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Cloning, expression, purification, and characterization of a glutamate-specific endopeptidase from Bacillus licheniformis.

Abstract：

:A gene encoding a glutamate-specific endopeptidase (GSE) from Bacillus licheniformis (BL) has been cloned in Escherichia coli cells. The recombinant protein was expressed as cytoplasmic insoluble inclusion bodies. Immobilized metal affinity chromatography was employed to purify the protein, and then a 27-kDa GSE intermediate was obtained by gradient urea dialysis. The remaining pro-peptide was completely removed by treatment with trypsin to obtain mature GSE-BL with a molecular weight of 26 kD at a final yield of up to 140.9 mg/L. With Z (benzyloxycarbomyl)-Phe-Leu-Glu-pNA (p-nitroanilide) as the substrate, the optimal temperature and pH conditions for the enzyme were 37 °C and 8.5, respectively, K(m) was 1.495 ± 0.034 mM, and V(max) was 50.237 μmol/mg min. The presence of calcium and ferrous ions enhanced the catalytic activity of GSE-BL. These results suggest that the recombinant protein is a relatively stable specific proteinase that could be effectively utilized in protein structure analysis, peptide synthesis, and the food industry.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Ye W,Liu J,Wang H,Wang J,Wang X

doi

10.1016/j.pep.2011.12.001

subject

Has Abstract

pub_date

2012-03-01 00:00:00

pages

138-43

issue

1

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(11)00347-0

journal_volume

82

pub_type

杂志文章

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