Carboxyl ester hydrolase from Penicillium expansum: cloning, characterization and overproduction by Penicillium griseoroseum.

摘要

Aims: In this study, a gene that encodes a carboxylesterase (carb) in Penicillium expansum GF was cloned, sequenced and overexpressed by Penicillium griseoroseum PG63, and the enzyme was characterized. Methods and Results: The recombinant strain, P. griseoroseum T55, obtained upon transformation using the plasmid pAN-52-1-carb, showed integration of the carb gene into at least two heterologous sites of the genome by Southern blotting. Furthermore, the recombinant strain T55 exhibited almost a fourfold increase in carboxylesterase activity compared with PG63 strain when both were cultured without inducers. Based on the secondary structure and multiple sequence alignments with carboxylesterases, cholinesterase and lipase, a three-dimensional model was obtained. The alpha / beta barrel topology, that is typical of esterases and lipases, was indicated for the CARB protein with Ser213-Glu341-His456 as the putative catalytic triad. CARB preferentially hydrolysed acyl chains with eight carbon atoms, and its activity was optimal at a pH of 7.0 and a temperature of 25 degrees C. CARB exhibited stability in alkaline pH, high activity under mesophilic conditions and stability in organic solvents. Conclusion: The CARB protein is potentially useful in bioremediation, food and chemical/pharmaceutical industries. Significance and Impact of the Study: This study is the first to report the development of a recombinant strain superproducing a Penicillium sp. carboxylesterase.