title: Engineering of a thermostable β-1,3-1,4-glucanase from Bacillus altitudinis YC-9 to improve its catalytic efficiency.
abstract: BACKGROUND
  Error-prone polymerase chain reaction (PCR) is frequently used in directed evolution of enzymes to modify their quality. In this study, error-prone PCR was used to improve the catalytic efficiency of β-1,3-1,4-glucanase from Bacillus altitudinis YC-9.
  
  
  RESULTS
  By screening, the mutant Glu-3060 with higher activity was selected among 5000 transformants. After induction with isopropyl β-D-1-thiogalactopyranoside (IPTG), the activity of the mutant Glu-3060 reached 474.6 U mL(-1), resulting in a 48.6% increment of the parent enzyme activity. Research on the characterization of the mutated enzyme showed the optimal pH of the mutated enzyme to be 5.0, which is lower than the parent enzyme, but thermal stability was almost the same between them. Sequence analysis of the mutated enzyme revealed that three amino acids were changed compared with the parent enzyme, including K142N, Q203L and N214D.
  
  
  CONCLUSIONS
  The three-dimensional structure predicted by SWISS-MODEL of the mutated enzyme Glu-3060 showed that the substitution of three amino acids had an effect on the catalytic activity, stability and optimal pH of the enzyme, through changing the charge properties or electron density, forming secondary keys, the acidity of the amino acids and the side chain group. The sum effects of all the factors were increased activity of the mutated enzyme and decreased optimal pH, while the same thermostability was maintained, thereby increasing the suitability of the enzyme for industrial use.
authors.affiliation: College of Food Science and Technology, Nanjing Agricultural University, Key Laboratory of Food Processing and Quality Control, Ministry of Agriculture of China, Nanjing 210095, People's Republic of China.|China Tobacco Jiangsu Industrial Co., Ltd., Nanjing 210000, People's Republic of China.
authors.forename: Shurui
authors.initials: S
authors.lastname: Mao
authors.affiliation: College of Food Science and Technology, Nanjing Agricultural University, Key Laboratory of Food Processing and Quality Control, Ministry of Agriculture of China, Nanjing 210095, People's Republic of China.
authors.forename: Peng
authors.initials: P
authors.lastname: Gao
authors.affiliation: College of Food Science and Technology, Nanjing Agricultural University, Key Laboratory of Food Processing and Quality Control, Ministry of Agriculture of China, Nanjing 210095, People's Republic of China.
authors.forename: Zhaoxin
authors.initials: Z
authors.lastname: Lu
authors.affiliation: College of Food Science and Technology, Nanjing Agricultural University, Key Laboratory of Food Processing and Quality Control, Ministry of Agriculture of China, Nanjing 210095, People's Republic of China.
authors.forename: Fengxia
authors.initials: F
authors.lastname: Lu
authors.affiliation: College of Food Science and Technology, Nanjing Agricultural University, Key Laboratory of Food Processing and Quality Control, Ministry of Agriculture of China, Nanjing 210095, People's Republic of China.
authors.forename: Chong
authors.initials: C
authors.lastname: Zhang
authors.affiliation: College of Food Science and Technology, Nanjing Agricultural University, Key Laboratory of Food Processing and Quality Control, Ministry of Agriculture of China, Nanjing 210095, People's Republic of China.
authors.forename: Haizhen
authors.initials: H
authors.lastname: Zhao
authors.affiliation: College of Food Science and Technology, Nanjing Agricultural University, Key Laboratory of Food Processing and Quality Control, Ministry of Agriculture of China, Nanjing 210095, People's Republic of China.
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Bie
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D003001:Cloning, Molecular; D044644:Endo-1,3(4)-beta-Glucanase / Q000737:chemistry / Q000235:genetics*; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D011487:Protein Conformation; D015202:Protein Engineering
chemical_list: D044644:Endo-1,3(4)-beta-Glucanase
keywords: Bacillus altitudinis; characterization; error-prone PCR; three-dimensional structure; β-1,3-1,4-glucanase