Improving the activity of L-aspartate-α-decarboxylase from Corynebacteriumjeikeium through semi-rational design and whole-cell catalytic synthesis of β-alanine

• 1、The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, JiangNan University, Wuxi 214122

Abstract：β-alanine is an important building block in the synthesis of many drugs. At present, β-alanine is synthesized mainly by decarboxylation of aspartic L-aspartate-α -decarboxylase (PanD)，however, the low activity of PanD enzyme is the bottleneck of whole cell catalytic synthesis of β-alanine. Therefore, in this study, through enzyme mining, PanD from Corynebacterium jeikeium was heterologous expressed in Escherichia coli. AlaphFold2 modeling and molecular docking were conducted by Corynebacterium gerii derived PanD. The mutation hotspot was determined by Rosetta virtual mutation, and the mutant L39A was finally screened by using thin layer chromatography (TLC) and re-screening after purification. Its specific enzyme activity was 13.45 U/mg, which was 1.4 times higher than that of wild type (9.6 U/mg). The results showed that the optimum pH of wild-type enzyme and L39A mutant was 6.5, and the enzyme activity was stable between pH 6.0 and 7.0. The optimum temperature of L39A and wild type was 55 ℃, but the thermal stability of L39A was higher than that of the wild type. The catalytic efficiency of the mutant was 1.4 times higher than that of the wild type. The structural analysis of the mutant showed that the 39th position was replaced by alanine with smaller side chain groups, which enhanced the hydrophilicity and increased the interaction between the key catalytic amino acid tyrosine at 58th position and other amino acids, which improved the stability of the region around the active center, thus improving the catalytic activity. Whole-cell catalytic data showed that L39A could convert 70% L-aspartic acid at 4 h, while the wild type could only convert about 50% L-aspartic acid at 4 h, and L39A could convert 90% L-aspartic acid at 10 h, and completely convert 1 mol/L L-aspartic acid at 12 h. The conversion efficiency of the mutant L39A was slightly better than that of the wild type The mutant screened in this study has the potential for industrial application, and a green and efficient β-alanine biosynthesis method has been established, which lays an important foundation for the industrialization of β-alanine biosynthesis.

Keywords： Bioengineering β-alanine Virtual mutation Whole cell catalysis