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Highly active metallocarboxypeptidase from newly isolated Geobacillus strain SBS-4S: Cloning and characterization

Paper ID Volume ID Publish Year Pages File Format Full-Text
21476 43224 2011 7 PDF Available
Title
Highly active metallocarboxypeptidase from newly isolated Geobacillus strain SBS-4S: Cloning and characterization
Abstract

The carboxypeptidase gene from Geobacillus SBS-4S was cloned and sequenced. The sequence analysis displayed the gene consists of an open reading frame of 1503 nucleotides encoding a protein of 500 amino acids (CBPSBS). The amino acid sequence comparison revealed that CBPSBS exhibited a highest homology of 41.6% (identity) with carboxypeptidase Taq from Thermus aquaticus among the characterized proteases. CBPSBS contained an active site motif 265HEXXH269 which is conserved in family-M32 of carboxypeptidases. The gene was expressed with His-Tag utilizing Escherichia coli expression system and purified to apparent homogeneity. The purified CBPSBS showed highest activity at pH 7.5 and 70°C. The enzyme activity was metal ion dependent. Among metal ions highest activity was found in the presence of Co2+. Thermostability studies of CBPSBS by circular dichroism spectroscopy demonstrated the melting temperature of the protein around 77°C. The enzyme exhibited Km and Vmax values of 14 mM and 10526 μmol min− 1 mg− 1 when carbobenzoxy–alanine–arginine was used as substrate. kcat and kcat/Km valves were 10175 s− 1 and 726 mM− 1 s− 1. To our knowledge this is the highest ever reported enzyme activity of a metallocarboxypeptidase and the first characterization of a metallocarboxypeptidase from genus Geobacillus.

Keywords
Thermophiles; Geobacillus SBS-4S; Metallocarboxypeptidase; Circular dichroism; Melting point; Thermostability
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Highly active metallocarboxypeptidase from newly isolated Geobacillus strain SBS-4S: Cloning and characterization
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 111, Issue 3, March 2011, Pages 259–265
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