Studies On Gene Screening, Cloning, Expression And Enzymatic Characterization Of Feruloyl Esterase From Escherichia Coli BL21

Ferulic acid esterases(FAEs, EC 3.1.1.73) draw the attention of the world owing to their broad application prospects in the papermaking, food, medicine, textile, feed processing, biosynthesis and energy development and other fields. Although microbial source of FAEs mainly concentrated in penicillium and aspergillus, bacteria such as lactobacillus can also produce, there is no information about FAE from Escherichia coli BL21. Excavation and research new enzymes are beneficial to enrich enzyme-producing strains source and find FAEs with strong specificity and good adaptability. In this study, genes may have FAE activity in the E. coli genome were cloned and expressed, the enzymatic properties were characterised. The main results were showed as follows:1) Preliminary identification of feruloyl esterase activity displayed by isolated Escherichia coli BL21 on ethyl ferulate culture medium. Escherichia coli BL21 is verified with ferulic acid esterase activity.2) There are twelve genes about hydrolases or esterases in BL21 screened by whole genome sequence analysis and conservative protein structure domain analysis. These genes are men D, mhp C, ybf F, ybh C, yjf P, aes, bio H, yqi A, yei G, pld B, rut D and yfh R. The corresponding fragments length are 759 bp, 882 bp, 765 bp, 1284 bp, 750 bp, 960 bp, 771 bp, 582 bp, 837 bp, 1023 bp, 801 bp and 882 bp respectively. After detecting activity of overexpressed proteins, mhp C, ybf F and bio H were identified as genes which had feruloyl esterase activity. The molecular of Mhp C, Ybf F and Bio H were predicted to be 32.6 k Da, 28.4 k Da and 28.5 k Da, respectively.3) The enzymatic property of purified Mhp C, Ybf F and Bio H were studied. The results showed that the substrate scope of these enzymes were extensive. Ferulic acid methyl ester used as the substrate, the optimal reaction temperature of Mhp C, Ybf F and Bio H were 60 oC, 40 oC and 40 oC respectively, and the optimum p H were 7.0, 9.0 and 9.0, respectively. The Km and the Kcat/Km of Mhp C for the best substrate Mp CA were 0.03 mmol·L-1 and 11.29×103 L·mol-1·s-1. The Km and the Kcat/Km of Ybf F for the best substrate MFA were 0.14 mmol·L-1 and 2.43×103 L·mol-1·s-1. The Km and the Kcat/Km of Bio H for the best substrate ethyl ferulate were 0.48 mmol·L-1 and 2.02×103 L·mol-1·s-1.