Prokaryotic expression, purification and characterization of Aspergillus sulphureus beta-mannanase and site-directed mutagenesis of the catalytic residues.
Wild type (WT) DNA sequence, which encoded a mature beta-mannanase of Aspergillus sulphureus, composed of 1,152 nucleotides (nt), was amplified from pUCm-T-mann by polymerase chain reaction (PCR). Based on this DNA fragment, mutants designated as E(206)G and E(314)G were constructed by overextension PCR (OE-PCR). Glutamic acids of the 206th and 314th sites in the amino acid sequence of beta-mannanase were separately replaced by glycine in these two mutants. The WT and mutant genes were ligated into prokaryotic vector pET-28a (+) and transformed into the Escherichia coli BL21 strain, respectively. The recombinant enzyme proteins were expressed by IPTG induction and detected by Western blot. The recombinant proteins purified with Ni-NTA column were dialyzed to correctly refold. The WT recombinant beta-mannanase showed optimal activity at 50 degrees C and pH 2.4. The kinetic parameters of K (m) and V (max) for purified beta-mannanase were 1.38 mg/ml and 72.99 U/mg, respectively. However, the mutant proteins did not show any activity. It was demonstrated that E(206) and E(314) were the catalytic residues of beta-mannanase.
Chen
Xiaoling
X
National Key Laboratory of Animal Nutrition, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing 100094, People's Republic of China.
Lu
Wenqing
W
Cao
Yunhe
Y
Li
Defa
D
D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics; D015153:Blotting, Western; D020134:Catalytic Domain; D015870:Gene Expression; D005818:Genetic Engineering; D005822:Genetic Vectors; D016297:Mutagenesis, Site-Directed; D044902:beta-Mannosidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D044902:beta-Mannosidase