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Characterization of the glutamate-specific endopeptidase from Bacillus licheniformis expressed in Escherichia coli

Paper ID Volume ID Publish Year Pages File Format Full-Text
23293 43430 2013 6 PDF Available
Title
Characterization of the glutamate-specific endopeptidase from Bacillus licheniformis expressed in Escherichia coli
Abstract

•Homologous model of GSE-BL was built based on GSE-BI crystal structure.•The catalytic triad of GSE-BL was demonstrated by site-directed mutagenesis.•Key amino acids involved in GSE-BL catalytic activity were confirmed.•Phe57 was replaced by Ala to improve the enzymatic activity of GSE-BL.•A new strategy of preparing GSE-BL without introducing protease was introduced.

Glutamate-specific endopeptidase from Bacillus licheniformis (GSE-BL) is widely used in peptide recovery and synthesis because of its unique substrate specificity. However, the mechanism underlying its specificity is still not thoroughly understood. In this study, the roles of the prosegment and key amino acids involved in the proteolytic activity of GSE-BL were investigated. Loss of the GSE-BL prosegment severely restricted enzymatic activity toward Z-Phe-Leu-Glu-pNA. A homologous model of GSE-BL revealed that it contains the catalytic triad “His47, Asp96 and Ser 167”, which was further confirmed by site-directed mutagenesis. In vitro mutagenesis further indicated that Val2, Arg89 and His190 are essential for enzymatic activity toward Z-Phe-Leu-Glu-pNA. Moreover, the catalytic efficiency of Phe57Ala GSE-BL toward Z-Phe-Leu-Glu-pNA was 50% higher than that of the native mature GSE-BL. This is the first study to fully elucidate the key amino acids for proteolytic activity of GSE-BL. Mature GSE-BL could be obtained through self-cleavage alone when Lys at -1 position was replaced by Glu, providing a new strategy for the preparation of mature GSE-BL. This study yielded some valuable insights into the substrate specificity of glutamate-specific endopeptidase, establishing a foundation for broadening the applications of GSE-BL.

Keywords
GSE-BL, glutamate-specific endopeptidase from Bacillus licheniformis; GSE-BI, glutamate-specific endopeptidase from Bacillus intermedius; GSE-SG, glutamate-specific endopeptidase from Streptomyces grieus; V8-GSE, glutamate-specific endopeptidase from Stap
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Characterization of the glutamate-specific endopeptidase from Bacillus licheniformis expressed in Escherichia coli
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 168, Issue 1, 10 October 2013, Pages 40–45
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