctgatg aatcccaggg ggagtttgag ctggttattc tgtttaccaa agccatgcag 240

ttggatagca tgttacagcg tatcaagcca ttactgccag ccgcgaaagt cgtgatgatt 300

ctatctaacg gtctgggaaa tattgaaacg ctggagaaat atgtcgatcg gcataaaatc 360

tatgcgggtg tgacgttatg gtccagcgaa ctggaggggg ctgggcatat tatggccacc 420

ggtaccggaa cgattgaact gcagccgatt gccagccagg attcggctca agaggctaag 480

gtcattgcca cccttaatag cgctggattg aatgctgaaa taagccctga cgtattatta 540

tcgatctgga agaaagcagc ctttaatagc gtaatgaaca cctattgcgc gctactggat 600

tgtaatatcg gcggatttgg tcagcggcct ggtgctttag atttagcgca agccgtagtt 660

gatgagtttg tgttagttgc tgccagccag aatatttcgt tgactgagca aatggtgatg 720

aatacggtga agaaagtgtt cgatccgcgt gagagcggcc accactatcc ttctatgcat 780

caggatttac ataaaggccg actgactgaa atcgactatt taaatggtgc gattgcgcga 840

atcggcgttc agaacaatat tgccgtaccg gttaacacac tcctgacgca attgattcac 900

gctaaagaag cgcaataa 918

<210> 12

<211> 305

<212> PRT

<213> Salmonella enterica

<400> 12

Met Lys Ile Ala Ile Ala Gly Ala Gly Ala Met Gly Cys Arg Phe Gly

1 5 10 15

Tyr Met Leu Leu Glu Ala Gly His Asp Val Thr Leu Ile Asp Ser Trp

20 25 30

Gln Glu His Val Asp Ala Ile Arg Ser Lys Gly Leu Phe Val Glu Thr

35 40 45

Glu Thr Thr Gln Lys Tyr Tyr Pro Ile Pro Ala Met Leu Ala Asp Glu

50 55 60

Ser Gln Gly Glu Phe Glu Leu Val Ile Leu Phe Thr Lys Ala Met Gln

65 70 75 80

Leu Asp Ser Met Leu Gln Arg Ile Lys Pro Leu Leu Pro Ala Ala Lys

85 90 95

Val Val Met Ile Leu Ser Asn Gly Leu Gly Asn Ile Glu Thr Leu Glu

100 105 110

Lys Tyr Val Asp Arg His Lys Ile Tyr Ala Gly Val Thr Leu Trp Ser

115 120 125

Ser Glu Leu Glu Gly Ala Gly His Ile Met Ala Thr Gly Thr Gly Thr

130 135 140

Ile Glu Leu Gln Pro Ile Ala Ser Gln Asp Ser Ala Gln Glu Ala Lys

145 150 155 160

Val Ile Ala Thr Leu Asn Ser Ala Gly Leu Asn Ala Glu Ile Ser Pro

165 170 175

Asp Val Leu Leu Ser Ile Trp Lys Lys Ala Ala Phe Asn Ser Val Met

180 185 190

Asn Thr Tyr Cys Ala Leu Leu Asp Cys Asn Ile Gly Gly Phe Gly Gln

195 200 205

Arg Pro Gly Ala Leu Asp Leu Ala Gln Ala Val Val Asp Glu Phe Val

210 215 220

Leu Val Ala Ala Ser Gln Asn Ile Ser Leu Thr Glu Gln Met Val Met

225 230 235 240

Asn Thr Val Lys Lys Val Phe Asp Pro Arg Glu Ser Gly His His Tyr

245 250 255

Pro Ser Met His Gln Asp Leu His Lys Gly Arg Leu Thr Glu Ile Asp

260 265 270

Tyr Leu Asn Gly Ala Ile Ala Arg Ile Gly Val Gln Asn Asn Ile Ala

275 280 285

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

290 295 300

Gln

305

<210> 13

<211> 1188

<212> DNA

<213> Pseudomonas aeruginosa (Pseudomonas aeruginosa)

<400> 13

atgtctcaga acctgttcaa cgttgaagac taccgtaaac tggctcagaa acgtctgccg 60

aaaatggttt acgactacct ggaaggtggt gctgaagacg aatacggtgt taaacacaac 120

cgtgacgttt tccagcagtg gcgtttcaaa ccaaagaggt tagttgacgt atcgcgtcgt 180

tctctgcagg ctgaagttct gggtaaacgt cagtctatgc cgctgctgat cggtccgacc 240

ggtctgaacg gtgctctgtg gccgaaaggt gacctggctc tggctcaggc tgctaccaaa 300

gctggtatcc cgttcgttct gtctaccgct tctaacatgt ctatcgaaga cctggctcgt 360

cagtgcgacg gtgacctgtg gttccagctg tacgttatcc accgtgaaat cgctcagggt 420

atggttctga aagctctgca ctctggttac accaccctgg ttctgaccac cgacgttgct 480

gttaacggtt accgtgaacg tgacctgcac aaccgtttca aaatgccgat gtcttacacc 540

ccgaaagtta tgctggacgg ttgcctgcac ccgcgttggt ctctggacct ggttcgtcac 600

ggtatgccgc agctggctaa cttcgtttct tctcagacct cttctctgga aatgcaggct 660

gctctgatgt ctaggcagat ggacgctagc ttcaactggg aagcgctgcg ttggctgcgt 720

gacctgtggc cgcacaaact gctggttaaa ggtctgctgt ctgctgaaga cgctgaccac 780

tgcatcgctg aaggtgctga cggtgttatc ctgtctaacc acggtggtcg tcagctggac 840

tgcgctgttt ctccgatgga agttctggct cagtctgttg ctaaaaccgg taaaccggtt 900

ctgatcgact ctggtttccg tcgtggttct gacatcgtta aagctctggc tctgggtgct 960

gaagctgttc tgctgggtcg tgctaccctg tacggtctgg ctgctcgtgg tgaaaccggt 1020

gttgacgaag ttctgaccct gctgaaagct gacatcgacc gtaccctggc tcagatcggt 1080

tgcccggaca tcacctctct gtctccggac tacctgcagt ctgaaggtgt tacctctacc 1140

gctccggttg accacctgat cggtaaaggt acccacgctc tcgagtga 1188

<210> 14

<211> 395

<212> PRT

<213> Pseudomonas aeruginosa (Pseudomonas aeruginosa)

<400> 14

Met Ser Gln Asn Leu Phe Asn Val Glu Asp Tyr Arg Lys Leu Ala Gln

1 5 10 15

Lys Arg Leu Pro Lys Met Val Tyr Asp Tyr Leu Glu Gly Gly Ala Glu

20 25 30

Asp Glu Tyr Gly Val Lys His Asn Arg Asp Val Phe Gln Gln Trp Arg

35 40 45

Phe Lys Pro Lys Arg Leu Val Asp Val Ser Arg Arg Ser Leu Gln Ala

50 55 60

Glu Val Leu Gly Lys Arg Gln Ser Met Pro Leu Leu Ile Gly Pro Thr

65 70 75 80

Gly Leu Asn Gly Ala Leu Trp Pro Lys Gly Asp Leu Ala Leu Ala Gln

85 90 95

Ala Ala Thr Lys Ala Gly Ile Pro Phe Val Leu Ser Thr Ala Ser Asn

100 105 110

Met Ser Ile Glu Asp Leu Ala Arg Gln Cys Asp Gly Asp Leu Trp Phe

115 120 125

Gln Leu Tyr Val Ile His Arg Glu Ile Ala Gln Gly Met Val Leu Lys

130 135 140

Ala Leu His Ser Gly Tyr Thr Thr Leu Val Leu Thr Thr Asp Val Ala

145 150 155 160

Val Asn Gly Tyr Arg Glu Arg Asp Leu His Asn Arg Phe Lys Met Pro

165 170 175

Met Ser Tyr Thr Pro Lys Val Met Leu Asp Gly Cys Leu His Pro Arg

180 185 190

Trp Ser Leu Asp Leu Val Arg His Gly Met Pro Gln Leu Ala Asn Phe

195 200 205

Val Ser Ser Gln Thr Ser Ser Leu Glu Met Gln Ala Ala Leu Met Ser

210 215 220

Arg Gln Met Asp Ala Ser Phe Asn Trp Glu Ala Leu Arg Trp Leu Arg

225 230 235 240

Asp Leu Trp Pro His Lys Leu Leu Val Lys Gly Leu Leu Ser Ala Glu

245 250 255

Asp Ala Asp His Cys Ile Ala Glu Gly Ala Asp Gly Val Ile Leu Ser

260 265 270

Asn His Gly Gly Arg Gln Leu Asp Cys Ala Val Ser Pro Met Glu Val

275 280 285

Leu Ala Gln Ser Val Ala Lys Thr Gly Lys Pro Val Leu Ile Asp Ser

290 295 300

Gly Phe Arg Arg Gly Ser Asp Ile Val Lys Ala Leu Ala Leu Gly Ala

305 310 315 320

Glu Ala Val Leu Leu Gly Arg Ala Thr Leu Tyr Gly Leu Ala Ala Arg

325 330 335

Gly Glu Thr Gly Val Asp Glu Val Leu Thr Leu Leu Lys Ala Asp Ile

340 345 350

Asp Arg Thr Leu Ala Gln Ile Gly Cys Pro Asp Ile Thr Ser Leu Ser

355 360 365

Pro Asp Tyr Leu Gln Ser Glu Gly Val Thr Ser Thr Ala Pro Val Asp

370 375 380

His Leu Ile Gly Lys Gly Thr His Ala Leu Glu

385 390 395

<210> 15

<211> 786

<212> DNA

<213> Microbacterium siberia (Exiguobacterium sibiricum)

<400> 15

atgtataatt ctctgaaagg caaagtcgcg attgttactg gtggtagcat gggcattggc 60

gaagcgatca tccgtcgcta tgcagaagaa ggcatgcgcg ttgttatcaa ctatcgtagc 120

catccggagg aagccaaaaa gatcgccgaa gatattaaac aggcaggtgg tgaagccctg 180

accgtccagg gtgacgtttc taaagaggaa gacatgatca acctggtgaa acagactgtt 240

gatcacttcg gtcagctgga cgtctttgtg aacaacgctg gcgttgagat gccttctccg 300

tcccacgaaa tgtccctgga agactggcag aaagtgatcg atgttaatct gacgggtgcg 360

ttcctgggcg ctcgtgaagc tctgaaatac ttcgttgaac ataacgtgaa aggcaacatt 420

atcaatatgt ctagcgtcca cgaaatcatc ccgtggccta ctttcgtaca ttacgctgct 480

tctaagggtg gcgttaaact gatgacccag actctggcta tggaatatgc accgaaaggt 540

atccgcatta acgctatcgg tccaggcgcg atcaacactc caattaatgc agaaaaattc 600

gaggatccga aacagcgtgc agacgtggaa agcatgatcc cgatgggcaa catcggcaag 660

ccagaggaga tttccgctgt cgcggcatgg ctggcttctg acgaagcgtc ttacgttacc 720

ggcatcaccc tgttcgcaga tggtggcatg accctgtacc cgagctttca ggctggccgt 780

ggttga 786

<210> 16

<211> 261

<212> PRT

<213> Microbacterium siberia (Exiguobacterium sibiricum)

<400> 16

Met Tyr Asn Ser Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Ser

1 5 10 15

Met Gly Ile Gly Glu Ala Ile Ile Arg Arg Tyr Ala Glu Glu Gly Met

20 25 30

Arg Val Val Ile Asn Tyr Arg Ser His Pro Glu Glu Ala Lys Lys Ile

35 40 45

Ala Glu Asp Ile Lys Gln Ala Gly Gly Glu Ala Leu Thr Val Gln Gly

50 55 60

Asp Val Ser Lys Glu Glu Asp Met Ile Asn Leu Val Lys Gln Thr Val

65 70 75 80

Asp His Phe Gly Gln Leu Asp Val Phe Val Asn Asn Ala Gly Val Glu

85 90 95

Met Pro Ser Pro Ser His Glu Met Ser Leu Glu Asp Trp Gln Lys Val

100 105 110

Ile Asp Val Asn Leu Thr Gly Ala Phe Leu Gly Ala Arg Glu Ala Leu

115 120 125

Lys Tyr Phe Val Glu His Asn Val Lys Gly Asn Ile Ile Asn Met Ser

130 135 140

Ser Val His Glu Ile Ile Pro Trp Pro Thr Phe Val His Tyr Ala Ala

145 150 155 160

Ser Lys Gly Gly Val Lys Leu Met Thr Gln Thr Leu Ala Met Glu Tyr

165 170 175

Ala Pro Lys Gly Ile Arg Ile Asn Ala Ile Gly Pro Gly Ala Ile Asn

180 185 190

Thr Pro Ile Asn Ala Glu Lys Phe Glu Asp Pro Lys Gln Arg Ala Asp

195 200 205

Val Glu Ser Met Ile Pro Met Gly Asn Ile Gly Lys Pro Glu Glu Ile

210 215 220

Ser Ala Val Ala Ala Trp Leu Ala Ser Asp Glu Ala Ser Tyr Val Thr

225 230 235 240

Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser Phe

245 250 255

Gln Ala Gly Arg Gly

260

Claims

1. An application of ketoacid reductase in catalytic synthesis of chiral aromatic 2-hydroxy acid is characterized in that the amino acid sequence of the ketoacid reductase is shown as SEQ ID NO. 10.

2. The use according to claim 1, wherein the nucleotide sequence of the gene encoding a ketoacid reductase is represented by SEQ ID No. 9.

3. The use according to claim 1, characterized in that the method of application is: taking supernatant of ketonic acid reductase obtained by ultrasonication of wet thallus obtained by fermentation culture of engineering bacteria containing ketonic acid reductase coding gene and supernatant of glucose dehydrogenase obtained by ultrasonication of wet thallus obtained by fermentation culture of engineering bacteria containing glucose dehydrogenase coding gene as catalysts, taking acetophenone acid as substrate, glucose as auxiliary substrate, and NAD⁺As coenzyme, KH of 100mM and pH7.0 is used₂PO₄-K₂HPO₄The buffer solution is used as a reaction medium, the reaction is carried out at 35 ℃ and 700rpm, and after the reaction is completed, a reaction solution containing (R) -mandelic acid is obtained; the dosage of the ketonic acid reductase supernatant is 800U/mL buffer calculated by ketonic acid reductase enzyme activityThe dosage of the glucose dehydrogenase supernatant is 800U/mL buffer solution in terms of glucose dehydrogenase activity, the dosage of glucose is 200-800 mM in terms of the volume of the buffer solution, the dosage of the substrate is 100-400 mM in terms of the volume of the buffer solution, and NAD (nicotinamide adenine dinucleotide)⁺The amount used was 0.5mM based on the volume of the buffer.

4. The use according to claim 1, characterized in that the method of application is: the method comprises the steps of taking wet thalli obtained by fermentation culture of engineering bacteria containing ketoreductase, 2-hydroxy acid dehydrogenase and glucose dehydrogenase coding genes as a catalyst, taking racemic aromatic 2-hydroxy acid as a substrate, taking glucose as an auxiliary substrate and taking a buffer solution with the pH value of 6.0-8.0 as a reaction medium to form a reaction system, and obtaining a conversion solution containing optically pure aromatic (R) -2-hydroxy acid after complete reaction at the temperature of 20-45 ℃ and the speed of 700 rpm.

5. Use according to claim 4, characterized in that the racemic aromatic 2-hydroxy acid is one of the following: mandelic acid, 2-fluoromandelic acid, 4-fluoromandelic acid, 2, 4-difluoromandelic acid, 3, 5-difluoromandelic acid, 2-chloromandelic acid, 3-chloromandelic acid, 4-chloromandelic acid, 2-bromomandelic acid, 3-bromomandelic acid, 4-methylmandelic acid, 4-trifluoromethylmandelic acid, 3-hydroxymandelic acid, 4-methoxymandelic acid, 3-methoxy-4-hydroxymandelic acid, 3-hydroxy-4-methylmandelic acid, 3-hydroxy-4-trifluoromethylmandelic acid, 3-methyl-4-methoxymandelic acid.

6. The use according to claim 4, wherein in the reaction system, the final concentration of the substrate is 20-300mM, the concentration of the co-substrate is 10-300mM, and the amount of the catalyst is 4-20g/L based on the dry weight of the wet cells.

7. The use according to claim 4, wherein the engineered bacterium is constructed by co-introducing genes encoding ketoacid reductase, 2-hydroxy acid dehydrogenase and glucose dehydrogenase into a host bacterium; the nucleotide sequence of the coding gene of the 2-hydroxy acid dehydrogenase is shown in SEQ ID NO.13, and the nucleotide sequence of the coding gene of the glucose dehydrogenase is shown in SEQ ID NO. 15.

8. The use according to claim 4, wherein the catalyst is prepared by the following process: inoculating engineering bacteria containing coding genes of ketoreductase, 2-hydroxy acid dehydrogenase and glucose dehydrogenase into LB liquid culture medium containing 50 mug/mL kanamycin and 50 mug/mL streptomycin, and performing shake culture at 37 ℃ and 150rpm for 8-10 h to obtain seed liquid; inoculating the seed solution into LB liquid medium containing 50. mu.g/mL kanamycin and 50. mu.g/mL streptomycin at an inoculum size of 2% by volume, and culturing at 37 ℃ and 150rpm with shaking to OD₆₀₀And (3) reaching 0.4-0.8, adding IPTG (isopropyl-beta-D-thiogalactoside) until the final concentration is 0.1mM, carrying out shake culture at 28 ℃ and 150rpm for 10-12 h, centrifugally collecting wet thalli, and washing twice with normal saline to obtain the wet thalli.