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Overexpression of a bacterial chymotrypsin: Application for l-amino acid ester hydrolysis

Paper ID Volume ID Publish Year Pages File Format Full-Text
17332 42660 2011 7 PDF Available
Title
Overexpression of a bacterial chymotrypsin: Application for l-amino acid ester hydrolysis
Abstract

In this work, a reliable protocol was designed to rapidly express and purify a microbial chymotrypsin(ogen) as a useful alternative to using animal proteases. The cDNA encoding for chymotrypsinogen from the deuteromycete Metarhizium anisopliae (chy1) was overexpressed in an Origami2(DE3) E. coli strain deficient in thioredoxin reductase and glutathione reductase activities, thus possibly allowing disulfide exchange. By using a quick purification protocol, in which the hexahistidine tag was added at the C-terminal end of the protease, the recombinant CHY1 protein could be purified in a single step on an Ni-NTA column as a mixture of 19.5- and 15-kDa mature active forms and did not require further activation/maturation steps. This expression and purification procedure offers an easier and faster means of producing recombinant CHY1 chymotrypsin than that previously described for Pichia pastoris. The kinetic properties could be characterized and CHY1 chymotrypsin was demonstrated to efficiently catalyze N-acetylated l-phenylalanine and l-tyrosine methyl ester hydrolysis.

Keywords
Chymotrypsin; Nonanimal protease; Overexpression; Amino acid ester hydrolysis
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Overexpression of a bacterial chymotrypsin: Application for l-amino acid ester hydrolysis
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 49, Issues 6–7, 10 December 2011, Pages 560–566
Authors
, , , , , , ,
Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
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Price was $35.95
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Price after discount Only $4.95
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Full-text PDF Download
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