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| Directional Molecular Rebuilding of β-mannanase MAN47 with Trypsin-resistance from Armillariella tabescens |
| HU Feng-juan1, WANG Xu-man1, LIU Da-ling1,2, YAO Dong-sheng1,3 |
1. Institute of Microbial Biotechnology, Jinan University, Guangzhou 510632, China;
2. Guangdong Provincial Key Laboratory of Bioengineering Medicine, Guangzhou 510632, China;
3. National Engineering Research Center of Genetic Medicine, Guangzhou 510632, China |
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Abstract The trypsin-resistance of β-mannanase MAN47 from Armillariella tabescens was improved by site-directed mutagenesis method. In order to determine the amino acid for mutation, homology modeling were used. Firstly, the structure of β-mannanase MAN47 was constructed by homology modeling and the contact of intramolecular trypsin cleavage sites (K and R) with the surrounding solvent (exposure extent) was analyzed on the basic principles of the catalytic characteristics of trypsin. Among those trypsin cleavage sites, K280 with most exposure extent to the surrounding solvent was chosen as the candidate mutation site. Then K280 was subject to simulating mutation. And K280N was determined as the suitable mutant after calculating the changes in H-bond length and the structure of enzymes before and after simulating mutation. Then the gene of the mutant K280N by site-directed mutagenesis were obtained, and it was cloned into secreted expression vector pYCα. The amplified recombinant plasmids in DH5α cells were transformed into Saccharomyces cerevisiae BJ5465 for the mutant enzyme expression. Subsequently, transformants were screened out by incubation with simulated intestinal fluid (pH 6.8 10mg/ml trypsin solution). And both the wild-type and mutated enzymes were characterized and evaluated on trypsin-resistance. Results showed that the mutant enzyme K280N had a half-life of 173 min after incubation with trypsin at 40°C, which was 74 min longer than that of the wild-type enzyme (99 min). But the optima pH and temperature of both the two enyzmes were similar. In conclusion, the trypsin-resistance of the β-mannanase MAN47 was successfully improved after site-directed mutagenesis.
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Received: 10 June 2011
Published: 25 October 2011
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