Hao Zhikui, Wu Hangui, Yang Meiling, Chen Jianjun, Xi Limin, Zhao Weijie, Yu Jialin, Liu Jiayang, Liao Xiangru, Huang Qingguo
Institute of Applied Biotechnology, Taizhou Vocational & Technical College, Taizhou 318000, China.
Department of Crop and Soil Sciences, University of Georgia, Griffin, GA 30223, USA.
Int J Mol Sci. 2016 May 26;17(6):825. doi: 10.3390/ijms17060825.
Two CHI genes from Chitinolyticbacter meiyuanensis SYBC-H1 encoding chitinases were identified and their protein 3D structures were predicted. According to the amino acid sequence alignment, CHI1 gene encoding 166 aa had a structural domain similar to the GH18 type II chitinase, and CHI2 gene encoding 383 aa had the same catalytic domain as the glycoside hydrolase family 19 chitinase. In this study, CHI2 chitinase were expressed in Escherichia coli BL21 cells, and this protein was purified by ammonium sulfate precipitation, DEAE-cellulose, and Sephadex G-100 chromatography. Optimal activity of CHI2 chitinase occurred at a temperature of 40 °C and a pH of 6.5. The presence of metal ions Fe(3+), Fe(2+), and Zn(2+) inhibited CHI2 chitinase activity, while Na⁺ and K⁺ promoted its activity. Furthermore, the presence of EGTA, EDTA, and β-mercaptoethanol significantly increased the stability of CHI2 chitinase. The CHI2 chitinase was active with p-NP-GlcNAc, with the Km and Vm values of 23.0 µmol/L and 9.1 mM/min at a temperature of 37 °C, respectively. Additionally, the CHI2 chitinase was characterized as an N-acetyl glucosaminidase based on the hydrolysate from chitin. Overall, our results demonstrated CHI2 chitinase with remarkable biochemical properties is suitable for bioconversion of chitin waste.
从美源几丁质分解菌SYBC-H1中鉴定出两个编码几丁质酶的CHI基因，并预测了它们的蛋白质三维结构。根据氨基酸序列比对，编码166个氨基酸的CHI1基因具有与GH18 II型几丁质酶相似的结构域，编码383个氨基酸的CHI2基因与糖苷水解酶家族19几丁质酶具有相同的催化结构域。在本研究中，CHI2几丁质酶在大肠杆菌BL21细胞中表达，并通过硫酸铵沉淀、DEAE-纤维素和Sephadex G-100层析进行纯化。CHI2几丁质酶的最佳活性出现在40℃和pH 6.5条件下。金属离子Fe(3+)、Fe(2+)和Zn(2+)的存在会抑制CHI2几丁质酶的活性，而Na⁺和K⁺则促进其活性。此外，EGTA、EDTA和β-巯基乙醇的存在显著提高了CHI2几丁质酶的稳定性。CHI2几丁质酶对p-NP-GlcNAc有活性，在37℃时Km和Vm值分别为23.0 µmol/L和9.1 mM/min。此外，基于几丁质的水解产物，CHI2几丁质酶被鉴定为N-乙酰葡糖胺酶。总体而言，我们的结果表明，具有显著生化特性的CHI2几丁质酶适用于几丁质废料的生物转化。
