Recombinant aspartate lyase and method for preparing R-3-aminobutyric acid with high repeated utilization rate

A technology of aspartic acid and aminobutyric acid, applied in the fields of enzyme engineering and biocatalysis, can solve the problems of inaccessibility, destruction of active centers, inactivation and denaturation, etc.

Active Publication Date: 2021-06-08
CHANGXING PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The advantage is that the enzyme is firmly combined with the carrier and has good stability; but the disadvantage is that the use of relatively strong reaction conditions will cause changes in the high-level structure of the enzyme protein and have a chance of destroying the active center, so it is often impossible to obtain high specific activity. M
After the aforementioned recombinant aspartase is immobilized with resin, the recovery rate of the enzyme activity is only 10%, and it is almost impossible to prepare the immobilized enzyme. It can be seen that the combination of the carrier and the enzyme protein will make it inactivated and denatured

Method used

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  • Recombinant aspartate lyase and method for preparing R-3-aminobutyric acid with high repeated utilization rate
  • Recombinant aspartate lyase and method for preparing R-3-aminobutyric acid with high repeated utilization rate
  • Recombinant aspartate lyase and method for preparing R-3-aminobutyric acid with high repeated utilization rate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] 1. Construction of recombinant aspartate lyase expression strain

[0033] A recombinant aspartate lyase, which has an amino acid sequence formed by sequentially concatenating SEQ ID NO.2, SEQ ID NO.1, and SEQ ID NO.3. The amino acid sequence of SEQ ID NO.2 is TCRKSYHKQGNRYQTYSRCKH; SEQ ID NO. The amino acid sequence of 3 is CHTSYGRYRKQRK. The nucleotide coding sequence of the entire amino acid sequence is shown in SEQ ID NO.4.

[0034] The hydrophilic distribution of the sequence SEQ ID NO.2 is as follows figure 1As shown in , the ordinate indicates the size of hydrophobicity (positive value indicates hydrophobicity, negative value indicates hydrophilicity); figure 2 The hydrophilic distribution of SEQ ID NO.3 is shown, and it can be seen that the proportion of polar amino acid residues is relatively large, and it has higher salt tolerance.

[0035] Entrust Shanghai Jierui Bioengineering Co., Ltd. to synthesize the gene, carry out double digestion and purification...

Embodiment 2

[0045] In this example, appropriate changes were made on the basis of Example 1, and the amino resin LX-1000HAA was replaced with epoxy resin LX-1000EP (Xi'an Lanxiao Technology New Materials Co., Ltd.), to obtain immobilized enzyme B.

[0046] If the enzyme solution disclosed in ZL 201810198044.8 is used to immobilize with LX-1000EP, the recovery rate of enzyme activity is 4.8%. The immobilized enzyme B in this example was immobilized with the carrier and reused 20 times, and the conversion rate was above 98%.

Embodiment 3

[0048] In this example, appropriate changes were made on the basis of Example 1, and the amino resin LX-1000HAA was replaced with epoxy resin LX-103B (Xi'an Lanxiao Technology New Materials Co., Ltd.) to obtain immobilized enzyme C.

[0049] For example, using the enzyme solution disclosed in ZL 201810198044.8 and LX-103B for immobilization, the recovery rate of enzyme activity is 5.3%. The immobilized enzyme C in this example was immobilized with the carrier and reused 20 times, and the conversion rate was above 98%.

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Abstract

The invention discloses recombinant aspartate lyase and a method for preparing R-3-aminobutyric acid with high repeated utilization rate. Through genetic engineering, a segment of polypeptide chain with a plurality of amino groups, namely polar amino acid residues, is added at each of two ends of an original gene, an enzyme solution is obtained through biological fermentation based on the sequence, and the immobilized enzyme is obtained through immobilizing with resin. According to the immobilized enzyme, the binding probability of covalent binding of an immobilized carrier and protein to an enzyme active center is reduced, so that the damage to enzyme activity after immobilization is reduced, the enzyme activity recovery rate can be increased to 50% or above, and due to the increase of the quantity of the polar amino acids, the concentration of a substrate which can be used for conversion is increased, and the cost of the enzyme is greatly reduced and the production process is saved in the industrial enzyme fermentation engineering.

Description

technical field [0001] The invention relates to the fields of enzyme engineering and biocatalysis, in particular to a recombinant aspartic acid lyase and a method for preparing R-3-aminobutyric acid with high reutilization rate. Background technique [0002] Chinese patent ZL 201810198044.8 (disclosure date: August 07, 2018) discloses a method for the preparation of R-3-aminobutyric acid. The method uses relatively high concentrations of crotonic acid and ammonium salts as substrates, adding magnesium ions The salt, under alkaline conditions, add recombinant aspartase for biocatalysis, and obtain a conversion rate of more than 98%. The recombinant aspartase is obtained based on the gene of Bacillus recombinant aspartase. [0003] The enzyme can be immobilized on the resin as a carrier, and the method is mainly that the enzyme is covalently bonded to the carrier, that is, the inactive site functional group on the enzyme molecule is covalently bonded to the carrier surface re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N11/091C12N11/089C12N15/70C12N1/21C12P13/00C12R1/19
CPCC12N9/88C12N11/091C12N11/089C12N15/70C12P13/005C12Y403/01001
Inventor 杨卫华张利坤谈聪唐云平李业彭汶铎钱敏帆严燕兵倪建洲吴怀春
Owner CHANGXING PHARMA
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