关键词: 特基拉芽孢杆菌, 大肠杆菌, L-天冬氨酸α-脱羧酶, 异源表达, β-丙氨酸

Abstract: L-aspartate α-decarboxylase is an important industrial enzyme which can stereo-selectively transform L-aspartate acid into β-alanine. In the present study, we cloned and expressed the L-aspartate α-decarboxylase gene (panD) from Bacillus tequilensis to construct an L-aspartate α-decarboxylase-producing Escherichia coli strain and optimized the culture conditions for high cell density fermentation. Using the genome of B. tequilensis PanD37 as template, the panD gene was amplified, and the recombinant plasmid pET32a(+)-panD was constructed and transformed into E. coli BL21(DE3) for expression. For high cell density growth and efficient L-aspartate α-decarboxylase expression, the optimum fermentation conditions in shake flasks were determined as follows: a pH-stat fed-batch culture was performed using glucose as the carbon source at an initial concentration of 5 g/L; after 8 h of culture at 37 ℃, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mmol/L as an inducer and the temperature was reduced to 26 ℃. Maximum L-aspartate α-decarboxylase activity of 1 109.8 U/mL and OD600 nm value of 106.3 were obtained when the fermentation was carried out in a 5 L fermentor. Whole-cell catalysis of 100 g/L L-aspartate gave a molar conversion rate of 99.2% after 10 h of reaction. This work can provides a promising basis for further application of L-aspartate α-decarboxylase in industrial β-alanine production.

Key words: Bacillus tequilensis, Escherichia coli, L-aspartate α-decarboxylase, heterelogous expression, β-alanine