Cloning of a beta-1,3-1,4-glucanase gene from Bacillus subtilis MA139 and its functional expression in Escherichia coli.

Abstract

A gene encoding beta-1,3-1,4-glucanase was cloned by polymerase chain reaction (PCR) from Bacillus subtilis MA139. Sequencing result showed 97% homology to the corresponding gene from Bacillus licheniformis. The open reading frame (ORF) of the gene contained 690 bp coding for a 226 amino-acid matured protein with the estimated molecular weight of 24.44 kDa. The beta-1,3-1,4-glucanase gene was subcloned into an expression vector of pET28a and expressed in Escherichia coli BL21 and then purified by metal affinity chromatography using a nickel-nitrilotriacetic acid (Ni-NTA) column. The purified beta-1,3-1,4-glucanase demonstrated 24.05 and 12.52 U ml(-1) activities for the substrates of barley beta-glucan and lichenan, respectively, and the specific activities were 728.79 and 379.1 U mg(-1) for them, respectively. The optimal temperature and pH of the purified enzyme were 40 degrees C and 6.4, respectively. When barley beta-glucan was used as the substrate, K (m) was 5.34 mg ml(-1), and K (cat) showed 7,206.71 S(-1), thus the ratio of K (cat) and K (m) was 1,349.67 ml s(-1) mg(-1). The activity of beta-1,3-1,4-glucanase was affected by a range of metal ions or ethylenediaminetetraacetic acid (EDTA).

• Authors: Jiayun Qiao, Bing Dong, Yihang Li, Bo Zhang, Yunhe Cao
• Journal: Applied biochemistry and biotechnology (Appl Biochem Biotechnol) Vol. 152 Issue 2 Pg. 334-42 (Feb 2009) ISSN: 1559-0291 [Electronic] United States
• PMID: 18365147 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Metals
• Recombinant Proteins
• Endo-1,3(4)-beta-Glucanase

Topics

• Amino Acid Sequence
• Bacillus subtilis (enzymology, genetics)
• Base Sequence
• Cloning, Molecular
• Endo-1,3(4)-beta-Glucanase (chemistry, genetics, isolation & purification, metabolism)
• Escherichia coli (genetics)
• Gene Expression
• Hydrogen-Ion Concentration
• Metals (pharmacology)
• Molecular Sequence Data
• Protein Stability
• Recombinant Proteins (chemistry, genetics, isolation & purification, metabolism)
• Temperature