title: Cloning, Expression and 3D Structure Prediction of Chitinase from Chitinolyticbacter meiyuanensis SYBC-H1.
abstract: 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.
authors.affiliation: Institute of Applied Biotechnology, Taizhou Vocational &amp; Technical College, Taizhou 318000, China. haozhikui123@126.com.
authors.forename: Zhikui
authors.initials: Z
authors.lastname: Hao
authors.affiliation: Institute of Applied Biotechnology, Taizhou Vocational &amp; Technical College, Taizhou 318000, China. wuhangui@163.com.
authors.forename: Hangui
authors.initials: H
authors.lastname: Wu
authors.affiliation: Institute of Applied Biotechnology, Taizhou Vocational &amp; Technical College, Taizhou 318000, China. evoh123@126.com.
authors.forename: Meiling
authors.initials: M
authors.lastname: Yang
authors.affiliation: Institute of Applied Biotechnology, Taizhou Vocational &amp; Technical College, Taizhou 318000, China. cjjkey@126.com.
authors.forename: Jianjun
authors.initials: J
authors.lastname: Chen
authors.affiliation: Institute of Applied Biotechnology, Taizhou Vocational &amp; Technical College, Taizhou 318000, China. xilm@tzvtc.com.
authors.forename: Limin
authors.initials: L
authors.lastname: Xi
authors.affiliation: Institute of Applied Biotechnology, Taizhou Vocational &amp; Technical College, Taizhou 318000, China. zhaowj79@126.com.
authors.forename: Weijie
authors.initials: W
authors.lastname: Zhao
authors.affiliation: Department of Crop and Soil Sciences, University of Georgia, Griffin, GA 30223, USA. yu3577@uga.edu.
authors.forename: Jialin
authors.initials: J
authors.lastname: Yu
authors.affiliation: Bioengineering Division, Huanghuai University, Zhumadian 463000, China. jyliu@uga.edu.
authors.forename: Jiayang
authors.initials: J
authors.lastname: Liu
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China. liaoxiangru@163.com.
authors.forename: Xiangru
authors.initials: X
authors.lastname: Liao
authors.affiliation: Department of Crop and Soil Sciences, University of Georgia, Griffin, GA 30223, USA. qhuang@uga.edu.
authors.forename: Qingguo
authors.initials: Q
authors.lastname: Huang
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D009346:Neisseriaceae / Q000737:chemistry / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification*; D010802:Phylogeny; D055550:Protein Stability; D017433:Protein Structure, Secondary; D020539:Sequence Analysis, Protein; D012988:Soil Microbiology; D040681:Structural Homology, Protein
chemical_list: D001426:Bacterial Proteins; D002688:Chitinases
keywords: 3D structure prediction; Chitinolyticbacter meiyuanensis; SYBC-H1; chitinase; purification; recombinant