CN-120060223-B - L-aspartic acid alpha-decarboxylase with improved substrate tolerance

Abstract

The invention discloses an L-aspartic acid alpha-decarboxylase with improved substrate tolerance, which is prepared by carrying out site-directed mutagenesis on the L-aspartic acid alpha-decarboxylase from bacillus subtilis, wherein phenylalanine at a No. 4 site is mutated into tryptophan, isoleucine at a No. 33 site is mutated into alanine, isoleucine at a No. 88 site is mutated into tryptophan, an enzyme mutant is obtained, a mutant recombinant plasmid is transformed into escherichia coli, and beta-alanine is generated by catalyzing substrate L-aspartic acid after induced expression. When the substrate addition concentration of the whole cell catalytic system is 60g/L, the conversion rate of three enzyme mutants T4W, I A and I88M can reach about 90%, the yields are 1.2 times, 1.3 times and 1.2 times of that of the unmutated strain respectively, the substrate tolerance is obviously improved, the combined mutation has a certain improvement on the substrate tolerance, and the discovery has important research value for industrially preparing beta-alanine.

Inventors

• LI NING
• XU LU
• Wu Ruisi
• WANG DAOBIN
• YUAN JUNWEN

Assignees

• 广西大学

Dates

Publication Date

20260512

Application Date

20250213

Claims (10)

1. 1. An L-aspartic acid- α -decarboxylase mutant, characterized in that the L-aspartic acid- α -decarboxylase mutant is specifically: The codon encoding amino acid 33 in the nucleotide sequence shown in SEQ ID NO. 1 is mutated from isoleucine to alanine, so that the isoleucine at position 33 in the translated amino acid sequence is replaced with alanine.
2. 2. The gene encoding the mutant L-aspartic acid- α -decarboxylase of claim 1.
3. 3. A recombinant expression vector comprising the coding gene according to claim 2.
4. 4. The recombinant expression vector of the coding gene according to claim 3, wherein the recombinant expression vector is framed by a pET28-a (+) vector.
5. 5. A genetically engineered bacterium comprising the coding gene of claim 2 or the recombinant expression vector of claim 3 or 4.
6. 6. The genetically engineered bacterium of claim 5, wherein the genetically engineered bacterium is escherichia coli BL21 (DE 3).
7. 7. Use of the mutant L-aspartic acid- α -decarboxylase of claim 1 for catalyzing the synthesis of β -alanine from L-aspartic acid.
8. 8. The use according to claim 7, characterized in that it is carried out by synthesizing beta-alanine by biocatalysis using L-aspartic acid as substrate and the genetically engineered bacterium according to claim 5 or 6 as whole-cell catalyst.
9. 9. The method according to claim 8, wherein the biocatalytic reaction is carried out in a 100 mM phosphate buffer solution containing 60 g/L L-aspartic acid, 50mM Fe2+, and pH 7.0, using wet cells having an OD 600 value as a catalytic host.
10. 10. The use according to claim 9, wherein the wet cell is prepared by centrifuging the fermentation broth, discarding the supernatant, and collecting the precipitate.

Description

L-aspartic acid alpha-decarboxylase with improved substrate tolerance Technical Field The invention belongs to the technical fields of genetic engineering and enzyme engineering, and particularly relates to L-aspartic acid alpha-decarboxylase with improved substrate tolerance. Background L-aspartic acid alpha-decarboxylase (PanD) is the key for synthesizing beta-alanine by an enzyme method and a microbial fermentation method, and the enzyme can catalyze L-aspartic acid to generate beta-alanine and is the key for influencing the yield of beta-alanine. Beta-alanine is widely used in the fields of medicine, food, chemical industry, environment, etc. First, many industrially important compounds such as 3-hydroxypropionic acid, poly-3-hydroxypropionate, pantothenic acid, carnosine, etc. are synthesized using β -alanine as an important precursor or intermediate. Secondly, in the food industry, beta-alanine is a food additive that improves both the taste of food and physical function as a nutritional supplement for athletes. In addition, the method can be directly used for producing the poly beta-alanine and is widely applied to the fields of cosmetics, water purification, construction and the like. The global demand for β -alanine series products in recent years is about 5 ten thousand tons and is still increasing, being called one of the 12 most developed potential tricarbonated products worldwide in the future. Beta-alanine is mainly produced by chemical methods, biological enzyme conversion methods and microbial fermentation methods. Although the chemical method is relatively mature compared with the biological synthesis, the waste treatment cost is high, the environmental pollution is serious, and the environmental protection standard is not met. Therefore, the method is environment-friendly and has mild conditions, and becomes a currently more promising method for preparing the beta-alanine. Currently, the L-aspartic acid alpha-decarboxylase used to produce beta-alanine is mainly derived from Corynebacterium glutamicum, bacillus subtilis, and Corynebacterium jejuni. However, wild-type L-aspartic acid alpha-decarboxylase and its mutants are generally subject to poor substrate tolerance. For example, li Huanhuan uses L-aspartic acid alpha-decarboxylase from Corynebacterium glutamicum, beta-alanine titer reaches 24.8g/L after 20h of bioconversion with a 40g/L L-aspartic acid yield of 92.6% in whole cell biocatalysts, but substrate inhibition occurs in whole cell biocatalyst systems when L-aspartic acid concentration is higher than 40g/L, wang Jing R12V site-directed mutagenesis is performed on Bacillus subtilis-derived L-aspartic acid alpha-decarboxylase, but when L-aspartic acid concentration reaches 100g/L, the conversion rate is only 67%. Therefore, the substrate tolerance of the enzyme is improved by utilizing the site-directed mutagenesis technology, and the method has important significance for industrial application of the biological method for preparing the beta-alanine. Disclosure of Invention In order to solve the problems, the invention provides an L-aspartic acid alpha-decarboxylase with improved substrate tolerance, which is designed to improve the tolerance and affinity of the L-aspartic acid alpha-decarboxylase from bacillus subtilis (Bacillus subtilis) to L-aspartic acid, so that the efficient production of beta-alanine is realized. In order to achieve the above purpose, the present invention adopts the following technical scheme: the L-aspartic acid alpha-decarboxylase mutant with improved substrate tolerance has the mutation of T4W, I33A, I88W, T W/I88W, I A/I88W compared with the L-aspartic acid alpha-decarboxylase encoded by panD gene derived from bacillus subtilis. The present invention provides a gene encoding the above L-aspartic acid alpha-decarboxylase mutant. The nucleotide sequence of the coded L-aspartic acid alpha-decarboxylase mutant is shown as SEQ ID NO. 1. The invention provides an expression vector carrying the gene. The expression vector is a pET-28a (+) vector. The present invention provides a host cell carrying the above gene or the above expression vector. The host cell is Escherichia coli BL21 (DE 3). The invention provides a preparation method of the L-aspartic acid alpha-decarboxylase mutant, which comprises the steps of inoculating the host cells into a fermentation medium for fermentation, collecting fermentation liquor obtained by fermentation after the fermentation is finished, centrifuging, and separating the L-aspartic acid alpha-decarboxylase mutant from cell sediment obtained by centrifuging after the centrifugation is finished. The invention provides the L-aspartic acid alpha-decarboxylase mutant prepared by the method. The invention provides a method for preparing beta-alanine, which takes the mutant or the host cell as a catalyst, takes L-aspartic acid as a substrate, and simultaneously adds 50mM Fe 2+ for catalytic reaction to prepare the beta-alanine. The ca