Authors: Xiaoling Chen Wenqing Lu Yunhe Cao Defa Li
Publish Date: 2007/09/12
Volume: 149, Issue: 2, Pages: 139-144
Abstract
Wild type WT DNA sequence which encoded a mature βmannanase of Aspergillus sulphureus composed of 1152 nucleotides nt was amplified from pUCmTmann by polymerase chain reaction PCR Based on this DNA fragment mutants designated as E206G and E314G were constructed by overextension PCR OEPCR Glutamic acids of the 206th and 314th sites in the amino acid sequence of βmannanase were separately replaced by glycine in these two mutants The WT and mutant genes were ligated into prokaryotic vector pET28a + 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 NiNTA column were dialyzed to correctly refold The WT recombinant βmannanase showed optimal activity at 50 °C and pH 24 The kinetic parameters of K m and V max for purified βmannanase were 138 mg/ml and 7299 U/mg respectively However the mutant proteins did not show any activity It was demonstrated that E206 and E314 were the catalytic residues of βmannanase
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