Abstract
Wild type (WT) DNA sequence, which encoded a mature β-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 E206G and E314G were constructed by overextension PCR (OE-PCR). Glutamic acids of the 206th and 314th sites in the amino acid sequence of β-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 β-mannanase showed optimal activity at 50 °C and pH 2.4. The kinetic parameters of K m and V max for purified β-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 E206 and E314 were the catalytic residues of β-mannanase.
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Acknowledgement
This research was supported by funds from National Natural Science Foundation of People’s Republic of China (No. 30600433) and National Basic Research Program (2004CB117503).
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Chen, X., Lu, W., Cao, Y. et al. Prokaryotic Expression, Purification and Characterization of Aspergillus sulphureus β-Mannanase and Site-Directed Mutagenesis of the Catalytic Residues. Appl Biochem Biotechnol 149, 139–144 (2008). https://doi.org/10.1007/s12010-007-8037-7
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DOI: https://doi.org/10.1007/s12010-007-8037-7