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BSF1 fibrinolytic enzyme from a marine bacterium Bacillus subtilis A26: Purification, biochemical and molecular characterization

Paper ID Volume ID Publish Year Pages File Format Full-Text
35095 45074 2009 8 PDF Available
Title
BSF1 fibrinolytic enzyme from a marine bacterium Bacillus subtilis A26: Purification, biochemical and molecular characterization
Abstract

A novel fibrinolytic enzyme, subtilisin BSF1, from a newly isolated Bacillus subtilis A26 was purified, characterized and the gene was isolated and sequenced. The subtilisin BSF1 was purified to homogeneity by five-step procedure with a 4.97-fold increase in specific activity and 6.28% recovery. The molecular weight of the purified enzyme was estimated to be 28 kDa by SDS-PAGE and gel filtration. The purified enzyme exhibited high fibrinolytic activity on fibrin agar plates.Interestingly, the enzyme was highly active over a wide range of pH from 7.0 to 12.0, with an optimum at pH 9.0. The relative activities at pH 10.0 and 11.0 were 97.8% and 85.2% of that at pH 9.0. The optimum temperature for enzyme activity was 60 °C. The activity of subtilisin BSF1 was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine protease. The N-terminal amino acid sequence of the first 11 amino acids (aa) of the purified fibrinolytic enzyme was AQSVPYGISQI.The bsf1 gene encoding the subtilisin BSF1 was isolated and its DNA sequence was determined. The bsf1 gene consisted of 1146 bp encoding a pre-pro-protein of 381 amino acids organized into a signal peptide (29 aa), a pro-peptide (77 aa) and a mature domain (275 aa). The deduced amino acids sequence of the mature enzyme (BSF1) differs from those of nattokinase from B. subtilis natto and subtilisin DFE from Bacillus amyloliquefaciens DC-4 by 5 and 39 amino acids, respectively.

Keywords
Bacillus subtilis; Subtilisin BSF1; Purification; Molecular characterization; DNA sequence
First Page Preview
BSF1 fibrinolytic enzyme from a marine bacterium Bacillus subtilis A26: Purification, biochemical and molecular characterization
Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 44, Issue 11, November 2009, Pages 1252–1259
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering