Molecular modification of l-aspartate-α-decarboxylase and multi-enzyme catalytic preparation of β-alanine

β-Alanine is an important non-essential amino acid and used in the manufacture of a wide range of drugs, in particular it plays a key role in the biosynthesis of carnosine and pantothenic acid. L-aspartate-α-decarboxylase (PanD) is the key enzyme in the enzymatic preparation of β-alanine. In this study, BsPanD* was modified by protein structure prediction and directed evolution, and a mutant BsPanD* PQ4 with good catalytic properties was obtained, which showed a 40 % increase in activity and remained stable in the pH 6.0–9.0 range. The strain E.coli pRSF Duet-1 - smaiA - ecaspA (pMA) was constructed for efficient co-expression of maleate cis - trans isomerase and L-aspartase, with an apparent activity of 253.4 U/g. The multi-enzyme cascade catalytic process for the preparation of β-alanine with maleate as the substrate was developed. The results showed that when the concentration of ammonium maleate was 1 mol/L, the substrate conversion rate was greater than 99.9 %, and the β-alanine concentration was 87.5 g/L with a yield of 98.3 %. This study not only solved the problem of low activity and poor stability of PanD, but also realized the preparation of β-alanine from maleate, a cheap chemical raw material, and laid the foundation for its industrial application.