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Substrate inactivation of bacterial L-aspartate α-decarboxylase from Corynebacterium jeikeium K411 and improvement of molecular stability by saturation mutagenesis
- Mo, Qin, Mao, An, Li, Youran, Shi, Guiyang
- World journal of microbiology & biotechnology 2019 v.35 no.4 pp. 62
- Corynebacterium jeikeium, aspartic acid, biocatalysts, biorefining, colorimetry, enzymes, manufacturing, mutagenesis, mutants, screening
- Bacterial L-aspartate α-decarboxylase (PanD) is a potential biocatalyst for the green production of β-alanine, an important block chemical for manufacturing nitrogen-containing chemicals in bio-refinery field. It was reported that the poor catalytic stability caused by substrate inactivation limited the large-scale application. Here, we investigated the characters of inactivation by L-aspartate of PanD from Corynebacterium jeikeium (PDCⱼₑᵢ), and found that L-aspartate induced a time-, and concentration-dependent inactivation of PDCⱼₑᵢ with the values of KI and kᵢₙₐcₜ being 288.4 mM and 0.235/min, respectively. To improve the catalytic stability of PDCⱼₑᵢ, conserved amino acid residues essential to catalytic stability were analyzed by comparing the discrepancy in the observed inactivation rate of various sources. By an efficient colorimetric high-throughput screening method, four mutants with 3.18–24.69% higher activity were obtained from mutant libraries. Among them, the best mutation (R3K) also performed 66.38% higher catalytic stability than the wild type, showing great potential for industrial bio-production of β-alanine.