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- Title
Catalytic Properties of the Active Pocket Key Tryptophan on Chitinase Chi304.
- Authors
TIAN Xiaoqian; LU Haiqiang; WU Ningfeng; TIAN Jian; GUAN Feifei
- Abstract
There are binding sites and hydrolytic active sites in chitinase, and the binding of key amino acids to substrate chitin can appropriately regulate the hydrolytic activity of the enzyme. The thermophilic chitinase Chi304 was as experimental material. Swiss-Model and analysis of its advanced structure revealed the presence of two tryptophans (W140 and W272) near its substrate binding pocket. And these two tryptophans were site-directed mutated to alanine. High performance liquid chromatography was used to detect the hydrolysis products of the enzyme. The ratio of product triacetyl chitosaccharide to diacetyl chitosaccharide (DP3/DP2) was used to evaluate the effect of the mutants. The mutants (W140A, W272A and W140/272A) hydrolyzed colloidal chitin, and the proportions of product (DP3/DP2) were increased by 23.3%, 45.7% and 80.0% compared with that of wild type, respectively. The results showed that W140 and W272 were the key amino acids affecting the binding of enzyme to substrate, and the mutation of alanine to Chi304 increased the endogenous activity and decreased the exogenous activity.
- Subjects
CHITIN; CHITINASE; HIGH performance liquid chromatography; HYDROLASES; TRYPTOPHAN; DIACETYL
- Publication
Journal of Agricultural Science & Technology (1008-0864), 2023, Vol 25, Issue 2, p76
- ISSN
1008-0864
- Publication type
Article
- DOI
10.13304/j.nykjdb.2021.0877