Preparation and application of L-amino acid deaminase mutant

An amino acid and deaminase technology, applied in the field of genetic engineering, can solve problems such as low yield, achieve high catalytic activity, improve production capacity, and be easy to popularize and apply.

Active Publication Date: 2020-06-12
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the current method of utilizing L-amino acid modification has improved the output and conversion rate of α-ketoisovaleric acid to a certain extent, but the output is still low. Therefore, in

Method used

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  • Preparation and application of L-amino acid deaminase mutant
  • Preparation and application of L-amino acid deaminase mutant
  • Preparation and application of L-amino acid deaminase mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Construction and screening of single mutants

[0037] (1) Mutant construction: primers for the 98th, 105th, 106th, and 341st mutation sites were designed, as shown in Table 1, and constructed by whole-plasmid PCR.

[0038] Table 1 Mutation primer sequence

[0039]

[0040]Construct the reaction PCR amplification system: PrimSTAR enzyme 0.5 μL, 5×PrimeSTAR Buffer 10 μL, dNTP 4 μL, two primers for each mutation site 1 μL, template (nucleotide sequence of pmirLAAD) 4 μL, water 32.5 μL; the reaction conditions are : ① 94°C for 3 minutes; ② 98°C for 10s; ③ 55°C for 30s; ④ 72°C for 3 minutes; ⑤ Cycle ② to ④ 29 times;

[0041] Incubate the above reaction system at 37°C for 3 hours to digest the plasmid template (digestion system: DpnI 0.5 μL, the above reaction PCR product 45 μL, 10×T Buffer 5 μL), and the digested product obtained after digestion is introduced into E. coli BL21 by chemical transformation method Competent cells, specific steps of chemical trans...

Embodiment 2

[0054] Example 2: Construction and screening of double, triple and quadruple mutants

[0055] (1) double mutation mutant construction: in the mutant PmirLAAD S98A On the basis of the mutation primers T105A-S and T105A-A, S106A-S and S106A-A, L341A-S and L341A-A, double mutation mutants were constructed respectively; in the mutant PmirLAAD T105A On the basis of the mutation primers S106A*-S and S106A*-A, L341A-S and L341A-A, double mutation mutants were constructed respectively; in the mutant PmirLAAD S106A On the basis of mutation primers L341A-S and L341A-A (Table 3); carry out double mutation mutant construction by whole plasmid PCR, for specific implementation, refer to step (1) in Example 1, and prepare 6 double mutation mutants Body PmirLAAD S98A / T105A 、PmirLAAD S98A / S106A 、PmirLAAD S98A / L341A 、PmirLAAD T105A / S106A 、PmirLAAD T105A / L341A 、PmirLAAD S106A / L341A .

[0056] Table 3 double mutant mutation primer sequence

[0057]

[0058] (2) Screening of double-mut...

Embodiment 3

[0066] Embodiment 3: Determination of kinetic parameters and product inhibition constants of parental enzymes and mutants

[0067] In order to evaluate the mutants, the present invention has determined the mutant parent PmirLAAD WT and mutant PmirLAAD M1 、PmirLAAD M2 、PmirLAAD M3 、PmirLAAD M4 Kinetic parameters at 25 °C.

[0068] k cat / K m Calculated by measuring the initial rate of α-ketoisovaleric acid produced by the hydrolysis of different concentrations of L-valine substrate at 25°C. The product inhibition constant of the parental enzyme and the mutant was determined by the assay of the product inhibition constant in the transformation process, and the PmirLAAD WT Parental enzyme strains and mutant strains were added to the reaction solution with wet cells at a final concentration of 10g / L, and 60mM L-valine was used as a substrate, and 10-100mM α-ketoisovaleric acid was added to the transformation system. Measure the initial reaction rate V after reacting for ab...

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Abstract

The invention discloses preparation and an application of an L-amino acid deaminase mutant, and belongs to the technical field of gene engineering. According to a modification method disclosed by theinvention, through protein modification of pmirLAAD, by analyzing a flexible Loop ring structure around a binding site of a pmirLAAD product and designing an optimal mutant, a defect that catalytic efficiency of a previous wild type enzyme is reduced due to product inhibition is overcome, and experimental verification is performed. And the optimal mutant pmirLAADM4 is obtained, and the catalytic efficiency (1.61 mM<-1>. Min<-1>) and a product inhibition constant (5.4 mM) are improved by 5.2 times and 5.7 times respectively compared with those of a contrast group. A yield of alpha-ketoisovaleric acid can reach 96.5 g/L, and a conversion rate is greater than 97%. By using the method provided by the invention, cost can be greatly reduced, and an industrial process of producing the alpha-ketoisovaleric acid by an enzymatic conversion method is accelerated.

Description

technical field [0001] The invention relates to the preparation and application of an L-amino acid deaminase mutant and belongs to the technical field of genetic engineering. Background technique [0002] L-amino acid deaminase (LAAD) is a flavin oxidase that can catalyze L-amino acid to generate α-keto acid. Researchers have done a lot of research on the spatial structure, substrate specificity, and catalytic ability of L-amino acid deaminase to unnatural substrates. Applications have gradually attracted the attention of researchers. [0003] α-keto acid is an important intermediate, which is mainly used in food, medicine, cosmetics and other fields, which makes the enzymatic conversion of α-keto acid widely used in industrial production. L-amino acid deaminase is a flavoproteinase, and the oxidative deamination reaction of L-amino acid can be divided into two steps: first, amino acid C α The hydrogen on the body is transferred to FAD, the amino acid becomes imino acid, ...

Claims

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Application Information

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IPC IPC(8): C12N9/06C12N15/53C12N15/70C12N1/21C12P7/40C12R1/19
CPCC12N9/0022C12N15/70C12P7/40C12Y104/03002C12Y103/99C12N9/0014
Inventor 吴静裴杉杉刘佳宋伟陈修来罗秋玲
Owner JIANGNAN UNIV
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