CN110904061B

CN110904061B - Alcohol dehydrogenase mutant with enhanced thermal stability and application thereof

Info

Publication number: CN110904061B
Application number: CN201911383100.6A
Authority: CN
Inventors: 丁雪峰; 王乾; 代兴兴
Original assignee: Nanjing Lang'en Biological Science & Technology Co ltd
Current assignee: Nanjing Lang'en Biological Science & Technology Co ltd
Priority date: 2019-12-28
Filing date: 2019-12-28
Publication date: 2021-05-18
Anticipated expiration: 2039-12-28
Also published as: CN110904061A

Abstract

The invention relates to an alcohol dehydrogenase mutant with enhanced thermostability and application thereof, wherein the alcohol dehydrogenase mutant is derived from wild-type alcohol dehydrogenase of Lactobacillus parabruchnei and has one or more of the following characteristics: a68T, N90S, E101D, S169V, a 235S. The sequence of the alcohol dehydrogenase mutant is SEQ ID NO.2, and the sequence of the polynucleotide for coding the alcohol dehydrogenase mutant is SEQ ID NO. 1. The recombinant alcohol dehydrogenase mutant has better thermal stability and is suitable for cyclic regeneration of NADH (NADPH) at higher temperature. The alcohol dehydrogenase is used for cyclic regeneration of NADH (NADPH), and has the advantages that inorganic salts are not introduced into the whole system, the substrate is various alcohols, and byproducts such as acetone and the like have low boiling point characteristics, can be directly separated from the product, and have no influence on the activity of the oxidoreductase. The alcohol dehydrogenase mutant of the invention has more lasting activity at 45-65 ℃ and better thermal stability, thus being more suitable for industrial application.

Description

Alcohol dehydrogenase mutant with enhanced thermal stability and application thereof

Technical Field

The invention relates to an alcohol dehydrogenase mutant, in particular to an alcohol dehydrogenase mutant with enhanced thermal stability and application thereof.

Background

Oxidoreductases are increasingly used in the preparation of chiral alcohols, amino acids and the like. However, nicotinamide adenine dinucleotide (NAD, NADH), or nicotinamide adenine dinucleotide phosphate (NADP, NADPH), is required as a coenzyme for 90% of the reactions catalyzed thereby. Therefore, there is a need for a coenzyme regeneration system that is low in cost and highly efficient in the application of oxidoreductases.

The enhanced thermal stability of the biocatalyst has many advantages. (1) Accelerate the kinetic reaction and effectively shorten the reaction period. (2) The enzyme extraction process is simplified, and the host cell protein is denatured, coagulated and precipitated at high temperature and is easy to separate from the target protein. (3) The cooling system for the reaction is not high in requirement, so that the energy consumption is reduced, and the cost is reduced. (4) The protein with improved stability can be transported and stored at room temperature, and the shelf life is effectively prolonged. (5) Under the condition of high-temperature catalytic reaction, the growth chance of mixed bacteria is avoided, thereby reducing the pollution of the metabolite of the bacteria to the product. (6) The high temperature helps to increase the solubility of the substrate and increases the production efficiency per unit volume.

Alcohol dehydrogenases are versatile catalysts for the enantioselective reduction of aldehydes or ketones to the corresponding alcohols. The (R) -specific alcohol dehydrogenases have different properties from the (S) -specific alcohol dehydrogenases, and these catalysts are used more and more frequently in the industrial synthesis of optically active alcohols. Optical activity is a prerequisite for the selective action of many pharmaceutically and pesticidally active compounds, in some cases one enantiomer having beneficial pharmaceutical activity and the other enantiomer having genotoxic effect. Therefore, in the synthesis of active compounds for pharmaceutical and agricultural chemicals, it is necessary to synthesize optically active alcohols using a catalyst having the required stereospecificity.

L-2-aminobutyric acid is an unnatural amino acid, which is a key chiral intermediate for the synthesis of some important drugs. For example, it can be converted to S-2-aminobutanamide, which is a direct precursor of the antiepileptic drugs levetiracetam and brivaracetam. L-2-aminobutyric acid can also be converted to S-2-aminobutanol, which is a chemical intermediate for the synthesis of ethambutol, an anti-tuberculosis compound. The coenzyme occupies a considerable proportion in the biological preparation of the L-2-aminobutyric acid, and taking formate dehydrogenase-ammonium formate coenzyme circulating process as an example, 1.2g-2g of NAD is consumed for preparing each kilogram of products, which is equivalent to 7-12 RMB/kilogram of products. Therefore, the improvement of the circulating efficiency of the coenzyme and the reduction of the dosage of the coenzyme have positive significance for reducing the production cost of the L-2-aminobutyric acid.

Atorvastatin calcium (trade name LIPITOR), rosuvastatin calcium (trade name CRESTOR) and pitavastatin (trade name Lipalo), are important cholesterol lowering statins. Chiral (S) -4-chloro-3-hydroxy-ethyl butyrate is an important key chiral intermediate of statins. Many of the known routes to these chiral intermediates, including chemical and enzymatic, have disadvantages. The enzymatic preparation and chiral (S) -4-chloro-3-hydroxy-ethyl butyrate require the use of a stereoselective ketoreductase, but the existing wild-type ketoreductase has low catalytic activity and insufficient coenzyme circulation efficiency, so that the wild-type ketoreductase is difficult to be an ideal method for commercial scale synthesis.

Atazanavir, sold under the trade name Reyataz, is an antiretroviral drug for the treatment and prevention of aids virus/aids, and is one of the major anti-aids drugs in the world today. It is the most important drug required by basic health systems on the basic drug list of the world health organization. Atazanavir (CN10282508C) was first developed by the centella asiatica company, and was approved by the FDA in the united states in 2003 and approved by china in 2007. The present main production process of (2R,3S) -1-chloro-3-tert-butyloxycarbonylamino-4-phenyl-2-butanol as key intermediate for preparing atazanavir includes two kinds of chemical synthesis and biological synthesis, wherein the biological synthesis can be obtained by biotransformation of 3S-1-chloro-3-tert-butyloxycarbonylamino-4-phenyl-2-butanol with corresponding alcohol dehydrogenase, and the preparation process involves not only stereospecific reduction of ketone, but also cyclic regeneration of coenzyme.

Duloxetine is a pharmaceutically active compound intended for use in the field of indications for depression and urinary incontinence. S-3-dimethylamino-1- (2-thienyl) -1-propanol is an important chiral alcohol and is a constituent unit in duloxetine synthesis. Several documents and patents describe synthetic routes to duloxetine. The disadvantage of these synthetic routes is that the synthesis first yields a racemic alcohol mixture, which requires subsequent resolution of the racemate by salt formation with an optically active counter ion, conversion of the racemate to a diastereomeric mixture, followed by physical separation of the diastereomers. This leads to high process costs due to repeated separation of solids and liquids, and increases the use of starting compounds due to the addition of optically active salts for the separation. Screening for a suitable alcohol dehydrogenase for stereospecific reduction of 3-methylamino-1- (2-thienyl) -propanone would provide a cheaper route to duloxetine.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a bus duct connector convenient to disassemble, and the specific technical scheme is as follows:

the recombinant alcohol dehydrogenase mutant has better thermal stability and is suitable for cyclic regeneration of NADH (NADPH) at higher temperature. The alcohol dehydrogenase is used for cyclic regeneration of NADH (NADPH), and has the advantages that inorganic salts are not introduced into the whole system, the substrate is various alcohols, and byproducts such as acetone and the like have low boiling point characteristics, can be directly separated from the product, and have no influence on the activity of the oxidoreductase. Compared with the alcohol dehydrogenase found at present, the alcohol dehydrogenase mutant of the invention has more durable activity at 45-65 ℃ and better thermal stability, thus being more suitable for industrial application.

The technical scheme for realizing the purpose of the invention is as follows: the wild-type alcohol dehydrogenase naturally present in the microorganism Lactobacillus parachusseri is capable of catalyzing the cyclic regeneration of the coenzyme NADH (NADPH). The inventors of the present disclosure have found that an alcohol dehydrogenase including a mutation at a certain position exhibits better thermostability than a wild-type alcohol dehydrogenase produced by Lactobacillus parachusseri (SEQ ID NO: 5). "wild-type alcohol dehydrogenase", "wild-type ADH enzyme" and "wild-type ADH alcohol dehydrogenase" refer to a polypeptide consisting of a polypeptide derived from Lactobacillus parachuticum having the sequence of SEQ ID NO: 5 amino acid sequence of an alcohol dehydrogenase. The enzyme is capable of catalyzing the cyclic regeneration of the coenzyme NADH (NADPH). "wild-type" refers to the same form of material or substance as found in nature. For example, a wild-type protein or nucleic acid sequence is the original sequence form that can be isolated from nature and exists in an organism without artificial modification. "increased thermostability" means that it exhibits better thermostability as measured in an in vitro or in vivo assay as compared to the wild-type alcohol dehydrogenase.

The invention provides an alcohol dehydrogenase mutant which is derived from wild-type alcohol dehydrogenase of Lactobacillus parachuteri and can catalyze cyclic regeneration of coenzyme NADH (NADPH). The alcohol dehydrogenase mutant shows stronger thermal stability compared with the wild-type alcohol dehydrogenase of SEQ ID NO. 5. Alcohol dehydrogenase mutants and polynucleotides encoding such mutants can be prepared using methods commonly used by those skilled in the art. Mutants can be obtained by in vitro recombination, polynucleotide mutagenesis, DNA shuffling, error-prone PCR and directed evolution methods etc. encoding the enzyme.

The alcohol dehydrogenase mutant has one or more mutations selected from the following characteristics: K49R, a68T, N90S, E101D, F147E, T152A, S169V, a 235S.

The alcohol dehydrogenase mutant is preferably selected from the sequences SEQ ID NO.2 and SEQ ID NO. 4. The full-length mutant alcohol dehydrogenase is not essential for maintaining the catalytic activity of the enzyme. Accordingly, truncated analogs and catalytically active fragments of alcohol dehydrogenase mutants are contemplated. For example, in some embodiments, several amino acids may be deleted from the C-terminus or N-terminus. Any particular truncated analog or fragment can be used in a corresponding assay to assess catalytic activity. Likewise, additional amino acid residues may be added to one or both termini without affecting catalytic activity. The additional sequences may be functional or non-functional. For example, the additional amino acid sequence may be used to aid in purification, as a marker, or to perform some other function. Thus, the alcohol dehydrogenase mutants of the present disclosure may be in the form of fusion proteins, wherein the alcohol dehydrogenase mutants (or fragments thereof) are fused to other proteins, such as by way of example and not limitation, a solubilizing tag (e.g., SUMO protein), a purification tag (e.g., metal-binding His tag), and a bacterial localization signal (e.g., secretion signal).

The present invention provides an alcohol dehydrogenase mutant having better thermostability than a wild-type alcohol dehydrogenase.

The above-mentioned alcohol dehydrogenase mutant coding sequence, which is preferably selected from SEQ ID NO.1, SEQ ID NO.3, has been sequence optimized for expression in E.coli. In some embodiments, the polynucleotide comprises codons optimized for expression in a particular type of host cell. The codon usage and preferences for each different type of microorganism are known as are optimized codons for the expression of a particular amino acid in these microorganisms. The present invention provides a recombinant plasmid, and in some embodiments, the control sequence includes a promoter, a leader sequence, a polyadenylation sequence, a propeptide sequence, a signal peptide sequence, a transcription terminator, and the like. For bacterial host cells, suitable promoters for directing transcription of the coding sequence include, but are not limited to, the genes selected from bacteriophage T5, bacteriophage T7, bacteriophage lambda, E.coli lacUV5 operon, E.coli trp operon, E.coli tac operon, and the like.

The invention has the beneficial effects that: the recombinant alcohol dehydrogenase mutant has better thermal stability and is suitable for cyclic regeneration of NADH (NADPH) at higher temperature. The alcohol dehydrogenase is used for cyclic regeneration of NADH (NADPH), and has the advantages that inorganic salts are not introduced into the whole system, the substrate is various alcohols, and byproducts such as acetone and the like have low boiling point characteristics, can be directly separated from the product, and have no influence on the activity of the oxidoreductase. Compared with the alcohol dehydrogenase found at present, the alcohol dehydrogenase mutant of the invention has more durable activity at 45-65 ℃ and better thermal stability, thus being more suitable for industrial application.

Drawings

FIG. 1 is an expression plasmid map of Lpa-a of the present invention

FIG. 2 is a 6-hour detection map of Lpa-a in the high-temperature biocatalytic preparation of L-2-aminobutyric acid in the invention.

Wherein, 3.6 minutes is threonine substrate; the product L-2-aminobutyric acid is obtained in 4.0 minutes; intermediate tetronic acid is obtained in 10.0 minutes.

FIG. 3 is a 12-hour detection map of Lpa-a in the high-temperature biocatalytic preparation of L-2-aminobutyric acid in the present invention.

FIG. 4 shows the 12-hour detection result of Lpa-a in the high-temperature biocatalytic preparation of (S) -4-chloro-3-hydroxy-ethyl butyrate.

Wherein, the upper color development band is substrate 4-chloroacetoacetic acid ethyl ester, and the lower color development band is product (S) -4-chloro-3-hydroxy-butyric acid ethyl ester. The reaction from left to right is as follows: reacting wild alcohol dehydrogenase at normal temperature; high-temperature reaction of wild alcohol dehydrogenase; lpa-a high temperature reaction.

FIG. 5 shows the 8-hour detection result of Lpa-a in the present invention for the high temperature biocatalytic preparation of (2R,3S) -1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol.

Wherein the upper color development band is substrate 3S-1-chloro-3-tert-butyloxycarbonylamino-4-phenyl-2-butanol, and the lower color development band is product (2R,3S) -1-chloro-3-tert-butyloxycarbonylamino-4-phenyl-2-butanol. The reaction from left to right is as follows: reacting wild alcohol dehydrogenase at normal temperature; high-temperature reaction of wild alcohol dehydrogenase; lpa-a high temperature reaction.

The specific implementation mode is as follows:

in order to better explain the invention, the invention is further illustrated below with reference to examples. The instruments and reagents used in the present examples are commercially available products unless otherwise specified.

Example 1

The secondary structure and codon preference of the gene are adjusted by a whole-gene synthesis method so as to realize high expression in escherichia coli. The Primer Premier (http:// Primer3.ut. ee /) and OPTIMIZER (http:// genes. urv. es/OPTIMIZER /) were used for design, and the Tm difference was kept within 3 ℃ and the Primer length was kept within 60base, and the obtained primers were dissolved in double distilled water and added to the following reaction system so that the final concentration of each Primer was 30nM and the final concentration of the head and tail primers was 0.6. mu.M.

2mM dNTP mix(2mM each dNTP)	5μl
		10×Pfu buffer	5μl
Pfu DNA polymerase(10U/μl)	0.5μl
		ddH2O	The total volume of the reaction system was adjusted to 50. mu.l

The prepared PCR reaction system is placed in a Bori XP cycler gene amplification instrument and amplified according to the following procedures: 30s at 98 ℃, 45s at 55 ℃, 120s at 72 ℃ and 35 x. The DNA fragment obtained by PCR was purified by gel cutting and cloned into the NdeI/XhoI site of pET30a by homologous recombination. Single clones were picked for sequencing. The DNA sequence successfully sequenced is SEQ ID NO.1 and is named as Lpa-a, and the corresponding amino acid sequence is SEQ ID NO. 2.

Example 2

The prepared PCR reaction system is placed in a Bori XP cycler gene amplification instrument and amplified according to the following procedures: 30s at 98 ℃, 45s at 55 ℃, 120s at 72 ℃ and 35 x. The DNA fragment obtained by PCR was purified by gel cutting and cloned into the NdeI/XhoI site of pET30a by homologous recombination. Single clones were picked for sequencing. The DNA sequence successfully sequenced is SEQ ID NO.3 and is named as Lpa-b, and the corresponding amino acid sequence is SEQ ID NO. 4.

Example 3

Synthesis of reference protein Lpa-CK gene sequence

According to the sequence of the wild-type protein from Lactobacillus parachuferi shown in WP _084973575, a Shanghai Czeri organism is entrusted to carry out whole-gene synthesis on the coding sequence of the protein, and the coding sequence is cloned into pET30a, so that a control protein expression plasmid Lpa-CK is obtained, and the corresponding amino acid sequence is SEQ ID NO. 5.

Example 4

Shake flask expression test

Coli single colonies containing the expression vector were picked and inoculated into 10ml of autoclaved medium: 10g/L tryptone, 5g/L yeast extract, 3.55g/L disodium hydrogen phosphate, 3.4g/L potassium dihydrogen phosphate, 2.68g/L ammonium chloride, 0.71g/L sodium sulfate, 0.493g/L magnesium sulfate heptahydrate, 0.027g/L ferric chloride hexahydrate, 5g/L glycerol, 0.8g/L glucose, and kanamycin to 50 mg/L. The culture was carried out at 30 ℃ and 250rpm overnight. Taking a 1L triangular flask the next day, and carrying out the following steps: 100 into 100ml of autoclaved medium: 10g/L tryptone, 5g/L yeast extract, 3.55g/L disodium hydrogen phosphate, 3.4g/L potassium dihydrogen phosphate, 2.68g/L ammonium chloride, 0.71g/L sodium sulfate, 0.493g/L magnesium sulfate heptahydrate, 0.027g/L ferric chloride hexahydrate, 5g/L glycerol, 0.3g/L glucose, and kanamycin to 50 mg/L. The cells were cultured at 30 ℃ until the OD 5-6 of the cells became zero, and the cells were immediately placed in a flask in a shaker at 25 ℃ and cultured at 250rpm for 1 hour. IPTG was added to a final concentration of 0.1mM and incubation was continued at 25 ℃ for 16 hours at 250 rpm. After completion of the culture, the culture was centrifuged at 12000g at 4 ℃ for 20 minutes to collect wet cells. Then the bacterial pellet is washed twice with distilled water, and the bacterial is collected and preserved at-70 ℃. Meanwhile, 2g of the thalli are added into 6ml of pure water for ultrasonic disruption, SDS-PAGE detection is carried out, and the crude enzyme liquid is stored at the temperature of minus 20 ℃.

Example 5

Fed-batch fermentation

The fed-batch fermentation was carried out in a computer-controlled bioreactor (Shanghai Seisaku) with a reactor capacity of 15L and a working volume of 8L, using 24g/L yeast extract, 12g/L peptone, 0.4% glucose, 2.31g/L catalase phosphate and 12.54g/L dipotassium hydrogen phosphate as the medium, pH 7.0. 200ml of culture was prepared for the primary inoculum and inoculated at OD 2.0. Throughout the fermentation, the temperature was maintained at 37 ℃, the dissolved oxygen concentration during fermentation was automatically controlled at 30% by the agitation rate (rpm) and aeration supply cascade, while the pH of the medium was maintained at 7.0 by 50% (v/v) orthophosphoric acid and 30% (v/v) aqueous ammonia. During the fermentation, when a large amount of dissolved oxygen rises, feeding is started. The feed solution contained 9% w/v peptone, 9% w/v yeast extract, 14% w/v glycerol. When OD600 was about 35.0 (wet weight about 60g/L), induction was carried out with 0.2mM IPTG for 16 hours. Taking 2g of thallus, adding 6ml of pure water, carrying out ultrasonic disruption, carrying out SDS-PAGE detection, and storing the crude enzyme liquid at-20 ℃.

Example 6

Sample Heat treatment

500ul of the alcohol dehydrogenase mutant Lpa-a prepared in example 1 and the alcohol dehydrogenase mutant Lpa-b prepared in example 2 were taken, added into a water bath kettle at 45 ℃, 55 ℃ and 65 ℃ for incubation for 60 minutes, taken out and placed at 4 ℃ for preservation to test enzyme activity residue, and the enzyme activity of an untreated sample was taken as 100%. Meanwhile, wild alcohol dehydrohydrin is used as a control after treatment under the same conditions.

Example 7

Enzyme activity detection

Taking 65 ml centrifuge tubes, respectively marking 1-6, respectively adding 3mM NADPH solution 0ul, 40ul, 80ul, 100ul, 120ul and 160ul, then supplementing 0.1M phosphate buffer solution with pH7.0 to 3ml each tube, mixing uniformly, detecting at 340nm and recording the absorbance value; obtaining a standard curve Y ═ k × X of NADPH according to the above measured values, wherein Y is the value of absorbance, X is the concentration (mM) of NADPH, and R2 of the curve is more than 99.5%; diluting the enzyme solution with pure water by a certain dilution ratio (reference dilution ratio: 600-1000 times), wherein the dilution ratio is suitable for changing the light absorption value per minute by 0.02-0.04; 5ml of centrifuge tube is taken, the samples are added into the centrifuge tube according to the following proportion, the mixture is quickly mixed, and the mixture is immediately poured into a cuvette.

Detection reagent	Dosage of
		Isopropanol (I-propanol)	500ul
2％NADP	100uL
		100mM PBS(pH 7.0)	2.35mL
Diluted enzyme solution	50uL

Detecting the change of absorbance at 340nm, recording a value every 1min, and keeping the change rate basically the same every minute, wherein the absorbance at 0min is S0, and the absorbance at 3min is S3;

the enzyme activity calculation formula is as follows:

enzyme activity (U/ml) [ (S0-S3) × 3ml × N ]/[ kXtime (t/min) × enzyme addition (ml) ]

Wherein N is the dilution multiple of the enzyme solution.

The detection results are as follows:

example 8

Application in preparation of L-2-aminobutyric acid

In a 250mL three-necked flask were added 24.0mL of water, 6g of feed grade L-threonine, and 4.8mL of analytically pure isopropanol in this order, the pH was adjusted to 8.8 with a small amount of 25% ammonia (w/w), and then 0.2mM coenzyme, 0.4mg of PLP, at a final concentration, were added and mixed well. Finally, 14ul of crude enzyme solution of heat-resistant threonine dehydrogenase, 140ul of crude enzyme solution of Lpa-a and 140ul of crude enzyme solution of heat-resistant leucine dehydrogenase are added. The flask is put into a water bath at 55 ℃ and stirred, the concentration of the substrate can reach more than 200g/L, and the dosage of the coenzyme is only 0.2 mM. Samples were taken at 6 hours and 12 hours of reaction, and the results are shown in FIG. 2 and FIG. 3. The detection conditions were as follows:

a chromatographic column: c185 μm 250mm × 4.6mm

Mobile phase: 4% methanol and 96% 0.1% H3PO4(pH3.0)

Flow rate: 0.7mL/min

Detection wavelength: 210nm

Column temperature: 30 deg.C

Example 9

Application in preparation of (S) -4-chloro-3-hydroxy-ethyl butyrate

In a 250mL three-necked flask were sequentially added 24mL of 0.1M PB buffer pH6.5, 4.9mL of analytically pure isopropanol, 10g of ethyl 4-chloroacetoacetate, and pH adjusted to 6.5 with 5M NaOH, placed in a 50 ℃ water bath for premixing for 10min, and finally 1.0g of Lpa-a crude enzyme solution was added to a final concentration of 0.086mM coenzyme. The flask was placed in a water bath at 50 ℃ and stirred, and the concentration of the substrate in 40ml of the total reaction system was 250 g/L. Meanwhile, the above reaction was repeated at 37 ℃ and 50 ℃ for Lpa-CK wild-type protein, respectively, as a control. Samples were taken at 12 hours of reaction and plate measurements were made, and the results are shown in FIG. 4. TLC detection conditions: extracting a small amount of reaction liquid by using ethyl acetate, developing agent petroleum ether: ethyl acetate 3:1, potassium permanganate colour development.

Example 10

Application in preparation of (2R,3S) -1-chloro-3-tert-butyloxycarbonylamino-4-phenyl-2-butanol

A500 ml three-necked beaker was charged with a magneton stirrer, and 2.7ml of toluene, 32ml of isopropanol, and 32g of 3S-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol were sequentially added thereto, and the mixture was mixed with the pre-melting substrate, 1mM MgCl2 and 0.1M PB (pH7.5) were added thereto to give a total of about 190ml, and the mixture was mixed with pH adjusted to 7.5. Finally, 21mg of NAD and 6.4ml of crude Lpa-a enzyme solution were added and subjected to a shake reaction at 53 ℃. 200ml of the total reaction system. Meanwhile, the above reaction was repeated at 37 ℃ and 53 ℃ for Lpa-CK wild-type protein, respectively, as a control. The reaction was carried out for 7 hours and the plate was sampled and examined, and the results are shown in FIG. 5. TLC detection conditions: petroleum ether: ethyl acetate ═ 3.5: and 1, developing by potassium permanganate.

Therefore, the alcohol dehydrogenase mutant with enhanced thermal stability provided by the invention has obviously enhanced enzyme activity residue under the high-temperature condition, and the reaction speed is faster than that at normal temperature. Compared with wild alcohol dehydrogenase, the reaction can be completed more quickly in the reaction for preparing L-2-aminobutyric acid by the L-threonine biological method, and the residual ketobutyric acid as the intermediate is very little; the reaction is faster in the preparation of (S) -4-chloro-3-hydroxy-ethyl butyrate by the 4-chloroacetoacetic acid ethyl ester biological method, the substrate residue is less, and the reaction is faster than the normal temperature reaction; and further, the substrate concentration of unit volume is increased, and the production efficiency is improved during industrial amplification.

The technical means disclosed by the scheme of the invention are not limited to the technical means disclosed by the technical means, and also comprise the technical scheme formed by equivalent replacement of the technical features. The present invention is not limited to the details given herein, but is within the ordinary knowledge of those skilled in the art.

Sequence listing

<110> Nanjing Langen Biotech Ltd

<120> alcohol dehydrogenase mutant with enhanced thermostability and application thereof

<130> 2019

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 759

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgaccgacc gtctgaaagg taaagttgct atcgttaccg gtggtaccct gggtatcggt 60

ctggctatcg ctgacaaatt cgttgaagaa ggtgctaaag ttgttatcac cggtcgtcac 120

gctgacgttg gtgaaaaagc tgctaaatct atcggtggtc cggacgttat ccgtttcgtt 180

cagcacgacg cttctgacga aaccggttgg accgaactgt tcgacaccac cgaaaacgct 240

ttcggtccgg ttaccaccgt tgttaactct gctggtatcg ctgtttctaa atctgttgaa 300

gacaccacca ccgaagaatg gcgtaaactg ctgtctgtta acctggacgg tgttttcttc 360

ggtacccgtc tgggtatcca gcgtatgaaa aacaaaggtc tgggtgcttc tatcatcaac 420

atgtcttcta tcgaaggttt cgttggtgac ccggctctgg gtgcttacaa cgcttctaaa 480

ggtgctgttc gtatcatgtc taaagttgct gctctggact gcgctctgaa agactacgac 540

gttcgtgtta acaccgttca cccgggttac atcaaaaccc cgctggttga cgacctggaa 600

ggtgctgaag aaatgatgtc tcagcgtacc aaaaccccga tgggtcacat cggtgaaccg 660

aacgacatcg cttggatctg cgtttacctg gcttctgacg aatctaaatt cgctaccggt 720

gctgaattcg ttgttgacgg tggttacacc gctcagtaa 759

<210> 2

<211> 252

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr

1 5 10 15

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

20 25 30

Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala

35 40 45

Lys Ser Ile Gly Gly Pro Asp Val Ile Arg Phe Val Gln His Asp Ala

50 55 60

Ser Asp Glu Thr Gly Trp Thr Glu Leu Phe Asp Thr Thr Glu Asn Ala

65 70 75 80

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

85 90 95

Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser

100 105 110

Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg

115 120 125

Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile

130 135 140

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

145 150 155 160

Gly Ala Val Arg Ile Met Ser Lys Val Ala Ala Leu Asp Cys Ala Leu

165 170 175

Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys

180 185 190

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

195 200 205

Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala

210 215 220

Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly

225 230 235 240

Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln

245 250

<210> 3

<211> 759

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgaccgacc gtctgaaagg taaagttgct atcgttaccg gtggtaccct gggtatcggt 60

ctggctatcg ctgacaaatt cgttgaagaa ggtgctaaag ttgttatcac cggtcgtcac 120

gctgacgttg gtgaaaaagc tgctcgttct atcggtggtc cggacgttat ccgtttcgtt 180

cagcacgacg cttctgacga agctggttgg accgaactgt tcgacaccac cgaaaacgct 240

ttcggtccgg ttaccaccgt tgttaactct gctggtatcg ctgtttctaa atctgttgaa 300

gaaaccacca ccgaagaatg gcgtaaactg ctgtctgtta acctggacgg tgttttcttc 360

ggtacccgtc tgggtatcca gcgtatgaaa aacaaaggtc tgggtgcttc tatcatcaac 420

atgtcttcta tcgaaggtga agttggtgac ccggctctgg gtgcttacaa cgcttctaaa 480

ggtgctgttc gtatcatgtc taaagttgct gctctggact gcgctctgaa agactacgac 540

gttcgtgtta acaccgttca cccgggttac atcaaaaccc cgctggttga cgacctggaa 600

ggtgctgaag aaatgatgtc tcagcgtacc aaaaccccga tgggtcacat cggtgaaccg 660

aacgacatcg cttggatctg cgtttacctg gcttctgacg aagctaaatt cgctaccggt 720

gctgaattcg ttgttgacgg tggttacacc gctcagtaa 759

<210> 4

<211> 252

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr

1 5 10 15

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

20 25 30

Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala

35 40 45

Arg Ser Ile Gly Gly Pro Asp Val Ile Arg Phe Val Gln His Asp Ala

50 55 60

Ser Asp Glu Ala Gly Trp Thr Glu Leu Phe Asp Thr Thr Glu Asn Ala

65 70 75 80

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

85 90 95

Lys Ser Val Glu Glu Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser

100 105 110

Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg

115 120 125

Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile

130 135 140

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

145 150 155 160

Gly Ala Val Arg Ile Met Ser Lys Val Ala Ala Leu Asp Cys Ala Leu

165 170 175

Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys

180 185 190

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

195 200 205

Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala

210 215 220

Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ala Lys Phe Ala Thr Gly

225 230 235 240

Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln

245 250

<210> 5

<211> 252

<212> PRT

<213> Lactobacillus parabuchneri

<400> 5

Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr

1 5 10 15

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

20 25 30

Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala

35 40 45

Lys Ser Ile Gly Gly Pro Asp Val Ile Arg Phe Val Gln His Asp Ala

50 55 60

Ser Asp Glu Ala Gly Trp Thr Glu Leu Phe Asp Thr Thr Glu Asn Ala

65 70 75 80

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

85 90 95

Lys Ser Val Glu Glu Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser

100 105 110

Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg

115 120 125

Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile

130 135 140

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

145 150 155 160

Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu

165 170 175

Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys

180 185 190

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

195 200 205

Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala

210 215 220

Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ala Lys Phe Ala Thr Gly

225 230 235 240

Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln

245 250

Claims

1. An alcohol dehydrogenase mutant, which is derived from Lactobacillus buchneri (L.)Lactobacillus parabuchneri) The sequence of the alcohol dehydrogenase mutant is shown as SEQ ID NO. 2.

2. A polynucleotide encoding the alcohol dehydrogenase mutant of claim 1.

3. The polynucleotide of claim 2, wherein the base sequence of said polynucleotide is set forth in SEQ ID No. 1.

4. A recombinant plasmid comprising the polynucleotide of claim 2.

5. A host cell comprising the recombinant plasmid of claim 4.

6. The host cell of claim 5, wherein the cell is an E.coli cell.

7. The host cell of claim 6, wherein the codons of the recombinant plasmid have been optimized for expression in the host cell.

8. A cyclic regeneration method of NADH, characterized in that NAD is catalyzed to generate NADH in the presence of the alcohol dehydrogenase mutant of claim 1 at 45-65 ℃.

9. A cyclic regeneration method of NADPH, characterized in that NADP is catalyzed to generate NADPH in the presence of the alcohol dehydrogenase mutant of claim 1 at 45-65 ℃.