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Isolation and characterization of a metagenome-derived and cold-active lipase with high stereospecificity for (R)-ibuprofen esters

Paper ID Volume ID Publish Year Pages File Format Full-Text
25189 43559 2007 8 PDF Available
Title
Isolation and characterization of a metagenome-derived and cold-active lipase with high stereospecificity for (R)-ibuprofen esters
Abstract

We report on the isolation and biochemical characterization of a novel, cold-active and metagenome-derived lipase with a high stereo-selectivity for pharmaceutically important substrates. The respective gene was isolated from a cosmid library derived from oil contaminated soil and designated lipCE. The deduced aa sequence indicates that the protein belongs to the lipase family l.3, with high similarity to Pseudomonas fluorescens lipases containing a C-terminal secretion signal for ABC dependent transport together with possible motifs for Ca2+-binding sites. The overexpressed protein revealed a molecular weight of 53.2 kDa and was purified by refolding from inclusion bodies after expression in Escherichia coli. The optimum temperature of LipCE was determined to be 30 °C. However, the enzyme still displayed 28% residual activity at 0 °C and 16% at −5 °C. Calcium ions strongly increased activity and thermal stability of the protein. Further detailed biochemical characterization of the recombinant enzyme showed an optimum pH of 7 and that it retained activity in the presence of a range of metal ions and solvents. A detailed analysis of the enzyme's substrate spectrum with more than 34 different substrates indicated that the enzyme was able to hydrolyze a wide variety of substrates including the conversion of long chain fatty acid substrates with maximum activity for pNP-caprate (C10). Furthermore LipCE was able to hydrolyze stereo-selectively ibuprofen-pNP ester with a high preference for the (R) enantiomer of >91% ee and it demonstrated selectivity for esters of primary alcohols, whereas esters of secondary or tertiary alcohols were nearly not converted.

Keywords
Lipase; Pseudomonas; Metagenome; Cold-active; Ibuprofen; Ca2+ activation
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Isolation and characterization of a metagenome-derived and cold-active lipase with high stereospecificity for (R)-ibuprofen esters
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 130, Issue 4, 15 July 2007, Pages 370–377
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us