Effects of inducible and constitutive expression vectors on heterologous expression of LEH mutants BE3 and BG5

Abstract Background Limonene epoxide hydrolase (LEH) is a class of enzymes in Epoxide hydrolases (EHs) that can form chiral products only by one step catalysis. High purity S or R chiral intermediates play an important role in the application of biomedical industry, and can be used to synthesize various drugs such as ibuprofen, linezoline, cilastatin, etc. Therefore, it has application value to find the expression system which can realize the stable and efficient conversion of LEH to produce high purity S and R chiral products. Results We explored the constitutive expression system of LEH for the first time, and tried to realize the expression of LEH in B.subtilis WB800. Firstly, the LEH mutant on the original inducible vector pBAD-Myc-HisA was obtained and connected to the constitutive vector pHY-p43 containing strong promoter p43. E. coli TOP10 and B.subtilis WB800 were used as the host bacteria to realize the non induced and secretory expression of LEH. Conclusions The results showed that the non induced expression of LEH could be achieved successfully by using the constitutive promoter vector pHY-p43, and the substrate affinity and enzyme catalytic efficiency of the mutants have increased. The catalytic decomposition of the substrate and the formation of chiral products by LEH were determined by GC-MS, which also had stable enzyme activity in the system. This study laid a foundation for large-scale fermentation of LEH and catalytic production of chiral products.