Leucine dehydrogenase mutants and their applications in the synthesis of non-natural α-amino acids

Abstract

The present invention discloses leucine dehydrogenase mutants and their applications in the synthesis of non-natural α-amino acids, belonging to the technical fields of enzyme engineering and microbial engineering. The kinetic parameters were determined using phenylglyoxylic acid as a substrate. The catalytic efficiency (k cat / K m ) of the double mutant T143G / L49G was 16.84 mM ‑1 ·s ‑1 , which was 2.8 times higher than that of the wild type. At high substrate concentrations, it could effectively catalyze the synthesis of L-phenylglycine from phenylglyoxylic acid, and the conversion rate could reach 99%. The kinetic parameters were determined using 2-oxobutyric acid as a substrate. The catalytic efficiency (k cat / K m ) of the double mutant T143L / V303L was 34.08 mM ‑1 ·s ‑1 , which was 13.4 times higher than that of the wild type. At high substrate concentrations, it could still effectively catalyze the synthesis of L-2-aminobutyric acid from 2-oxobutyric acid, and the conversion rate could reach 99%. On the basis of successfully improving the catalytic efficiency (k cat / K m ), the double mutants T143G / L49G and T143L / V303L in the present invention still maintained the good temperature stability of the wild-type strain and had more practical industrial application value.

Description

Technical Field

[0001] The present invention relates to leucine dehydrogenase mutants and their applications in the synthesis of non-natural α-amino acids, belonging to the technical fields of enzyme engineering and microbial engineering. Background Art

[0002] Chiral non-natural α-amino acids are widely used as building blocks and synthetic precursors in the synthesis of polypeptides, drug active ingredients, bioactive molecules, etc. For example, L-2-aminobutyric acid can be used as a precursor for the synthesis of the anticonvulsant drug Levetiracetam and the antituberculosis drug Ethambutol; L-tert-leucine can be used as a precursor for the synthesis of the antiretroviral drug Atazanavir; L-phenylglycine can be used for the synthesis of the polypeptide Pasireotide for treating Cushing's syndrome; L-homophenylalanine is a common intermediate for approximately 20 currently available antihypertensive drugs in the world, including Benazepril, Captopril, Delapril, and Imidapril. In addition, non-natural α-amino acids can be easily converted into other valuable compounds, such as the chiral β-amino alcohol molecular family. Therefore, the green and efficient synthesis of non-natural α-amino acids as building blocks and synthetic precursors has great practical application value.

[0003] The α-amino acid dehydrogenase family can catalyze the direct asymmetric reductive amination reaction of α-keto acid substrates. When coupled with a coenzyme recycling system, α-amino acid dehydrogenase can catalyze the formation of α-amino acids from α-keto acids in one step by only consuming inexpensive reducing agents, and its by-product is only water, which is very green, and the optical purity (ee value) of its product can reach 99%. Therefore, the asymmetric reductive amination reaction of α-keto acids catalyzed by α-amino acid dehydrogenase is the most promising method for producing chiral non-natural α-amino acids ( Figure 1 ).

[0004] Among the α-amino acid dehydrogenase family, glutamate dehydrogenase (GluDH) has strict substrate specificity and can only catalyze the formation of the corresponding L-glutamic acid from the α-ketoglutaric acid substrate with a carboxyl group in the side chain, and it is rarely used in the synthesis of non-natural α-amino acids; phenylalanine dehydrogenase (PheDH) can catalyze the preparation of α-phenylpyruvic acid and its derivatives with a benzene ring in the side chain to produce the corresponding L-phenylalanine and its homologues, but its substrate specificity is also limited to a certain range.

[0005] Leucine dehydrogenase (LeuDH, EC 1.4.1.9) can catalyze the direct asymmetric synthesis of chiral α-amino acids from a series of aliphatic and aromatic α-keto acids. Its substrate range is wider than that of glutamate dehydrogenase and phenylalanine dehydrogenase, and it is a potential enzyme for the industrial synthesis of non-natural α-amino acids. However, wild-type leucine dehydrogenase can only efficiently catalyze the synthesis of L-leucine from its natural substrate 4-methyl-2-oxopentanoic acid, and the synthesis efficiency of a series of non-natural α-amino acid products with industrial application value is greatly reduced.

[0006] Therefore, there is an urgent need to obtain leucine dehydrogenase mutants with higher synthesis efficiency for a series of chiral non-natural α-amino acids with industrial application value to solve the defects existing in the synthesis of existing non-natural α-amino acids. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a leucine dehydrogenase mutant (or several) with high synthesis efficiency for a series of non-natural α-amino acids with industrial application value.

[0008] The present invention provides a leucine dehydrogenase mutant, which is obtained by mutating the threonine at position 143 of the leucine dehydrogenase with the amino acid sequence shown in SEQ ID No. 1 into glycine, and is named mutant T143G;

[0009] Or, the mutant is obtained by mutating the threonine at position 143 of the leucine dehydrogenase with the amino acid sequence shown in SEQ ID No. 1 into glycine, and the leucine at position 49 into glycine. It is named mutant T143G / L49G;

[0010] Or, the mutant is obtained by mutating the threonine at position 143 of the leucine dehydrogenase with the amino acid sequence shown in SEQ ID No. 1 into leucine, and is named mutant T143L;

[0011] Or, the mutant is obtained by mutating the threonine at position 143 of the leucine dehydrogenase with the amino acid sequence shown in SEQ ID No. 1 into leucine, and the valine at position 303 into leucine. It is named mutant T143L / V303L.

[0012] The present invention also provides a gene encoding the above leucine dehydrogenase mutant.

[0013] The present invention also provides a vector carrying the above gene.

[0014] The present invention also provides a recombinant cell carrying the above gene or the above vector.

[0015] In one embodiment, the recombinant cell uses bacteria or fungi as host cells.

[0016] The present invention also provides a recombinant Escherichia coli expressing the above-mentioned leucine dehydrogenase mutant.

[0017] In one embodiment, Escherichia coli E. coli BL21(DE3) is used as the host, and pET28(+) is used as the expression vector.

[0018] The present invention also provides a method for catalytic synthesis of L-phenylglycine. The method uses phenylglyoxylic acid as the substrate and couples the above-mentioned mutant T143G or mutant T143G / L49G with glucose dehydrogenase to synthesize L-phenylglycine.

[0019] Alternatively, the method uses phenylglyoxylic acid as the substrate and couples the recombinant cell expressing mutant T143G or mutant T143G / L49G with the recombinant cell expressing glucose dehydrogenase to synthesize L-phenylglycine.

[0020] The present invention also provides a method for catalytic synthesis of L-2-aminobutyric acid. The method uses 2-oxobutyric acid as the substrate and couples the above-mentioned mutant T143L or mutant T143L / V303L with glucose dehydrogenase to synthesize L-2-aminobutyric acid.

[0021] Alternatively, the method uses 2-oxobutyric acid as the substrate and couples the recombinant cell expressing the above-mentioned mutant T143L or mutant T143L / V303L with the recombinant cell expressing glucose dehydrogenase to synthesize L-2-aminobutyric acid.

[0022] The present invention also provides the use of the above-mentioned leucine dehydrogenase mutant, or the above-mentioned gene, or the above-mentioned vector, or the above-mentioned recombinant cell, or the above-mentioned recombinant Escherichia coli in the preparation of non-natural α-amino acids.

[0023] In one embodiment, the non-natural α-amino acid includes L-2-aminobutyric acid or L-phenylglycine.

[0024] The present invention also provides the use of the above-mentioned leucine dehydrogenase mutant, or the above-mentioned gene, or the above-mentioned vector, or the above-mentioned recombinant cell, or the above-mentioned recombinant Escherichia coli in the pharmaceutical field.

[0025] Beneficial effects:

[0026] (1) When kinetic parameters were measured using phenylglyoxylic acid as the substrate, the catalytic efficiency (k cat / K m ) of the wild-type leucine dehydrogenase was 6.12 mM -1 ·s -1 , and the catalytic efficiency (k cat / K m) is 16.84 mM -1 ·s -1 , achieving a 2.8-fold increase in catalytic efficiency (k cat / K m ). The catalytic efficiency (k cat / K m ) of the single-point mutant T143G is 15.40 mM -1 ·s -1 , achieving a 2.5-fold increase in catalytic efficiency (k cat / K m ).

[0027] (2) When measuring the kinetic parameters using 2-oxobutyric acid as the substrate, the catalytic efficiency (k cat / K m ) of the wild-type leucine dehydrogenase is 2.54 mM -1 ·s -1 . The catalytic efficiency (k cat / K m ) of the double-point mutant T143L / V303L is 34.08 mM -1 ·s -1 , achieving a 13.4-fold increase in catalytic efficiency (k cat / K m ). The catalytic efficiency (k cat / K m ) of the single-point mutant T143L is 12.21 mM -1 ·s -1 , achieving a 4.8-fold increase in catalytic efficiency (k cat / K m ).

[0028] (3) Compared with the reported wild-type leucine dehydrogenase that catalyzes the synthesis of L-phenylglycine from phenylglyoxylic acid, the double-point mutant T143G / L49G obtained in the present invention can still effectively catalyze the synthesis of L-phenylglycine from phenylglyoxylic acid at a high substrate concentration (200 mmol / L), and the conversion rate can reach 99%. Under the same conditions, the substrate conversion rate of the wild-type when catalyzing phenylglyoxylic acid is only 84%.

[0029] (4) Compared with the reported wild-type leucine dehydrogenase that catalyzes the synthesis of L-2-aminobutyric acid from 2-oxobutyric acid, the double-point mutant T143L / V303L obtained in the present invention can still effectively catalyze the synthesis of L-2-aminobutyric acid from 2-oxobutyric acid at a high substrate concentration (300 mmol / L), and the conversion rate can reach 99%. Under the same conditions, the substrate conversion rate of the wild-type when catalyzing 2-oxobutyric acid is only 56%.

[0030] (5) The double-point mutants T143G / L49G and T143L / V303L obtained in the present invention, while successfully improving the catalytic efficiency (k cat / K m ), still maintain the good temperature stability of the wild-type strain and have more practical industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 : Flow chart of preparing α-amino acid from α-keto acid by coupling α-amino acid dehydrogenase with glucose dehydrogenase.

[0032] Figure 2 : Determination of the conversion rates of phenylglyoxylic acid and 2-oxobutyric acid catalyzed by the whole cells of mutants T143G / L49G and T143L / V303L.

[0033] Figure 3 : Determination of the ee value of L-phenylglycine synthesized by the whole cells of mutant T143G / L49G.

[0034] Figure 4 : Determination of the ee value of L-2-aminobutyric acid synthesized by the whole cells of mutant T143L / V303L. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0035] Escherichia coli E. coli BL21(DE3) involved in the following examples was purchased from Beina Biology, the plasmid pET-28a(+) was purchased from Novagen, glucose dehydrogenase (GluDH) was purchased from Merck, and NAD + , NADH were purchased from Solarbio Science & Technology Co., Ltd.

[0036] The media involved in the following examples are as follows:

[0037] LB liquid medium: Yeast extract 5.0 g·L -1 , Tryptone 10.0 g·L -1 , NaCl 10.0 g·L -1 , Kanamycin 50 mg·L -1 .

[0038] LB solid medium: Yeast extract 5.0 g·L -1 , Tryptone 10.0 g·L -1 , NaCl 10.0 g·L -1 , Agar powder 20 g·L -1 , Kanamycin 50 mg·L -1 .

[0039] Example 1: Preparation of the gene sequence of leucine dehydrogenase mutant

[0040] The specific steps are as follows:

[0041] Chemically synthesize the gene of leucine dehydrogenase with the encoded amino acid sequence as shown in SEQ ID No.1 (the nucleotide sequence of the gene is as shown in SEQ ID No.2), and clone it between the BamHⅠ and XhoⅠ restriction enzyme sites of the pET-28a(+) plasmid to obtain the recombinant plasmid pET28a-LeuDH. Transform the recombinant plasmid pET28a-LeuDH into Escherichia coli E.coli BL21 to obtain the recombinant Escherichia coli pET28a-LeuDH / E.coli BL21 (refer to the literature "Jing Li, Jiang Pan, Jie Zhang, et al. Stereoselective synthesis of l-tert-leucine by a newly cloned leucine dehydrogenase from Exiguobacterium sibiricum[J]. J MOL CATAL B-ENZYM, 2014, 105:11-17.").

[0042] Using the whole plasmid PCR technique, perform site-directed mutagenesis with the obtained recombinant plasmid pET28a-LeuDH as the template to obtain the mutant plasmids pET28a-T143G and pET28a-T143L. Then, using the mutant plasmids pET28a-T143G and pET28a-T143L as templates respectively, obtain the mutant plasmids T143G / L49G and T143L / V303L;

[0043] Among them, the primers used for the mutation T143G are as follows:

[0044] T143G-For: CATGGAAACAGACTTCGTAGSCGGTG (SEQ ID No.3);

[0045] T143G-Rev: CACCGSCTACGAAGTCTGTTTCCATG (SEQ ID No.4);

[0046] The primers used for the mutation L49G (T143G / L49G mutant) are as follows:

[0047] L49G-For: CGGACCAGCAGBWGGCGGACTCCGTATG (SEQ ID No.5);

[0048] L49G-Rev: CWVCTGCTGGTCCGAGTGTCGTATCATGAA (SEQ ID No.6);

[0049] The primers used for the mutation T143L are as follows:

[0050] T143L-For: CTTCGTABKKGGTGTCAGCCCGGCATTCGGAT (SEQ ID No.7);

[0051] T143L-Rev: TGACACCMMVTACGAAGTCTGTTTCCATGTG (SEQ ID No.8);

[0052] The primers used for the mutation V303L (T143L / V303L mutant) are as follows:

[0053] V303L-For: CATCAATBKKGCCGACGAACTCGACGGG (SEQ ID No.9);

[0054] V303L-Rev: CGTCGGCMMAATTGATGACACCTCCTGCG (SEQ ID No.10);

[0055] The PCR reaction was carried out in a 50 μL system. The reaction conditions were pre-denaturation at 94 °C for 4 min; then 30 cycles were entered: denaturation at 98 °C for 10 s, annealing at 55 °C for 5 s, extension at 72 °C for 8 min; finally, extension at 72 °C for 10 min and incubation at 4 °C.

[0056] The PCR amplification products were detected by 1% agarose gel electrophoresis. After the detection, 0.5 μL of methylation template digestion enzyme (Dpn I) was added to 10 μL of the amplification products, and they were mixed by pipetting. The reaction was carried out at 37 °C for 1.5 h. The amplification products treated with Dpn I were transformed into Escherichia coli E. coli BL21(DE3), and spread on LB solid medium. They were cultured at 37 °C for 8 - 10 h. 20 transformants were picked from the LB solid medium, inoculated into LB liquid medium for culture, and cultured at 37 °C for 10 h. Then the plasmids were extracted. When the sequencing was correct, the recombinant Escherichia coli pET28a-T143G / E. coli BL21, pET28a-T143L / E. coli BL21, T143G / L49G / E. coli BL21, T143L / V303L / E. coli BL21 containing the genes encoding the mutants T143G, T143G / L49G, T143L, and T143L / V303L were obtained.

[0057] Example 2: Inductive culture and protein purification of leucine dehydrogenase mutants

[0058] Using the leucine dehydrogenase with the amino acid sequence shown in SEQ ID No.1 as the wild type, the obtained recombinant Escherichia coli pET28a-LeuDH / E.coli BL21 and recombinant Escherichia coli pET28a-T143G / E.coli BL21, pET28a-T143L / E.coli BL21, T143G / L49G / E.coli BL21, T143L / V303L / E.coli BL21 were respectively spread on LB solid medium and cultured at 37°C for 8 - 10 h to obtain single colonies; single colonies were picked and inoculated into LB liquid medium and cultured at 37°C, 200 rpm for 6 - 8 h to obtain seed liquid; the seed liquid was inoculated into LB liquid medium at an inoculation amount of 2% (v / v), cultured at 37°C, 200 rpm for 2 - 3 h, then IPTG with a final concentration of 0.1 mmol / L was added, and the induction culture was continued at 17°C, 200 rpm for 12 - 17 h to obtain fermentation broth; the fermentation broth was centrifuged at 4°C, 8000 rpm for 5 min, the supernatant was discarded, and the precipitate was washed twice with 9% normal saline to obtain the wet cells of the wild type, mutant T143G, T143G / L49G, T143L, and T143L / V303L.

[0059] The wet cells were resuspended in buffer A (100 mmol / L Tris, 150 mmol / L NaCl, 20 mmol / L imidazole, pH 7.5) and ultrasonically disrupted, then centrifuged at 10000 rpm, 4°C for 30 min to obtain crude enzyme solution; after filtering with a 0.22 μm aqueous filter membrane, the sample was slowly loaded onto a Ni-NAT affinity chromatography column. After loading, it was first washed with buffer A and then gradient eluted with buffer B (100 mmol / L Tris, 150 mmol / L NaCl, 500 mmol / L imidazole, pH 7.5), and the elution peak corresponding to 300 mmol / L imidazole was collected to obtain the pure enzymes of the wild type, mutant T143G, T143G / L49G, T143L, and T143L / V303L. Subsequently, the imidazole in the pure enzyme was removed with a desalting column, and centrifugal concentration was carried out at 4000 rpm with an ultrafiltration tube (molecular weight cut-off 30 kDa) to obtain the concentrated pure enzymes of the wild type, mutant T143G, T143G / L49G, T143L, and T143L / V303L.

[0060] Example 3: Catalytic efficiency of different leucine dehydrogenase mutants on phenylglyoxylic acid

[0061] In an NH4Cl-NH4OH buffer solution (1 mol / L, pH 9.0), add mandelic acid (0.1 - 25 mmol / L) and NADH (0.5 mmol / L) to obtain a reaction system; incubate the reaction system at 30 °C for 2 min, then add 20 μl of the concentrated pure enzymes of the wild type, mutant T143G, and T143G / L49G obtained in Example 2 to start the reaction. The control group does not contain the concentrated enzyme solution, and other components are the same; allow the reaction to proceed at 30 °C for 5 min, record the absorbance change at 340 nm every 10 s, and obtain the catalytic activities of the wild type, mutant T143L, and T143L / V303L towards mandelic acid.

[0062] Catalytic activity (U / mg) = Ew × V / 6220 / L × protein concentration of the concentrated enzyme solution; where Ew is the absorbance change value at 340 nm within 1 min; V is the volume of the enzyme activity assay reaction system, in mL, which is 0.2 here; 6200 is the molar extinction coefficient, in L / mol / cm; L is the optical path length, in cm, which is 0.5 here; the protein concentration of the concentrated enzyme solution is measured using a Bradford protein assay kit (for measuring protein concentration with a Bradford protein assay kit, see the reference: Zhou-Pan X R, E Sérée, Zhou X J, et al. Involvement of Human Liver Cytochrome P450 3A in Vinblastine Metabolism: Drug Interactions1[J]. Cancer Research, 1993, 53(21): 5121 - 5126.), in mg / mL. Use Origin software to perform non-linear fitting on the calculated catalytic activity and the corresponding substrate concentration to obtain the catalytic efficiency (k cat / K m ) of the enzyme.

[0063] The results are as follows: The catalytic efficiency (k cat / K m ) of the wild type towards mandelic acid is 6.12 mM -1 ·s -1 ; the catalytic efficiency (k cat / K m ) of the single-point mutant T143G towards mandelic acid is 15.40 mM -1 ·s -1 ; the catalytic efficiency (k cat / K m ) of the double-point mutant T143G / L49G towards mandelic acid is 16.84 mM -1 ·s -1 .

[0064] Example 4: Catalytic efficiency of different leucine dehydrogenase mutants towards 2-oxobutyric acid

[0065] 2-Oxobutyric acid (0.1 - 25 mmol / L) and NADH (0.5 mmol / L) were added to an NH4Cl-NH4OH buffer (1 mol / L, pH 9.0) to obtain a reaction system; after incubating the reaction system at 30 °C for 2 min, 20 μL of the concentrated pure enzymes of the wild type, mutant T143L, and T143L / V303L obtained in Example 2 were added to start the reaction. The control group did not contain the concentrated enzyme solution, and other components were the same; the reaction was carried out at 30 °C for 5 min, and the absorbance change at 340 nm was recorded every 10 s to obtain the catalytic activities of the wild type, mutant T143L, and T143L / V303L towards 2-oxobutyric acid.

[0066] Catalytic activity (U / mg) = Ew×V / 6220 / L×protein concentration of the concentrated enzyme solution; where Ew is the absorbance change value at 340 nm within 1 min; V is the volume of the enzyme activity assay reaction system, in mL, which is 0.2 here; 6200 is the molar extinction coefficient, in L / mol / cm; L is the optical path length, in cm, which is 0.5 here; the protein concentration of the concentrated enzyme solution was measured using a Bradford protein assay kit, in mg / mL. The Origin software was used to perform non-linear fitting on the calculated catalytic activities and the corresponding substrate concentrations to obtain the catalytic efficiency (k cat / K m ) of the enzyme.

[0067] The results were as follows: The catalytic efficiency (k cat / K m ) of the wild type towards 2-oxobutyric acid was 2.54 mM -1 ·s -1 ; the catalytic efficiency (k cat / K m ) of the single mutant T143L towards 2-oxobutyric acid was 12.21 mM -1 ·s -1 ; the catalytic efficiency (k cat / K m ) of the double mutant T143L / V303L towards 2-oxobutyric acid was 34.08 mM -1 ·s -1 .

[0068] Example 5: Thermal stability of different leucine dehydrogenase mutants

[0069] The wild-type, double-point mutants T143G / L49G and T143L / V303L concentrated enzyme solutions obtained in Example 2 were incubated in a water bath at a temperature range of 30 - 70 °C for 30 min. The catalytic activities of the wild-type, mutant T143G / L49G, and T143L / V303L towards 2-oxobutyric acid were measured at 30 °C after incubation for 30 min. Taking the activity before incubation at different temperatures as 100%, the relative activity was calculated by comparing with the remaining activity after incubation to investigate the temperature stability of the wild-type, double-point mutants T143G / L49G, and T143L / V303L.

[0070] The results were as follows: The T 50 30 (temperature corresponding to the activity after 30 min of incubation being half of the activity before incubation) values of mutants T143G / L49G and T143L / V303L were 60 °C and 59 °C respectively, and the T 50 15 value of the wild-type under the same conditions was 62 °C. It can be seen that mutants T143G / L49G and T143L / V303L still maintain excellent temperature stability on the basis of improving the catalytic efficiency.

[0071] Example 6: Leucine dehydrogenase mutant T143G / L49G does not catalyze the reductive amination to synthesize L-phenylglycine

[0072] Leucine dehydrogenase mutant T143G / L49G is used for the synthesis of L-phenylglycine by coupling with glucose dehydrogenase (GluDH).

[0073] The volume of the reaction system was 200 ml, which contained the wet cells of T143L / V303L (4 g) obtained in Example 2, wet cells of GluDH (4.8 g), phenylglyoxylate (200 mmol / L), glucose (240 mmol / L), NAD + (0.1 mmol / L), NH4Cl - NH4OH buffer solution (2 M, pH 8.5).

[0074] The reaction mixture was reacted at 30 °C and 200 rpm. Samples (1 mL) were taken regularly during the reaction, and the samples were quenched with NaOH aqueous solution (200 μL, 10 M).

[0075] 600 μL of the quenched sample was taken, and the concentration of the remaining phenylglyoxylate in the reaction system was measured by liquid chromatography to calculate the substrate conversion rate. The measurement conditions were as follows: Diamonsil C18(2) liquid chromatography column (5 μm; 250 mm × 4.6 mm); injection volume, 10 μL; mobile phase A (55% methanol plus 0.1% trifluoroacetic acid), 20 min at 30 °C; flow rate, 0.8 mL / min; wavelength 230 nm.

[0076] Take 600 μL of the quenched sample, determine the configuration and concentration of the product phenylglycine in the reaction system by liquid chromatography, and calculate the ee value of the product. Determination conditions: ODS-UG-5 liquid chromatography column (5 μm; 150 mm × 4.6 mm); injection volume, 20 μL; mobile phase B (5% acetonitrile plus 0.1% trifluoroacetic acid), mobile phase C (60% acetonitrile plus 0.1% trifluoroacetic acid), linear gradient 0 - 100% C; 45 min at 30 °C; flow rate, 1 mL / min; wavelength 340 nm.

[0077] The results were as follows: The substrate conversion rate of T143G / L49G catalyzing 200 mmol / L phenylglyoxylic acid could reach 99% ( Figure 2 ), and the ee value of the product L-phenylglycine could reach 99% ( Figure 3 ). Under the same conditions, the substrate conversion rates of the wild type and the mutant T143G catalyzing 200 mmol / L phenylglyoxylic acid were only 84% and 89%, respectively.

[0078] Example 7: Asymmetric reductive amination of leucine dehydrogenase mutant T143L / V303L to synthesize L-2-aminobutyric acid

[0079] The leucine dehydrogenase mutant T143L / V303L was used for the synthesis of L-2-aminobutyric acid by coupling with glucose dehydrogenase (GluDH).

[0080] The volume of the reaction system was 200 ml, which contained the wet cells of T143L / V303L (4 g) obtained in Example 2, the wet cells of GluDH (4.8 g), 2-oxobutyric acid (300 mmol / L), glucose (360 mmol / L), NAD + (0.1 mmol / L), NH4Cl-NH4OH buffer solution (2 M, pH 8.5).

[0081] The reaction mixture was reacted at 30 °C and 200 rpm. Samples (1 mL) were taken regularly during the reaction, and the samples were quenched with NaOH aqueous solution (200 μL, 10 M).

[0082] Take 600 μL of the quenched sample, determine the concentration of the remaining 2-oxobutyric acid in the reaction system by liquid chromatography, and calculate the substrate conversion rate. Determination conditions: Diamonsil C18(2) liquid chromatography column (5 μm; 250 mm × 4.6 mm); injection volume, 10 μL; mobile phase A (55% methanol plus 0.1% trifluoroacetic acid), 20 min at 30 °C; flow rate, 0.8 mL / min; wavelength 230 nm.

[0083] Take 600 μL of the quenched sample, and determine the configuration and concentration of the product 2-aminobutyric acid in the reaction system by liquid chromatography, and calculate the ee value of the product. The determination conditions are as follows: ODS-UG-5 liquid chromatography column (5 μm; 150 mm × 4.6 mm); injection volume, 20 μL; mobile phase B (5% acetonitrile plus 0.1% trifluoroacetic acid), mobile phase C (60% acetonitrile plus 0.1% trifluoroacetic acid), linear gradient is 0-100% C; 45 min at 30 °C; flow rate, 1 mL / min; wavelength 340 nm.

[0084] The results are as follows: When T143L / V303L catalyzes 300 mmol / L 2-oxobutyric acid, the substrate conversion rate can reach 99% ( Figure 2 ), and the ee value of the product L-2-aminobutyric acid can reach 99% ( Figure 4 ). Under the same conditions, the substrate conversion rates of the wild type and the mutant T143L when catalyzing 300 mmol / L 2-oxobutyric acid are only 56% and 77%, respectively.

[0085] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A leucine dehydrogenase mutant, characterized in that, The mutant is obtained by mutating the threonine at position 143 and the leucine at position 49 of the leucine dehydrogenase with the amino acid sequence shown in SEQ ID No. 1 into glycine, and is named mutant T143G / L49G.

2. A gene encoding the leucine dehydrogenase mutant according to claim 1.

3. A vector carrying the gene according to claim 2.

4. A recombinant microbial cell carrying the gene according to claim 2 or the vector according to claim 3.

5. A recombinant Escherichia coli, characterized in that, Express the leucine dehydrogenase mutant according to claim 1.

6. The recombinant Escherichia coli according to claim 5, wherein Using Escherichia coli E. coli BL21(DE3) as the host and pET28(+) as the expression vector.

7. A method for catalytic synthesis of L-phenylglycine, characterized in that, The method is to use the mutant T143G / L49G according to claim 1 to couple with glucose dehydrogenase to synthesize L-phenylglycine using phenylglyoxylic acid as a substrate; Alternatively, the method is to use a recombinant cell expressing the mutant T143G / L49G according to claim 1 to couple with a recombinant cell expressing glucose dehydrogenase to synthesize L-phenylglycine using phenylglyoxylic acid as a substrate.

8. Use of the leucine dehydrogenase mutant according to claim 1, or the gene according to claim 2, or the vector according to claim 3, or the recombinant microbial cell according to claim 4, or the recombinant Escherichia coli according to claim 5 or 6 in the preparation of L-phenylalanine.

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