Cloning of a beta-1,3-1,4-glucanase gene from Bacillus subtilis MA139 and its functional expression in Escherichia coli.
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).
Qiao
Jiayun
J
National Key Laboratory of Animal Nutrition, China Agricultural University, Beijing, People's Republic of China.
Dong
Bing
B
Li
Yihang
Y
Zhang
Bo
B
Cao
Yunhe
Y
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D044644:Endo-1,3(4)-beta-Glucanase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
D008670:Metals; D011994:Recombinant Proteins; D044644:Endo-1,3(4)-beta-Glucanase