Carboxyl ester hydrolase from P enicillium expansum : cloning, characterization and overproduction by P enicillium griseoroseum

Aims In this study, a gene that encodes a carboxylesterase ( carb ) in P enicillium expansum GF was cloned, sequenced and overexpressed by P enicillium griseoroseum PG 63, and the enzyme was characterized. Methods and Results The recombinant strain, P . griseoroseum T 55, 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 S outhern blotting. Furthermore, the recombinant strain T 55 exhibited almost a fourfold increase in carboxylesterase activity compared with PG 63 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 α/β barrel topology, that is typical of esterases and lipases, was indicated for the CARB protein with S er 213 ‐ G lu 341 ‐ H is 456 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°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 P enicillium sp. carboxylesterase.