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Alternative pig liver esterase (APLE) – Cloning, identification and functional expression in Pichia pastoris of a versatile new biocatalyst

Paper ID Volume ID Publish Year Pages File Format Full-Text
24805 43538 2008 10 PDF Available
Title
Alternative pig liver esterase (APLE) – Cloning, identification and functional expression in Pichia pastoris of a versatile new biocatalyst
Abstract

Isolated from pig liver as a crude, inhomogeneous enzyme fraction, pig liver esterase (PLE) was found to metabolize a wide range of substrates; often in a highly stereoselective manner. This crude esterase preparation, however, contains several iso-enzymes at proportions varying from batch to batch. Racemic methyl-(4E)-5-chloro-2-isopropyl-4-pentenoate is cleaved enantioselectively by crude PLE, but not by recombinantly expressed γ-isoform of PLE. Concluding that another PLE iso-enzyme must carry the relevant activity, we cloned and sequenced cDNAs of several PLE isoforms and functionally expressed them in Pichia pastoris. One novel isoform termed alternative pig liver esterase (APLE) was found to hydrolyze methyl-(2R,4E)-5-chloro-2-isopropyl-4-pentenoate in a highly stereoselective manner (E > 200). When heterologously expressed and directed for secretion in P. pastoris, APLE was found to be localized in the periplasm. The presence or absence of a putative C-terminal ER retention signal did neither influence functional expression nor cellular localization. The recombinant enzyme, purified by ion exchange chromatography, had a specific activity of 36 U (mg protein)−1 towards racemic methyl-(4E)-5-chloro-2-isopropyl-4-pentenoate.

Keywords
Biocatalysis; Pig liver esterase; Kinetic resolution; Heterologous expression; Pichia pastoris; Periplasm
First Page Preview
Alternative pig liver esterase (APLE) – Cloning, identification and functional expression in Pichia pastoris of a versatile new biocatalyst
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 133, Issue 3, 1 February 2008, Pages 301–310
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering