CN112592913B

CN112592913B - Thermally stable threonine deaminase and application thereof

Info

Publication number: CN112592913B
Application number: CN202011384201.8A
Authority: CN
Inventors: 丁雪峰; 钱明; 代兴兴
Original assignee: Nanjing Nuoyun Biotechnology Co ltd
Current assignee: Nanjing Nuoyun Biotechnology Co ltd
Priority date: 2020-11-30
Filing date: 2020-11-30
Publication date: 2022-04-22
Anticipated expiration: 2040-11-30
Also published as: CN112592913A

Abstract

The embodiment of the invention discloses a thermostable threonine deaminase and application thereof, belonging to the technical field of biochemistry, the thermostable threonine deaminase is derived from a wild type threonine deaminase of Chryseobacterium takakiae, can catalyze L threonine at high temperature to generate 2-ketobutyrate, and can be applied to high-temperature conversion of L threonine to prepare L-2-aminobutyric acid. The embodiment of the invention discloses that the wild type threonine deamination from Chryseobacterium takakiae shows better thermal stability compared with threonine deaminase from Escherichia coli, the threonine deaminase mutant has more durable activity at 45-60 ℃, is used for preparing 2-ketobutyrate and L-2-aminobutyric acid, has higher reaction temperature and higher substrate solubility, can effectively increase the substrate concentration, and can effectively accelerate the kinetic reaction and improve the production efficiency.

Description

Thermally stable threonine deaminase and application thereof

Technical Field

The invention belongs to the technical field of biochemistry, and particularly relates to thermally stable threonine deaminase and application thereof.

Background

L-2-aminobutyric acid (L-ABA) is a non-protein amino acid and has the effects of inhibiting human body neural information transmission, enhancing the activity of glucose phosphatase and promoting brain cell metabolism. The derivative (S) -2-aminobutanamide of the L-2-aminobutanoic acid can be used as a key intermediate of novel antiepileptic drugs such as levetiracetam, brivaracetam and other chiral drugs, and the other derivative (S) -2-aminobutanol of the L-2-aminobutanoic acid is a key intermediate of ethambutol hydrochloride of a bacteriostatic antituberculous drug. The optical purity of these drugs is critical to the safety and efficacy of the treatment. The R-enantiomer of levetiracetam has no antiepileptic activity and the (R, R) -form of amphetamine can cause blindness. Although ethambutol and levetiracetam are now counterfeit drugs, in many countries the high drug prices have led to the inability of most epileptic patients to be treated with levetiracetam.

At present, with the expiration of the patents of medicaments such as levetiracetam and the like, the market popularity is improved, and the demand on the key intermediate L-2-aminobutyric acid is increased.

The production method of L-2-aminobutyric acid mainly comprises a fermentation method, a chemical synthesis method and a biological catalysis method. The fermentation method has the biggest defects of low yield and insufficient product purity. The existing fermentation method is mainly used for modifying a threonine metabolism path, so that the metabolism flows to 2-ketobutyric acid and 2-aminobutyric acid, but the reported data are less than 50g/L, and the existing fermentation titer is not very significant in production amplification by combining the current market price situation (the four-quarter quoted price in 2018 is less than 15 dollars) and the market capacity (hundred-ton grade of global market) of the L-2-aminobutyric acid. For example, in patent CN201510208576.1, 100g/L threonine and 10g/L glutamic acid are added in the late stage of fermentation, the reaction is still essentially whole-cell biocatalytic, the highest record of the product is only about 57g/L, and the conversion rate is low. CN201210015308.4 was fermented to produce a product by overexpressing threonine deaminase and leucine dehydrogenase on the basis of a threonine producing strain, but this method requires 60 hours of fermentation time and the highest score of the product was only 20 g/L. In addition, a large amount of metabolites are generated in the microbial fermentation process, and a large amount of organic culture medium and inorganic salt are brought into the fermentation culture medium, so that the separation is influenced to a certain extent, and the purity and chiral purity of the final product are lower than those of an in vitro catalysis approach. For example, in the patent of CN201610208600.6, after the fermentation is finished, the product is filtered by a ceramic membrane and an ultrafiltration membrane, and then is adsorbed and eluted by ion exchange resin, the purity is only 97.3%, which is still lower than the purity requirement of 98.5% of common-grade L-2-aminobutyric acid, and the use of the ion exchange resin has no practical significance for the current price of L-2-aminobutyric acid.

Most of products synthesized by a chemical method are racemes, and further resolution is needed. In addition, the chemical method needs expensive reagents, byproducts are easy to generate, and a plurality of organic solvents are used, so that the cost of environment-friendly treatment is further increased.

The biocatalytic method mainly includes an amino acylase method, an amino acid oxidase method, an amidase method, a nitrilase method, a transaminase method, an amino acid dehydrogenase method and the like. The concentration and production efficiency of the catalytic substrate of the transaminase method and the amino acid dehydrogenase method are high, and the two types of catalytic substrate are mainly used in the existing biological catalytic methods in the market. The transaminase method is to synthesize 2-aminobutyric acid by 2-ketobutyric acid under the catalysis of transaminase. When the alpha-aminotransferase is used for synthesis, only 58 to 62% of yield is obtained due to reversibility of the reaction, and the process is complicated, and acetolactate synthase, alanine racemase and D-amino acid oxidase need to be introduced to remove by-products. L-2-aminobutyric acid can also be synthesized by using omega-transaminase with benzylamine or isopropylamine as an amino donor, which is irreversible, but the currently reported omega-transaminase method generally has low substrate concentration. Patent CN201510900727.X uses amine and ketone compounds as raw materials, and produces chiral amine through stereoselectivity transamination, but the conversion concentration is low, the highest data is only 46g/L, and the difference from industrial scale-up production is still not small.

Amino acid dehydrogenase is the most deeply studied biocatalytic pathway at present, and patents CN201210066624.4 and CN201010139227.6 report that L-threonine is used as a raw material, threonine deaminase is firstly converted into 2-ketobutyric acid, and leucine dehydrogenase is used for catalyzing 2-ketobutyric acid to prepare L-2-aminobutyric acid, but CN201210066624.4 is used for catalyzing 1000g of threonine and 600g of whole cell thallus is needed, so that the production significance in fermentation cost is not achieved. The coenzyme regeneration system of CN201010139227.6 is glucose, and as the dosage of glucose is too large and the molecular weight of gluconic acid and the product L-2-aminobutyric acid is similar, the mature and simple membrane separation method can not effectively separate the product and the byproduct, thereby increasing the separation cost; the reaction of the enzyme in the patent is not more than 30 ℃, the reaction temperature is low, the reaction speed is slow, and the conversion efficiency is low. In addition, the amount of coenzyme used in the existing reports: the substrate dosage is almost all more than 1: 1000, that is, at least 1 kg of coenzyme is consumed when 1 ton of substrate is put into practical production, and the price of the coenzyme is high (such as the price of 95 percent standard NAD imported from Oriental yeast is about 8000 yuan/kg), so that the production cost of the L-2-aminobutyric acid is indirectly increased.

Disclosure of Invention

The invention aims to solve the technical problems of poor thermal stability, low conversion efficiency and low L-2-aminobutyric acid yield in the process of preparing L-2-aminobutyric acid by a biological catalysis method in the prior art.

The technical scheme provided by the invention is as follows:

a thermostable threonine deaminase is derived from wild type threonine deaminase of Chryseobacterium takakiae, can catalyze L threonine at high temperature to generate 2-ketobutyrate, and can be applied to high-temperature conversion of L threonine to prepare L-2-aminobutyric acid.

The thermally stable threonine deaminase is preferably selected from the sequence SEQ ID No. 2.

The thermally stable threonine deaminase has higher thermal stability than the threonine deaminase of Escherichia coli SEQ ID No. 3.

A polynucleotide encoding a recombinant polypeptide as hereinbefore described.

The polynucleotide is preferably selected from SEQ ID NO. 1.

A recombinant plasmid comprising an expression vector linked to said polynucleotide.

A host cell comprising the recombinant plasmid.

Preferably, the host cell is an E.coli.

The host cell, wherein the codons and secondary structure of the recombinant plasmid have been optimized for expression in the 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.

A method for catalyzing L threonine to generate 2-ketobutyric acid and then generating L-2-aminobutyric acid, which comprises the step of converting L threonine into 2-ketobutyric acid and then converting the L-ketobutyric acid into the L-2-aminobutyric acid under the high-temperature condition of 45-60 ℃ in the presence of threonine deaminase and leucine dehydrogenase and alcohol dehydrogenase.

By adopting the technical scheme, the invention achieves the following technical effects:

1. the embodiment of the invention discloses that the wild threonine deamination derived from Chryseobacterium takakiae shows better thermal stability compared with threonine deaminase derived from Escherichia coli, and the threonine deaminase mutant has more durable activity at 45-60 ℃.

2. 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.

3. The threonine deaminase disclosed by the embodiment of the invention is used for preparing 2-ketobutyric acid and L-2-aminobutyric acid, has higher reaction temperature and higher substrate solubility, can effectively increase the substrate concentration, can effectively accelerate the kinetic reaction and improve the production efficiency.

4. The threonine deaminase with enhanced thermal stability has wide application prospect in the fields of chemical industry, food, pharmacy, energy development and the like.

Drawings

FIG. 1 is an expression plasmid map of TD223 wt;

FIG. 2 is an expression plasmid map of TD 223;

FIG. 3 is a protein gel map of TD223 wt;

FIG. 4 is a protein gel map of TD 223;

FIG. 5 shows the results of 20-hour thin-layer chromatography of example 9 for the high-temperature biocatalytic preparation of L-2-aminobutyric acid. The upper color development band is the product L-2-aminobutyric acid, and the lower color development band is the substrate threonine. From left to right are: reaction samples; substrate threonine control;

FIG. 6 shows the results of 20-hour thin-layer chromatography of example 10 for the high-temperature biocatalytic preparation of L-2-aminobutyric acid. The upper color development band is the product L-2-aminobutyric acid, and the lower color development band is the substrate threonine. From left to right are: reaction samples; substrate threonine control;

FIG. 7 is a 12 hour HPLC chromatogram of example 12 TD233 used for the high temperature biocatalytic preparation of L-2-aminobutyric acid. 3.146 min to obtain the product L-2-aminobutyric acid;

FIG. 8 is a 16 hour HPLC chromatogram of example 13 TD233wt for the high temperature biocatalytic preparation of L-2-aminobutyric acid. 2.691 min as substrate threonine; 3.167 min to obtain the product L-2-aminobutyric acid;

FIG. 9 is a liquid phase chromatogram of a substrate, a product, and an intermediate standard. 2.671 min as substrate threonine; 3.151 min to obtain the product L-2-aminobutyric acid; 7.529 min as intermediate tetronic acid;

FIG. 10 shows the results of thin layer chromatography on the substrate and the product standard. The upper color development band is the product L-2-aminobutyric acid, and the lower color development band is the substrate threonine.

Detailed Description

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 acquisition of wild-type threonine deaminase Gene sequence

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 design was carried out using Primer Premier (http:// Primer3.ut. ee /) and OPTIMIZER (http:// genes. urv. es/OPTIMIZER /), and the difference of annealing temperatures (Tm) was kept within 3 ℃ and the length of the primers 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 μ 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 TD223wt, the expression plasmid map and the protein glue map of the DNA sequence are respectively shown in figure 1 and figure 3, and the corresponding amino acid sequence is SEQ ID NO. 2.

EXAMPLE 2 acquisition of Gene sequences of threonine deaminase mutants

The threonine deaminase mutant provided by the invention is derived from wild type threonine deaminase of Chryseobacterium takakiae, and can catalyze L threonine to generate 2-ketobutyrate and then generate L-2-aminobutyric acid. The threonine deaminase mutant exhibits greater thermostability than the wild-type threonine deaminase of SEQ ID No. 2. Threonine deaminase 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 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.4 and is named as TD223, the expression plasmid map and the protein glue map of the DNA sequence are respectively shown in fig. 2 and 4, and the corresponding amino acid sequence is SEQ ID NO. 5.

EXAMPLE 3 obtaining of threonine deaminase Gene sequence derived from Escherichia coli

According to the sequence of the wild-type protein derived from Escherichia coli shown in QJZ14922, the Suzhou Jinzhi organism is entrusted with the whole-gene synthesis of the coding sequence of the protein, and the coding sequence is cloned into pET30a, so as to obtain a control protein expression plasmid EC07, the corresponding amino acid sequence of which is SEQ ID NO. 3.

Example 4 Shake flask expression assay

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, a small amount of thallus is taken for SDS-PAGE detection.

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 50.0 (wet weight was about 100g/L), the temperature was controlled at 28 ℃ and expression was induced with 0.15mM IPTG.

Example 6 thermal stability experiment

The threonine deaminase prepared in the embodiment 4 is added into a water bath kettle with the temperature of 45 ℃ and the temperature of 55 ℃ for incubation for 120 minutes, the threonine deaminase is taken out and placed at the temperature of 4 ℃ for preservation to test the enzyme activity residue, and the enzyme activity of an untreated sample is taken as 100%. Meanwhile, the wild threonine deaminase derived from Escherichia coli was treated under the same conditions as the control.

Example 7 enzyme Activity detection

Taking 6 5ml centrifuge tubes, respectively marking 1-6, respectively adding diluted butanone acid solution 0 μ l, 40 μ l, 80 μ l, 100 μ l, 120 μ l and 160 μ l, then supplementing 0.1M phosphate buffer solution with pH of 7.0 to 3ml each tube, mixing uniformly, detecting at 230nm and recording absorbance value; obtaining a standard curve Y ═ k × X of tetronic acid according to the above measured values, wherein Y is the value of absorbance, X is the concentration (umol) of tetronic acid, and R of the curve is²>99.5 percent; diluting the enzyme solution with pure water by a certain dilution ratio (reference dilution ratio: 40-100 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
		Threonine 40mM	2.95mL
Diluted enzyme solution	50μl

Detecting the change in absorbance at 230nm, recording the value every 1min, and the rate of change is substantially the same every minute, wherein the absorbance at 0min is S₀And absorbance at 3min is S₃

Definition of enzyme activity: one unit of enzyme activity is defined as the amount of enzyme required to produce 1umol of 2-ketobutyric acid per minute under the assay conditions. The enzyme activity detection results are as follows:

example 8 bioconversion reactions

5ml of pure water, 2.2g of feed-grade threonine (content > 98%), 1.6ml of industrial isopropanol are added to a 100ml triangular flask, a small amount of 25% ammonia (w/w) is taken to adjust the pH to 8.9, and a crude NAD (10% content, from Nanjing Langen Biotech Co., Ltd.) with a final concentration of 0.2mM, 1mg of PLP are added and mixed well. Finally, 120. mu.l TD223, 100. mu.l leucine dehydrogenase LD161 (available from Nanjing Langen Biotechnology Co., Ltd.), and 100. mu.l alcohol dehydrogenase AD151 (available from Nanjing Langen Biotechnology Co., Ltd.) were added thereto, and the mixture was reacted at 50 ℃ with a shaker speed set at 180 rpm. The total system is about 10ml, and the substrate concentration is more than 220 g/L. A large amount of insoluble substances are at the bottom of the bottle at the beginning of the reaction, but no obvious substrate precipitation exists in 3 hours, the whole reaction becomes clear, and the reaction speed of the used threonine deaminase is very high in the first 3 hours; at this time, after the reaction solution is taken out and cooled, more crystals are precipitated, which is caused by the crystallization of the product L-2-aminobutyric acid due to the decrease in solubility at low temperature.

Example 9 bioconversion reactions

In a 100ml triangular flask, 7.0ml pure water, 2.5g feed grade threonine (content > 98%), 2.0ml industrial isopropanol, a small amount of 25% ammonia (w/w) was added to adjust the pH to 8.8, and then 0.25mM NAD crude product, 0.166mg PLP (13. mu.l of 1% PLP mother liquor) were added and mixed well. Finally, 63. mu.l TD223, 84. mu.l leucine dehydrogenase LD161, and 84. mu.l alcohol dehydrogenase AD151 were added thereto, and the mixture was reacted at 50 ℃ with a shaker speed of 180 rpm. The total system is about 10ml, and the substrate concentration is about 250 g/L. As shown in FIG. 10, FIG. 10 shows the results of thin layer chromatography of the substrate and the product standard, wherein the upper color band is the product L-2-aminobutyric acid, and the lower color band is the substrate threonine. As shown in FIG. 5, when compared with FIG. 10, the upper colored band is the product L-2-aminobutyric acid, and the lower colored band is the substrate threonine. From left to right are: reaction samples; substrate threonine control; the plaque was free of substrate at 20 hours.

Example 10 bioconversion reactions

In a 100ml triangular flask, 7.0ml pure water, 3g feed grade threonine (content > 98%), 2.4ml industrial isopropanol were added, a small amount of 25% ammonia (w/w) was added to adjust the pH to 8.8, and then the final concentration of 0.3mM NAD crude product, 0.2mg PLP (20. mu.l of 1% PLP mother liquor) were added and mixed well. Finally, 100. mu.l TD223, 100. mu.l leucine dehydrogenase LD161, and 100. mu.l alcohol dehydrogenase AD151 were added, and the mixture was reacted at 50 ℃ with a shaker speed of 180 rpm. The total system is about 10ml, the substrate concentration is about 300g/L, and a large amount of substrate precipitates on the bottom of the bottle and is not dissolved. After the reaction is carried out for 2 hours, no obvious precipitate exists, the reaction system is in a clear and transparent state, and a large amount of pasty precipitate appears at the bottom of a shake flask after 5.5 hours, so that the generated L-2-aminobutyric acid is separated out when the concentration exceeds the solubility. As shown in FIG. 6, when compared with FIG. 10, the upper colored band is the product L-2-aminobutyric acid, and the lower colored band is the substrate threonine. From left to right are: reaction samples; substrate threonine control; the plaque was free of substrate at 20 hours.

Example 11 bioconversion reactions

In a 100ml triangular flask, 7.9ml pure water, 1.2g feed grade threonine (content > 98%), 0.5ml industrial isopropanol were added, a small amount of 25% ammonia (w/w) was added to adjust the pH to 8.0, then the final concentration of 0.2mM NAD crude, 0.05mg PLP were added and mixed well. Finally, 40. mu.l TD223, 33. mu.l leucine dehydrogenase LD161, and 33. mu.l alcohol dehydrogenase AD151 were added, and the mixture was reacted at 50 ℃ with a shaker speed of 180 rpm. The total system is about 10ml, and the substrate concentration is about 120 g/L. After 3 hours of reaction, 0.3ml of isopropanol was added. And (3) sampling for 10 hours, and detecting by a liquid phase, wherein the concentration of the L-2-aminobutyric acid is about 97 g/L.

Example 12 bioconversion reactions

In a 100ml triangular flask, 7.2ml pure water, 1.5g feed grade threonine (content > 98%), 1.2ml industrial isopropanol were added, a small amount of 25% ammonia (w/w) was taken to adjust the pH to 8.9, then the final concentration of 0.15mM NAD crude, 0.75mg PLP were added and mixed well. Finally, 90. mu.l TD223, 75. mu.l leucine dehydrogenase LD161, and 75. mu.l alcohol dehydrogenase AD151 were added, and the mixture was reacted at 50 ℃ with a shaker speed of 180 rpm. The total system is about 10ml, and the substrate concentration is about 150 g/L. After reaction for 6 hours and 12 hours, sampling and detecting, the results show that the concentrations of the L-2-aminobutyric acid are 109g/L and 123g/L respectively. FIG. 7 is a 12-hour HPLC chromatogram for the high-temperature biocatalytic preparation of L-2-aminobutyric acid in the present example, and FIG. 9 is a liquid phase chromatogram of a substrate, a product, and an intermediate standard. As can be seen in FIG. 9, 2.671 min is the substrate threonine; 3.151 min to obtain the product L-2-aminobutyric acid; 7.529 min gave the intermediate tetronic acid. Comparing FIG. 9, it can be seen from FIG. 7 that 3.146 min is the product L-2-aminobutyric acid; the peaks of the substrate threonine and the intermediate tetronic acid are not obviously detected; after reacting for 12 hours, the substrate and the intermediate are basically converted into the L-2-aminobutyric acid.

Example 13 bioconversion reactions

In a 100ml triangular flask, 7.2ml pure water, 1.5g feed grade threonine (content > 98%), 1.2ml industrial isopropanol were added, a small amount of 25% ammonia (w/w) was taken to adjust the pH to 8.9, then the final concentration of 0.15mM NAD crude, 0.75mg PLP were added and mixed well. Finally, 90. mu.l TD223wt, 75. mu.l leucine dehydrogenase LD161, and 75. mu.l alcohol dehydrogenase AD151 were added, and the mixture was reacted at 50 ℃ with a shaker speed of 180 rpm. The total system is about 10ml, and the substrate concentration is about 150 g/L. After 6 hours, 12 hours and 16 hours of reaction, sampling and detecting, the results show that the concentrations of the L-2-aminobutyric acid are 98g/L, 112g/L and 122g/L respectively. Comparing FIG. 9, it can be seen from FIG. 8 that 3.167 min is the product L-2-aminobutyric acid; the peaks of the substrate threonine and the intermediate tetronic acid are not obviously detected, and the substrate is basically and completely converted into the L-2-aminobutyric acid.

As can be seen from examples 12 and 13, the threonine deaminase mutants disclosed in the present example have higher enzyme-catalyzed conversion efficiency than the wild-type threonine deaminase derived from Chryseobacterium takakiae under the same high-temperature catalytic conditions.

Example 14 bioconversion reactions

In a 100ml triangular flask, 7.2ml pure water, 1.5g feed grade threonine (content > 98%), 1.2ml industrial isopropanol were added, a small amount of 25% ammonia (w/w) was taken to adjust the pH to 8.9, then the final concentration of 0.15mM NAD crude, 0.75mg PLP were added and mixed well. Finally, 90. mu.l of Escherichia coli-derived threonine deaminase, 75. mu.l of leucine dehydrogenase LD161 and 75. mu.l of alcohol dehydrogenase AD151 were added, and the mixture was reacted at 50 ℃ with a shaker speed of 180 rpm. The total system is about 10ml, and the substrate concentration is about 150 g/L. After the reaction is carried out for 6 hours and 12 hours, sampling detection is carried out, and the results show that the concentrations of the L-2-aminobutyric acid are 6g/L and 6g/L respectively. It was demonstrated that threonine dehydrogenase catalyzing the first-step reaction was rapidly inactivated under high temperature conditions, thereby stopping the subsequent catalytic reaction. Meanwhile, the threonine deaminase from escherichia coli is proved to be incapable of being effectively applied to high-temperature catalytic reaction.

EXAMPLE 15 production of amplification reaction System

14 tons of tap water and 2.7 tons of feed grade threonine (the content is more than 98 percent) are added into a 20 cubic reaction tank, 2.15 cubic isopropanol is used for adjusting the pH to 8.9 by 25 percent ammonia water (w/w), then 1.78 kilograms of NAD coenzyme and 0.3 kilogram of pyridoxal phosphate are added, and the temperature is controlled to 48-52 ℃ after uniform mixing. Finally 160 kg of TD223, 135 kg of leucine dehydrogenase LD161 and 135 kg of alcohol dehydrogenase AD151 are added, and finally a small amount of tap water is added until the volume is 18 cubic meters, and the stirring speed is set to be 30 r/min. The total system was about 18 cubic and the substrate concentration was about 150 g/L. Sampling and detecting after 10 hours of reaction, and the result shows that the concentration of the L-2-aminobutyric acid reaches 118 g/L. It can be seen that due to the high temperature reaction, even the coenzyme amount: the ratio of the substrate dosage is only 1:1500, and the product can still be effectively generated.

Example 16 bioconversion reactions

2100 ml triangular shake flasks were taken and added with 7.2ml pure water, 1.5g feed grade threonine (content > 98%), 1.2ml industrial isopropanol, a small amount of 25% ammonia (w/w) was taken to adjust the pH to 8.9, then the final concentration of 0.15mM NAD crude product, 0.75mg PLP were added and mixed well. Finally, 90. mu.l TD223, 75. mu.l leucine dehydrogenase LD161, and 75. mu.l alcohol dehydrogenase AD151 were added, and the mixture was reacted at 45 ℃ and 60 ℃ with a shaker speed of 180 rpm. The total system is about 10ml, and the substrate concentration is about 150 g/L. After the reaction for 9 hours, sampling detection is carried out, and the results show that the concentrations of the L-2-aminobutyric acid are 121g/L and 119g/L respectively.

Example 16 thin layer chromatography dot plate

The reaction solution (100. mu.l) was diluted with purified water by a certain ratio. Take 1. mu.l of supernatant TLC spot plate, developing n-butanol: acetic acid: water-4: 1: 1. The running board reaches 4/5, is taken out and dried, and is subjected to ninhydrin color development. The results are shown in FIG. 10, with the bottom band being the substrate and the top band being the product.

Example 17 HPLC detection conditions

A chromatographic column: c185 μm 250mm × 4.6 mm;

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

Flow rate: 0.7 mL/min;

detection wavelength: 210 nm;

column temperature: at 30 ℃.

The above description is only for the purpose of illustrating the present invention and is not intended to limit the scope of the present invention, and any person skilled in the art can substitute or change the technical solution of the present invention and its conception within the scope of the present invention.

SEQUENCE LISTING

<110> Nanjing Nuo cloud Biotechnology Ltd

<120> a thermostable threonine deaminase and use thereof

<130> 2020

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 1263

<212> DNA

<213> Artificial sequence

<400> 1

atgaacaaca ccctgacctt cccgaccctg gaatctatca tccaggctgg taaatctatc 60

gaaaacgttg ttaactacac cccgctgcag tacaacgctc gtctgtctga aaaattcggt 120

gctcacatct acctgaaacg tgaagacctg cagccggttc gttcttacaa actgcgtggt 180

gcttaccaca aaatcaaatc tctgttcaac gaaggtaaaa cctctgaagg tatcgtttgc 240

gcttctgctg gtaaccacgc tcagggtgtt gctttctctt gcaaacagct gcagatcaaa 300

ggtaccatct tcatgccggt taccaccccg aaacagaaac tggaacaggt tgaaatgttc 360

ggtggtcact tcgttgaaat caaactgttc ggtgacacct tcgacgcttc taaaaacgct 420

gctctggact tcgctgaaac cttcggtgct gctttcatcc acccgttcga cgacgttcag 480

atcatcgaag gtcaggctac cgttgctctg gaaatcctgg gtcagcagaa agaagctatg 540

gacttcgttt tcatcccgat cggtggtggt ggtctggctt ctggtatctc taccgttttc 600

aaagaactgt ctgctgaaac ccgtctgatc ggtgttgaac cgaaaggtgc tccgtctatg 660

aaaatctcta tcgaaaacaa aatcaacacc gaactgccgg aaatcgaccg tttcgttgac 720

ggtgctgctg ttaaaaaagt tggtgacctg accttcgaaa tctgccgtaa caccctgtct 780

gaatgcatct ctgttgacga aggtaaaatc tgcaacacca tcctgcagct gtacaacaaa 840

gacgctgttg ttctggaacc ggctggtgct ctgtctatct ctgctctgga ccagttccgt 900

aaccgtatca aaggtaaaaa cgttgtttgc atcgtttctg gttctaacaa cgacatcacc 960

cgtatggaag aaatcaaaga acgtgctctg ctgtacaacg gtctgaaaca ctacttcatg 1020

gttaaattcc cgcagcgtcc gggtgctctg aaagacttcg ttctgaacgt tctgggtgtt 1080

aacgacgaca tcacccactt cgaatacacc aaaaaaaact ctcgtgaaac cgctctggct 1140

atcgttggta tcgaactgtc tgacccgtct gacttcgaag gtctgcgtca gcgtatgcag 1200

gctctggact acctggaatc ttacctgaac gaaaacccgg acgttctgaa catgctggtt 1260

taa 1263

<210> 2

<211> 420

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<213> Chryseobacterium takakiae

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Met Asn Asn Thr Leu Thr Phe Pro Thr Leu Glu Ser Ile Ile Gln Ala

1 5 10 15

Gly Lys Ser Ile Glu Asn Val Val Asn Tyr Thr Pro Leu Gln Tyr Asn

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Ala Arg Leu Ser Glu Lys Phe Gly Ala His Ile Tyr Leu Lys Arg Glu

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Asp Leu Gln Pro Val Arg Ser Tyr Lys Leu Arg Gly Ala Tyr His Lys

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Ile Lys Ser Leu Phe Asn Glu Gly Lys Thr Ser Glu Gly Ile Val Cys

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Ala Ser Ala Gly Asn His Ala Gln Gly Val Ala Phe Ser Cys Lys Gln

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Leu Gln Ile Lys Gly Thr Ile Phe Met Pro Val Thr Thr Pro Lys Gln

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Lys Leu Glu Gln Val Glu Met Phe Gly Gly His Phe Val Glu Ile Lys

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Leu Phe Gly Asp Thr Phe Asp Ala Ser Lys Asn Ala Ala Leu Asp Phe

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Ala Glu Thr Phe Gly Ala Ala Phe Ile His Pro Phe Asp Asp Val Gln

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

165 170 175

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

180 185 190

Ala Ser Gly Ile Ser Thr Val Phe Lys Glu Leu Ser Ala Glu Thr Arg

195 200 205

Leu Ile Gly Val Glu Pro Lys Gly Ala Pro Ser Met Lys Ile Ser Ile

210 215 220

Glu Asn Lys Ile Asn Thr Glu Leu Pro Glu Ile Asp Arg Phe Val Asp

225 230 235 240

Gly Ala Ala Val Lys Lys Val Gly Asp Leu Thr Phe Glu Ile Cys Arg

245 250 255

Asn Thr Leu Ser Glu Cys Ile Ser Val Asp Glu Gly Lys Ile Cys Asn

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Thr Ile Leu Gln Leu Tyr Asn Lys Asp Ala Val Val Leu Glu Pro Ala

275 280 285

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

290 295 300

Gly Lys Asn Val Val Cys Ile Val Ser Gly Ser Asn Asn Asp Ile Thr

305 310 315 320

Arg Met Glu Glu Ile Lys Glu Arg Ala Leu Leu Tyr Asn Gly Leu Lys

325 330 335

His Tyr Phe Met Val Lys Phe Pro Gln Arg Pro Gly Ala Leu Lys Asp

340 345 350

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

355 360 365

Tyr Thr Lys Lys Asn Ser Arg Glu Thr Ala Leu Ala Ile Val Gly Ile

370 375 380

Glu Leu Ser Asp Pro Ser Asp Phe Glu Gly Leu Arg Gln Arg Met Gln

385 390 395 400

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

405 410 415

Asn Met Leu Val

420

<210> 3

<211> 514

<212> PRT

<213> Escherichia coli

<400> 3

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

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Leu Arg Ala Val Leu Arg Ala Pro Val Tyr Glu Ala Ala Gln Val Thr

20 25 30

Pro Leu Gln Lys Met Glu Lys Leu Ser Ser Arg Leu Asp Asn Val Ile

35 40 45

Leu Val Lys Arg Glu Asp Arg Gln Pro Val His Ser Phe Lys Leu Arg

50 55 60

Gly Ala Tyr Ala Met Met Ala Gly Leu Thr Glu Glu Gln Lys Ala His

65 70 75 80

Gly Val Ile Thr Ala Ser Ala Gly Asn His Ala Gln Gly Val Ala Phe

85 90 95

Ser Ser Ala Arg Leu Gly Val Lys Ala Leu Ile Val Met Pro Thr Ala

100 105 110

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

115 120 125

Leu Leu His Gly Ala Asn Phe Asp Glu Ala Lys Ala Lys Ala Ile Glu

130 135 140

Leu Ser Gln Gln Gln Gly Phe Thr Trp Val Pro Pro Phe Asp His Pro

145 150 155 160

Met Val Ile Ala Gly Gln Gly Thr Leu Ala Leu Glu Leu Leu Gln Gln

165 170 175

Asp Ala His Leu Asp Arg Val Phe Val Pro Val Gly Gly Gly Gly Leu

180 185 190

Ala Ala Gly Val Ala Val Leu Ile Lys Gln Leu Met Pro Gln Ile Lys

195 200 205

Val Ile Ala Val Glu Ala Glu Asp Ser Ala Cys Leu Lys Ala Ala Leu

210 215 220

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

225 230 235 240

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

245 250 255

Glu Tyr Leu Asp Asp Ile Ile Thr Val Asp Ser Asp Ala Ile Cys Ala

260 265 270

Ala Met Lys Asp Leu Phe Glu Asp Val Arg Ala Val Ala Glu Pro Ser

275 280 285

Gly Ala Leu Ala Leu Ala Gly Met Lys Lys Tyr Ile Ala Leu His Asn

290 295 300

Ile Arg Gly Glu Arg Leu Ala His Ile Leu Ser Gly Ala Asn Val Asn

305 310 315 320

Phe His Gly Leu Arg Tyr Val Ser Glu Arg Cys Glu Leu Gly Glu Gln

325 330 335

Arg Glu Ala Leu Leu Ala Val Thr Ile Pro Glu Glu Lys Gly Ser Phe

340 345 350

Leu Lys Phe Cys Gln Leu Leu Gly Gly Arg Ser Val Thr Glu Phe Asn

355 360 365

Tyr Arg Phe Ala Asp Ala Lys Asn Ala Cys Ile Phe Val Gly Val Arg

370 375 380

Leu Ser Arg Gly Leu Glu Glu Arg Lys Glu Ile Leu Gln Met Leu Asn

385 390 395 400

Asp Gly Gly Tyr Ser Val Val Asp Leu Ser Asp Asp Glu Met Ala Lys

405 410 415

Leu His Val Arg Tyr Met Val Gly Gly Arg Pro Ser His Pro Leu Gln

420 425 430

Glu Arg Leu Tyr Ser Phe Glu Phe Pro Glu Ser Pro Gly Ala Leu Leu

435 440 445

Arg Phe Leu Asn Thr Leu Gly Thr Tyr Trp Asn Ile Ser Leu Phe His

450 455 460

Tyr Arg Ser His Gly Thr Asp Tyr Gly Arg Val Leu Ala Ala Phe Glu

465 470 475 480

Leu Gly Asp His Glu Pro Asp Phe Glu Thr Arg Leu Asn Glu Leu Gly

485 490 495

Tyr Asp Cys His Asp Glu Thr Asn Asn Pro Ala Phe Arg Phe Phe Leu

500 505 510

Ala Gly

<210> 4

<211> 1263

<212> DNA

<213> Artificial sequence

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atgaacaaca ccctgacctt cccgaccctg gaatctatca tccaggctgg taaatctatc 60

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gctcacatct acctgaaacg tgaagacctg cagccggttc gttcttacaa actgcgtggt 180

gcttacaaca aaatcaaatc tctgttcaac gaaggtaaaa cctctgaagg tgttgtttgc 240

gcttctgctg gtaaccacgc tcagggtgtt gctttctctt gcaaacagct gcagatcaaa 300

ggtaccatct tcatgccggt taccaccccg aaacagaaac tggaacaggt tgaaatgttc 360

ggtggtcact tcgttgaaat caaactgttc ggtgacacct tcgacgcttc taaaaacgct 420

gctctggact tcgctgaaac cttcggtgct gctttcatcc acccgttcga cgacgttcag 480

atcatcgaag gtcaggctac cgttgctctg gaaatcctgg gtcagcagaa agaagctatg 540

gacttcgttt tcatcccgat cggtggtggt ggtctggctt ctggtatctc taccgttttc 600

aaatctctgt ctgctgaaac ccgtctgatc ggtgttgaac cgaaaggtgc tccgtctatg 660

aaaatctcta tcgaaaacaa aatcaacacc gaactgccgg aaatcgaccg tttcgttgac 720

ggtgctgctg ttaaaaaagt tggtgacctg accttcgaaa tctgccgtaa caccctgtct 780

gaatgcatct ctgttgacga aggtaaaatc tgcaacacca tcctgcagct gtacaacaaa 840

gacgctgttg ttctggaacc ggctggtgct ctgtctatct ctgctctgga ccagttccgt 900

aaccgtatca aaggtaaaaa cgttgtttgc atcgtttctg gttctaacaa cgacatcacc 960

cgtatggaag aaatcaaaga acgtgctctg ctgtacaacg gtctgaaaca ctacttcatg 1020

gttaaattcc cgcagcgtcc gggtgctctg aaagacttcg ttctgaacgt tctgggtgtt 1080

aacgacgaca tcacccactt cgaatacacc aaaaaaaact ctaaagaaac cgctctggct 1140

atcgttggta tcgaactgtc tgacccgtct gacttcgaag gtctgcgtca gcgtatgcag 1200

gctctggact acctggaatc ttacctgaac gaaaacccgg acgttctgaa catgctggtt 1260

taa 1263

<210> 5

<211> 420

<212> PRT

<213> Artificial sequence

<400> 5

Met Asn Asn Thr Leu Thr Phe Pro Thr Leu Glu Ser Ile Ile Gln Ala

1 5 10 15

Gly Lys Ser Ile Glu Asn Val Val Asn Tyr Thr Pro Leu Gln Tyr Asn

20 25 30

Ala Arg Leu Ser Glu Lys Phe Gly Ala His Ile Tyr Leu Lys Arg Glu

35 40 45

Asp Leu Gln Pro Val Arg Ser Tyr Lys Leu Arg Gly Ala Tyr Asn Lys

50 55 60

Ile Lys Ser Leu Phe Asn Glu Gly Lys Thr Ser Glu Gly Val Val Cys

65 70 75 80

Ala Ser Ala Gly Asn His Ala Gln Gly Val Ala Phe Ser Cys Lys Gln

85 90 95

Leu Gln Ile Lys Gly Thr Ile Phe Met Pro Val Thr Thr Pro Lys Gln

100 105 110

Lys Leu Glu Gln Val Glu Met Phe Gly Gly His Phe Val Glu Ile Lys

115 120 125

Leu Phe Gly Asp Thr Phe Asp Ala Ser Lys Asn Ala Ala Leu Asp Phe

130 135 140

Ala Glu Thr Phe Gly Ala Ala Phe Ile His Pro Phe Asp Asp Val Gln

145 150 155 160

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

165 170 175

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

180 185 190

Ala Ser Gly Ile Ser Thr Val Phe Lys Ser Leu Ser Ala Glu Thr Arg

195 200 205

Leu Ile Gly Val Glu Pro Lys Gly Ala Pro Ser Met Lys Ile Ser Ile

210 215 220

Glu Asn Lys Ile Asn Thr Glu Leu Pro Glu Ile Asp Arg Phe Val Asp

225 230 235 240

Gly Ala Ala Val Lys Lys Val Gly Asp Leu Thr Phe Glu Ile Cys Arg

245 250 255

Asn Thr Leu Ser Glu Cys Ile Ser Val Asp Glu Gly Lys Ile Cys Asn

260 265 270

Thr Ile Leu Gln Leu Tyr Asn Lys Asp Ala Val Val Leu Glu Pro Ala

275 280 285

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

290 295 300

Gly Lys Asn Val Val Cys Ile Val Ser Gly Ser Asn Asn Asp Ile Thr

305 310 315 320

Arg Met Glu Glu Ile Lys Glu Arg Ala Leu Leu Tyr Asn Gly Leu Lys

325 330 335

His Tyr Phe Met Val Lys Phe Pro Gln Arg Pro Gly Ala Leu Lys Asp

340 345 350

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

355 360 365

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

370 375 380

Glu Leu Ser Asp Pro Ser Asp Phe Glu Gly Leu Arg Gln Arg Met Gln

385 390 395 400

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

405 410 415

Asn Met Leu Val

420

Claims

1. A method for catalyzing L threonine to generate 2-ketobutyric acid and then generating L-2-aminobutyric acid, which comprises the steps of converting L threonine into 2-ketobutyric acid and then converting the L-ketobutyric acid into L-2-aminobutyric acid under the conditions of high temperature of 45-60 ℃ in the presence of threonine deaminase and leucine dehydrogenase and alcohol dehydrogenase; wherein, the threonine deaminase sequence is shown as SEQ ID NO. 2.