Phenylalanine dehydrogenase mutant and application thereof in synthesis of L-homophenylalanine

A technology of phenylalanine dehydrogenase and high phenylalanine, which is applied in the field of enzyme engineering and microbial engineering to achieve good temperature stability

Pending Publication Date: 2021-12-21
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, there is no report on the use of protein engineering to improve the catalytic efficiency of phenylalanine dehydrogenase to prepare L-homophenylalanine

Method used

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  • Phenylalanine dehydrogenase mutant and application thereof in synthesis of L-homophenylalanine
  • Phenylalanine dehydrogenase mutant and application thereof in synthesis of L-homophenylalanine
  • Phenylalanine dehydrogenase mutant and application thereof in synthesis of L-homophenylalanine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Preparation of Phenylalanine Dehydrogenase mutant gene sequence

[0052] Specific steps are as follows:

[0053] Refer to the literature "Asano Y, Nakazawa A, Endo K, et al.Phenylana manufacturer Dehydrogenaseof Bacillus Badius.purification, Characterization and Gene Cloning [J] .EUR JBIOCHEM, 1987, 168 (1): 153-159." Chemical synthetic amino acid sequence such as The wild-type phenylalanine dehydrogenase shown in SEQ ID NO.1, the nucleotide sequence encoding the wild-type phenylanine dehydrogenase gene is shown in SEQ ID NO.2.

[0054] The obtained gene encoding the wild-type phenylalanine dehydrogenase was ligated by both the PET-28A (+) plasmid after bisase digestion (XHO I, BamHi), and the recombinant plasmid PET28A-PHEDH was obtained, and E. coli E was transformed. Coli BL21 (DE3) obtained recombinant E. coli PET28A-PHEDH / E.COLI BL21.

[0055] Using the total plasmid PCR technology, the obtained recombinant plasmid PET28A-PHEDH was used as a template to ob...

Embodiment 2

[0073] Example 2: Inducing culture and protein purification of phenylalanine dehydrogenase mutants

[0074] The recombinant E. coli PET28A-PHEDH / E.Coli BL21 obtained in Example 1 and a gene encoding mutant L306V, V309A, V309G, V309G / L50V, V309G / L306V, V309G / L306V / V144A, and V309G / L306V / V144G. The recombinant E. coli was applied to the LB solid medium, cultured from 8 to 10 h at 37 ° C to obtain a single colony; picking a single colony into an LB liquid medium, from 37 ° C, 200 rpm culture 6 to 8 h, obtained seed fluid; After 2% (V / V) of the inoculation amount, the LB liquid medium was added to 37 ° C, 200 rpm in 2 to 3 h hours, IPTG of the final concentration of 0.1 mmol / L, 2 ° C, 200 rpm continued to induce the culture 12 To 17h, a fermentationery was obtained; the fermentationery solution was centrifuged at 4 ° C, 8000 rpm for 5 minutes, discarded, washed with 9% saline, washed twice, and obtained wild type, mutant L306V, V309A, V309G, V309G / L50V, V309 g / l3...

Embodiment 3

[0076] Example 3: Catalytic efficiency of different phenylalanine dehydrogenase mutants on 2-oxo-4-phenylbousic acid

[0077] Specific steps are as follows:

[0078] Different benzenes were obtained at different concentrations (0.1 to 25 mmol / L2-oxo-4-phenylbutyric acid). Catalytic efficiency of alanine dehydrogenase.

[0079] In NH 4 CL-NH 4 2-oxo-4-phenylbucic acid (0.1 to 25 mmol / L) and NADH (0.5 mmol / L) were added to the OH buffer (1 mol / L, pH 9.0), and the reaction system was obtained; the reaction system was constructed in 30 After 2 min at ° C, 20 μl of the wild type, mutant, V309 g / L306V / V144A, and V309G / L306V / V1444, and V309 g / L306V / V144 g, and V309 g / L306V / V144 g, respectively. The concentrated enzyme solution is not contained, the other components are the same; 50 ° C reaction 5 min, each 10s recording the absorbance of the reaction system at 340 nm at 340 nm, and calculates the wild type, mutant L306V, V309A, V309G, V309G / L50V, V309G / The cat...

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Abstract

The invention discloses a phenylalanine dehydrogenase mutant and application thereof in synthesis of L-homophenylalanine, and belongs to the technical field of enzyme engineering and microbial engineering. According to the mutant, the mutant is obtained by mutating one or more of amino acids at the 306th site, the 309th site, the 144th site and the 50th site of phenylalanine dehydrogenase with an amino acid sequence as shown in SEQ ID No.1. The phenylalanine dehydrogenase mutant disclosed by the invention shows relatively high catalytic efficiency to 2-oxo-4-phenylbutyric acid, the highest catalytic efficiency can reach 219.16 mM <-1 >. S <-1 >, under a high-concentration substrate, the conversion rate can reach 99%, and the specific space-time conversion rate can reach 30.9 mmol.g <-1 >. L <-1 >. H <-1 >; and the phenylalanine dehydrogenase mutant disclosed by the invention still has good temperature stability. By adopting the method disclosed by the invention, the production capacity of the unit catalyst and the reaction efficiency of the catalyst are improved, and the reaction cost is reduced.

Description

Technical field [0001] The present invention relates to a phenylalanine dehydrogenase mutant and its application in L-highenzophenylalanine synthesis, belonging to the field of enzyme engineering and microbial engineering. Background technique [0002] L-high-2-amino-4-phenylbutyric acid is a non-natural chiral amino acid, which is more than a common L-phenylalanine. base( figure 1 . [0003] Optical pure L-high recombinantine is an important raw material for the preparation of vascular tension reducing enzyme inhibitor drugs, and is a common structural block of approximately 20 anti-hypertension drugs in the world. On the one hand, it can be used directly to make Benazepril, ENALAPRIL, Cilazapril (Slapley), Lisinopril, etc., on the other hand, To produce antihypertensive drugs, such as QuinaPril, DelaPril, etc. (DELAPRIL) figure 1 . Such drugs can be used to treat or relieve different degrees of hypertension, and patients with myocardial infarction, myocardial infarction, left h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12N15/70C12P13/22C12N1/21C12R1/19
CPCC12N9/0018C12N15/70C12P13/222C12Y104/0102
Inventor 穆晓清吴涛徐岩
Owner JIANGNAN UNIV
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