Esterase BioH is a critical enzyme for Biotin synthesis in Escherichia coli , which has been previously found to be active in the acylation of secondary alcohols and amines. Directed evolution towards improved acylation activity requires a high-throughput screening method. The aim of this study is to explore whether the acylation activity of BioH can be improved by directed evolution of its hydrolysis activity. A colorimetric method based on p -nitrophenyl butyrate hydrolysis was adopted in the high-throughput determination of hydrolysis activity. The wild-type BioH showed a hydrolysis activity of 18 U/mg, and the specific activities for α-phenylethanol and α-phenylethylamine acylation were 12.8 U/mg and 3.5 U/mg, respectively. After two rounds of directed evolution, seven mutants with improved hydrolysis activity were obtained, among which, K213E, Q70L/M170T and M197L/K213E also showed improvement in acylation activity. To further improve the acylation activity, site mutations were generated in different combinations at the four hot spots Q70L, M170T, M197L and K213E. Among the resulting mutants, Q70L/M197L/K213E showed the highest activity in α-phenylethylamine acylation with a 120% improvement, while Q70L/K213E had the highest α-phenylethanol acylation activity, which was improved by 70%. The results demonstrated that directed evolution of the hydrolysis activity might be a possible approach to improve the acylation activity of the esterase BioH.
After analyzing protein surface structure, the opposite side of the activity site of esterase BioH was observed to be hydrophilic. Hence subsequently, a hydrophilic-modified solid support was selected to immobilize enzyme through oriented adsorption. The immobilized enzyme remained 86% of its original activity under the acylation of 1-phenylethanol, much more than those immobilized on other solid supports with low hydrophilicity (20–35.6%), as the mode of the immobilization could facilitate enzyme to expose its active site to the substrate. This demonstrated that the strategy to select solid support based on the analysis of protein surface structure was successful. It was also noticed that the substrate of the reaction was obviously adsorbed onto the support, therefore the adsorption effect could not be ignored and it was necessary to obtain the adsorption isotherm of the substrate for a more authentic result. The parameter K m shifted from 884.56 to 296.09 mM after immobilization. This was possibly mainly caused by the increment of substrate concentration around the enzyme due to the substrate adsorbing on the support. It could be another explanation for why those parameters were changed after enzyme immobilization in addition to some existing proposed reasons such as stabilizing conformation, facilitating dispersion of enzyme, etc.
危险品化学品经营许可证（带存储）