A membrane-bound L-amino acid deaminase mutant and its application

Abstract

The present invention discloses a membrane-bound L-amino acid deaminase mutant and its application. The membrane-bound L-amino acid deaminase mutant is obtained by mutating the wild-type PM1 enzyme derived from Proteus mirabilis. The amino acid sequence of the wild-type PM1 enzyme is shown in SEQ ID No. 1, and the mutation mode is any one of the following: (1) G259W; (2) D340N. The PM1 enzyme mutant of the present invention is obtained by mutating the wild-type PM1 enzyme derived from Proteus mirabilis. The turnover numbers of the whole cells of the two mutants G259W and D340N for catalyzing L-phenylalanine are increased by 1.3 times and 1.2 times respectively compared with that of the WT.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a membrane-bound L-amino acid deaminase mutant and its application. Background Art

[0002] α-Phenylpyruvic acid (PPA) is an important metabolic intermediate of phenylalanine in organisms. It is widely used in the pharmaceutical, food, and chemical industries. It is a key precursor for the biological preparation of D-phenylalanine, and also a precursor for the preparation of chiral drugs and the common food sweetener aspartame. Additionally, phenylpyruvic acid can be hydrogenated and reduced to obtain the natural antibacterial substance 3-phenyllactic acid (PLA).

[0003] Currently, the main chemical synthesis methods of PPA include the hydrolysis method of α-acetamidocinnamic acid, the hydantoin method, the dicarbonylation method, and the amino acid biotransformation method. The chemical synthesis method of PPA has low yield and is prone to produce toxic and harmful products.

[0004] Microbial fermentation mainly relies on the metabolism within the cells to synthesize PPA. Since the metabolic pathway for synthesizing PPA within the cells is long and the enzyme activity is not high, it takes a long time, and the yield and synthesis efficiency are low, which is not conducive to industrial production.

[0005] In contrast, using inexpensive L-phenylalanine as a raw material, the preparation of PPA by biotransformation has the characteristics of mild reaction conditions and environmental friendliness.

[0006] Currently, the main enzymes capable of biotransforming and synthesizing α-keto acids are: aminotransferase (AT), amino acid dehydrogenases (ADH), L-amino acid oxidase (L-AAO), and membrane-bound L-amino acid deaminase (mL-AAD).

[0007] Among the above four enzymes, the reaction catalyzed by AT is reversible. It requires the additional addition of the coenzyme factor pyridoxal phosphate (PLP) and another α-keto acid as the amino acceptor, resulting in a high cost and being unsuitable for industrial production.

[0008] Similarly, ADH also catalyzes a reversible reaction, and the reaction requires the coenzyme factor NADP +, but for cost considerations, a coenzyme regeneration system (adding NADPH oxidase) is often selected. However, the optimal pH of NADPH oxidase and ADH differ significantly. The optimal pH of NADPH oxidase is 6.0 - 7.5, while the optimal pH for ADH catalysis is greater than 10. Therefore, neither of them can work at their optimal pH, resulting in low catalytic efficiency and being unsuitable for industrial production.

[0009] The catalytic reactions of L-AAO and mL-AAD are both irreversible reactions with relatively high conversion rates. However, when L-AAO catalyzes the production of α-keto acids, by-products NH3 and H2O2 are generated. A large amount of H2O2 not only has a toxic effect on cells, affecting the activity of L-AAO, but also degrades the generated α-keto acids. Therefore, catalase is often added to the system to digest H2O2, so the cost increases. mL-AAD does not require the addition of coenzyme factors or extra amino group acceptors during catalysis, does not produce H2O2, does not degrade the product, and is also easier to heterologously express. Therefore, mL-AAD is the first choice enzyme for preparing PPA. Therefore, improving the catalytic activity of mL-AAD towards L-phenylalanine has important practical significance for the preparation of PPA by biological methods. Summary of the Invention

[0010] The present invention provides two mutants of the enzyme Proteus mirabilis mL-AAD (PM1). These two enzymes have higher enzyme activity than the wild-type PM1 and can more efficiently catalyze the synthesis of α-phenylpyruvic acid.

[0011] The present invention first provides a membrane-bound L-amino acid deaminase mutant, which is obtained by mutating the wild-type PM1 enzyme derived from Proteus mirabilis. The amino acid sequence of the wild-type PM1 enzyme is shown in SEQ ID No.1, and the mutation method is any one of the following:

[0012] (1) G259W;

[0013] (2) D340N.

[0014] The present invention also provides a gene encoding the membrane-bound L-amino acid deaminase mutant of claim 1. Preferably, the gene sequence when the mutation site is G259W is shown in SEQ ID No.3; the gene sequence when the mutation site is D340N is shown in SEQ ID No.4.

[0015] The present invention also provides a recombinant plasmid containing the said gene.

[0016] The present invention also provides a genetically engineered cell containing the said gene. Preferably, the genetically engineered cell includes a host cell and the said gene introduced into the host cell, and the host cell is Escherichia coli for protein expression. For example, the host cell can be Escherichia coli BL21(DE3).

[0017] The present invention also provides the application of the said membrane-bound L-amino acid deaminase mutant in catalyzing the production of α-ketophenylpyruvic acid from L-phenylalanine.

[0018] The present invention also provides the application of the said genetically engineered cell in catalyzing the production of α-ketophenylpyruvic acid from L-phenylalanine.

[0019] The present invention also provides a method for catalyzing the production of α-ketophenylpyruvic acid from L-phenylalanine, comprising the following steps: using L-phenylalanine as a substrate and adding the said membrane-bound L-amino acid deaminase mutant to carry out a catalytic reaction to produce α-ketophenylpyruvic acid.

[0020] The present invention also provides a method for catalyzing the production of α-ketophenylpyruvic acid from L-phenylalanine, comprising the following steps: using L-phenylalanine as a substrate and adding the said genetically engineered cell to carry out a catalytic reaction to produce α-ketophenylpyruvic acid.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] The PM1 enzyme mutant of the present invention is obtained by mutating the wild-type PM1 enzyme derived from Proteus mirabilis, and the turnover numbers of the whole-cell catalysis of L-phenylalanine by the two mutants G259W and D340N are increased by 1.3 times and 1.2 times respectively compared with that of WT. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a schematic diagram of the deamination reaction of the membrane-bound L-amino acid deaminase of the present invention, wherein mL-ADD refers to the membrane-bound L-amino acid deaminase. DETAILED DESCRIPTION OF THE INVENTION

[0024] Based on PM1 (EC 1.4.3.2) of Proteus mirabilis, the present invention introduces mutations at sites 259 or 340 in the substrate pocket domain by site-directed mutagenesis PCR to obtain mutants G259W and D340N with better catalytic efficiency for L-phenylalanine than WT.

[0025] The amino acid sequence of wild-type membrane-bound L-amino acid deaminase (PM1) is shown in SEQ ID No. 1. The nucleotide sequence encoding wild-type membrane-bound L-amino acid deaminase (PM1) is shown in SEQ ID No. 2, the nucleotide sequence encoding the G259W mutant is shown in SEQ ID No. 3, and the nucleotide sequence encoding the D340N mutant is shown in SEQ ID No. 4.

[0026] Figure 1 This is a schematic diagram of the deamination reaction of the membrane-bound L-amino acid deaminase of the present invention.

[0027] Example 1: Construction of mutants

[0028] I. Obtaining site-directed mutagenesis PCR products.

[0029] According to the PM1 gene sequence (GenBank: ACD36582.1), this gene sequence was synthesized by Shanghai Sangon Biotech Co., Ltd. and inserted into the Nco I and BamH I sites of plasmid pET28a to obtain the pET28a-mlaad plasmid. Using this plasmid as a template, site-directed PCR amplification was carried out. The PCR amplification used TransStart FastPfu DNA Polymerase (Catalog number: AP221-01). FastPfu DNA Polymerase (Catalog number: AP221-01).

[0030] The PCR reaction system was: 25 μL of 2×PCR Buffer, 2 μL of upstream primer (10 mmol / L), 2 μL of downstream primer (10 mmol / L), 1 μL of plasmid template (50 ng / μL), 1 μL of FastPfu DNA polymerase, and sterile ultrapure water was added to make up the total volume to 50 μL.

[0031] The PCR program was as follows: 94°C, 5 min; (94°C, 30 s; 50°C, 30 s; 72°C, 4 min) × 25 cycles; 72°C, 5 min; finally cooled to 4°C for storage.

[0032] The primers used for G259W mutation were:

[0033] Upstream primer: 5′-TGTCGCCGGTGGTGTT T GGTCACGTTT-3′;

[0034] Downstream primer: 5′-AAACGTGACC A AACACCACCGGCGACA-3′,

[0035] The primers used for D340N mutation were:

[0036] 5′-CCTCTGCTGGCTTTACCT A ATTTCCCAGT-3′;

[0037] 5′-ACTGGGAAAT T AGGTAAAGCCAGCAGAGG-3′,

[0038] Among them, the underlined letters represent the substituted bases.

[0039] II. Transformation and verification of site-directed mutagenesis PCR products.

[0040] Digest the obtained site-directed mutagenesis PCR products with DMT enzyme to recognize and cut adenine methylation. The digestion system is as follows: 50 μL of PCR products, 1 μL of DMT. After gently pipetting and mixing, centrifuge briefly to collect at the bottom of the tube, and place it in a constant temperature reaction at 37°C for 1.5 h.

[0041] Transform the digested products into Escherichia coli BL21(DE3) by the CaCl2 method. The specific operation is as follows:

[0042] (1) Take 100 μL of competent cell suspension from the -70°C refrigerator and thaw it on ice;

[0043] (2) Add 5 μL of site-directed mutagenesis PCR digestion products, mix gently, and let it stand on ice for 1 h;

[0044] (3) Heat shock in a 42°C water bath for 60 s, and then immediately place it on ice for 2 min;

[0045] (4) Add 400 μL of LB medium preheated in a 42°C water bath, mix well, and culture at 37°C with shaking at 200 r min -1 for 1 h to resuscitate the cells;

[0046] (5) Centrifuge at 4000 r min -1 for 30 s, discard a part of the supernatant, shake the remaining bacterial liquid well, take an appropriate amount and spread it on an LB plate containing 50 μg / mL kanamycin, place it face up for half an hour, wait for the bacterial liquid to be absorbed by the medium, then invert the plate and culture it overnight at 37°C.

[0047] (6) After the transformants grow out, randomly select several clones, extract the plasmids, and perform full-automatic DNA sequencing to confirm that the expected mutation sites 259 have successfully mutated from G to W and the 340th site has successfully mutated from D to N.

[0048] Example 2: Determination of the whole-cell catalytic performance of WT and mutants

[0049] Pick single colonies of wild type and mutant enzymes from the LB solid medium and inoculate them into 5 mL of LB liquid medium containing 50 μg / mL kanamycin antibiotic. Incubate overnight at 37 °C and 200 rpm. Inoculate the seed liquid into the LB medium containing kanamycin antibiotic for scale-up culture at an inoculation amount of 1%. Incubate at 37 °C and 200 rpm until OD 600 When it reaches 0.6 - 0.8, add IPTG with a final concentration of 0.5 μM and incubate at 30 °C and 150 rpm for 10 h to induce protein expression. Wash 1 mL of the induced bacterial cells twice with phosphate buffer at pH = 7.2, and resuspend the collected bacterial cells in the same buffer. Take 1 mL of the suspension and add 1 mL of 0.1 mol / L L-phenylalanine solution. After mixing, react at 37 °C and 200 rpm for 2 h. After the reaction, add 100 μL of the whole-cell catalytic product to 90 μL of 20% TCA solution to terminate the reaction. Determine the concentration of PPA generated by the method of color development with 2,4-dinitrophenylhydrazine. Under the same treatment conditions, after reacting for 10 h, the yields of mutant enzymes G259W and D340N are 1.218 mg / L and 1.125 mg / L respectively, which are higher than the yield of wild type PM1 (1.00 mg / L).

[0050] Example 3: Determination of kinetic parameters of mutant enzymes

[0051] Determine the initial reaction rate at different substrate concentrations (0.1 - 0.001 mM).

[0052] Using phenylalanine as the substrate, under the conditions of pH 7.2 and 37 °C, determine the Michaelis kinetic parameters of the whole-cell catalysts of wild type enzyme, mutant enzyme G259W and mutant enzyme D340N expression strains respectively. Determine the catalytic rate under the condition of substrate concentration of 0.1 - 0.001 mM. Perform non-linear regression on the Michaelis-Menten equation to determine the apparent kinetic parameters. The calculation results are shown in Table 1.

[0053] Table 1

[0054] mutation <![CDATA[K m (mM)]]> <![CDATA[K cat (s -1 )]]> <![CDATA[K cat / K m (s -1 mM -1 )]]> WT 11.916 0.120 0.010 G259W 10.541 0.135 0.013 D340N 9.426 0.110 0.012

[0055] K m Represents the affinity between the enzyme and the substrate.

[0056] K cat Refers to the turnover number or catalytic constant, which represents the number of substrate molecules converted per second per molecule of enzyme or each enzyme active center when the enzyme is saturated with the substrate. K cat The larger the value, the higher the catalytic efficiency of the enzyme.

[0057] K cat / K mis the apparent second-order rate constant for the reaction of an enzyme with a substrate, and its magnitude can be used to compare the catalytic efficiency of enzymes.

[0058] As can be seen from the results in Table 1, the K cat / K m of mutants G259W and D340N are 1.3 and 1.2 times that of WT, respectively, indicating that the catalytic efficiencies of mutant enzymes G259W and D340N are greater than that of wild-type PM1.

Claims

1. A membrane-bound L-amino acid deaminase mutant, characterized in that, It is obtained by mutating the wild-type membrane-bound L-amino acid deaminase PM1 derived from Proteus mirabilis. The amino acid sequence of the wild-type PM1 enzyme is shown in SEQ ID No. 1, and the mutation method is any one of the following: (1) G259W; (2) D340N.

2. A gene encoding the membrane-bound L-amino acid deaminase mutant according to claim 1.

3. The gene according to claim 2, wherein When the mutation site is G259W, the gene sequence is shown in SEQ ID No. 3; When the mutation site is D340N, the gene sequence is shown in SEQ ID No.

4.

4. A recombinant plasmid containing the gene according to claim 2 or 3.

5. A genetically engineered cell containing the gene according to claim 2 or 3; The genetically engineered cell includes a host cell and the gene introduced into the host cell, and the host cell is Escherichia coli for protein expression.

6. Use of the membrane-bound L-amino acid deaminase mutant according to claim 1 in catalyzing the production of α-ketophenylpyruvic acid from L-phenylalanine.

7. Use of the genetically engineered cell according to claim 5 in catalyzing the production of α-ketophenylpyruvic acid from L-phenylalanine.

8. A method for catalyzing the formation of α-phenylpyruvic acid from L-phenylalanine, characterized in that, It includes the following steps: Using L-phenylalanine as a substrate, adding the membrane-bound L-amino acid deaminase mutant according to claim 1 for a catalytic reaction to produce α-ketophenylpyruvic acid.

9. A method for catalyzing the production of α-phenylpyruvic acid from L-phenylalanine, characterized in that, It includes the following steps: Using L-phenylalanine as a substrate, adding the genetically engineered cell according to claim 5 for a catalytic reaction to produce α-ketophenylpyruvic acid.

Images