Abstract
Chitosanases EAG1, a classical glycoside hydrolase from Bacillus ehimensis, is relatively unstable with higher temperature. This shortcoming seriously restricts its industrial application. Therefore, it is crucial to clarify the theoretical basis of thermo stability and to produce enzymes with high activity and stability. Using the structural modeling and molecular dynamical simulation, residues Leu84, Gly113, Asp116, Ala207 and Leu286 were believed to be the key residues for structural stability. Then the predicted residue Leu84 was mutated to ALA. It was shown that the L84A mutation can improve the thermal stability of chitosanases EAG1. Together with previous studies, mutations of G113C, D116C, A207C and L286C forms two sulfur bonds can change the thermal stability of EAG1. The results suggest that the thermal stability of EAG1 could be engineered by site-directed mutagenesis on the conserved residues. This protocol could be employed for improving thermal stability of other chitosanases EAG1.
Highlights
Chitosanases, enzymes that catalyze the endo-hydrolysis of glycolytic links in chitosan, belong to glycoside hydrolase families
We focused on the investigation the theoretical basis for thermo stability of EAG1 by using the structural modeling and molecular dynamical simulation
The blast result showed that EAG1 shared 91% sequence identity with a chitosanase from bacillus circulans (PDBid: 1QGI), it was selected as the template
Summary
Chitosanases, enzymes that catalyze the endo-hydrolysis of glycolytic links in chitosan, belong to glycoside hydrolase families. In order to improve the thermo stability of proteins, a number of protein engineering techniques have been developed, such as random mutagenesis, DNA shuffling, truncation, circularization [6] and so on [7, 8]. Are there any other residues that can play this role?
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