The thermostable esterase Aaeo1 displays a low expression level and forms a great amount of inclusion bodies in E. coli. Herein, a split-GFP system was established in which the fluorescence intensity exhibited a good linear correlation with the soluble protein expression level and the esterase activity. In the primary high-throughput screening, the mutant library was screened by flow cytometry via detection of a split-GFP reporter. Then, through a secondary screening against esterase activity, two mutants with improved soluble expression and catalytic activity were obtained. The soluble expression of the mutant enzymes in E. coli was improved by 2-fold. The kcat/ Km values of the mutant enzymes were 2-fold higher than that of the parent. We explored the relationship between the amino acid mutations in the two mutants and the enzyme activity. The enzyme activity of mutant I51V-E170D was 4.5 times higher than that of the parent.
Keywords: catalytic activity; high-throughput screening; soluble expression; split-GFP; thermostable esterase.