title: [Improving thermal stability of Geobacillus sp. ZH1 carboxylesterase by error-prone PCR].
abstract: OBJECTIVE
  This study was aimed to improve the thermal stability of carboxylesterase from Geobacillus sp. ZH1 by directed evolution.
  
  
  METHODS
  A library of carboxylesterase mutants was constructed by introducing random mutagenesis using error-prone PCR to screen mutant enzymes with improved thermostability. After induction, expression and purification, the mutant enzyme was characterized.
  
  
  RESULTS
  After screening, one mutant strain 65 was obtained with improved carboxylesterase thermal stability. Sequence analysis revealed two amino acid substitutions, including T113S and M160K. According to homologous modeling, T113S was located on the fifth β-sheet. Another mutant site M160K was located on a loop between the fifth and the sixth α-helix, being on the surface of the mutant enzyme. The mutated Lys160 formed an extra hydrogen bond with nearby Thr162. The half-life of mutant enzyme 65 and the parent enzyme at 90 degrees C was 3.1 h and 1.9 h, respectively. The mutant enzyme 65 had a better thermal stability than the parent enzyme.
  
  
  CONCLUSIONS
  Directed evolution by error-prone PCR of Geobacillus sp. ZH1 carboxylesterase gene is effective to improve the thermal stability.
authors.forename: Han
authors.initials: H
authors.lastname: Liu
authors.forename: Liyun
authors.initials: L
authors.lastname: Wu
authors.forename: He
authors.initials: H
authors.lastname: Gao
authors.forename: Hui
authors.initials: H
authors.lastname: Ni
authors.forename: Huinong
authors.initials: H
authors.lastname: Cai
authors.forename: Yanbing
authors.initials: Y
authors.lastname: Zhu
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D056506:Geobacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D002265:Carboxylic Ester Hydrolases