title: Improvement in the thermostability of chitosanase from Bacillus ehimensis by introducing artificial disulfide bonds.
abstract: OBJECTIVE
  To determine the effects of artificial disulfide bridges on the thermostability and catalytic efficiency of chitosanase EAG1.
  
  
  RESULTS
  Five artificial disulfide bridges were designed based on the structural information derived from the three-dimensional (3-D) model of chitosanase EAG1. Two beneficial mutants (G113C/D116C, A207C-L286C) were located in the flexible surface loop region, whereas the similar substitutions introduced in α-helices regions had a negligible effect. Mut5, the most active mutant, had a longer half-life at 50 °C (from 10.5 to 69.3 min) and a 200 % higher catalytic efficiency (K cat/K m) than that of the original EAG1.
  
  
  CONCLUSIONS
  The contribution of disulfide bridges to enzyme thermostability is mainly dependent on its location within the polypeptide chain. Strategical placement of a disulfide bridge in flexible regions provides a rigid support and creation of a protected microenvironment, which is effective in improving enzyme's thermostability and catalytic efficiency.
authors.affiliation: Laboratory of Enzyme Engineering,Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, 266071, People's Republic of China. shengjun@ysfri.ac.cn.
authors.forename: Jun
authors.initials: J
authors.lastname: Sheng
authors.affiliation: Laboratory of Enzyme Engineering,Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, 266071, People's Republic of China.
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Ji
authors.affiliation: Laboratory of Enzyme Engineering,Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, 266071, People's Republic of China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Zheng
authors.affiliation: Laboratory of Enzyme Engineering,Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, 266071, People's Republic of China.
authors.forename: Zhipeng
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Laboratory of Enzyme Engineering,Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, 266071, People's Republic of China.
authors.forename: Mi
authors.initials: M
authors.lastname: Sun
mesh_terms: D001407:Bacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain; D004220:Disulfides / Q000737:chemistry*; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics; D006207:Half-Life; D006358:Hot Temperature; D008958:Models, Molecular; D009154:Mutation; D017433:Protein Structure, Secondary
chemical_list: D001426:Bacterial Proteins; D004220:Disulfides; D006026:Glycoside Hydrolases; C019868:chitosanase
keywords: Bacillus ehimensis; Chitosanase; Disulfide bridge; Homology modeling; Kinetic parameters; Thermostability