title: Enhanced Thermostability of Lipoxygenase from Anabaena sp. PCC 7120 by Site-Directed Mutagenesis Based on Computer-Aided Rational Design.
abstract: Lipoxygenase from Anabaena sp. PCC 7120 (Ana-LOX) was thermally unstable. So, improving the thermostability of the enzyme was quite essential. The target site of Ana-LOX selected for site-directed mutagenesis was based on computer-aided rational design. The thermostability and specific activity of Ana-LOX were improved with replacing valine with alanine at the target site 421 and the site 40. Compared to the wild-type enzyme which has a half-life (T 1/2) of inactivation of 3.8 min at 50 °C, the T 1/2 of mutant enzymes with V421A and V40A substitution increased to 4.4 and 7.0 min, respectively. The double mutant V421A/V40A showed a synergistic effect with a T 1/2 value of 8.3 min, resulting in a 1.18-fold improvement compared to the original Ana-LOX. V421A, V40A, and V421A/V40A also obtained 4.83, 41.58, and 80.07 % increase in specific activity, respectively. This study provides useful theoretical reference for enzyme molecular modification and computer-aided rational design.
authors.affiliation: Laboratory of Enzyme Engineering, College of Food Science and Technology, Nanjing Agriculture University, Nanjing, 210095, People's Republic of China.
authors.forename: Hanwen
authors.initials: H
authors.lastname: Diao
authors.affiliation: Laboratory of Enzyme Engineering, College of Food Science and Technology, Nanjing Agriculture University, Nanjing, 210095, People's Republic of China.
authors.forename: Chong
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Laboratory of Enzyme Engineering, College of Food Science and Technology, Nanjing Agriculture University, Nanjing, 210095, People's Republic of China.
authors.forename: Shuicheng
authors.initials: S
authors.lastname: Wang
authors.affiliation: Laboratory of Enzyme Engineering, College of Food Science and Technology, Nanjing Agriculture University, Nanjing, 210095, People's Republic of China.
authors.forename: Fengxia
authors.initials: F
authors.lastname: Lu
authors.affiliation: Laboratory of Enzyme Engineering, College of Food Science and Technology, Nanjing Agriculture University, Nanjing, 210095, People's Republic of China. fmb@njau.edu.cn.
authors.forename: Zhaoxin
authors.initials: Z
authors.lastname: Lu
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics*; D017033:Anabaena / Q000201:enzymology*; D001665:Binding Sites / Q000235:genetics; D017076:Computer-Aided Design; D004795:Enzyme Stability / Q000235:genetics*; D007700:Kinetics; D008084:Lipoxygenase / Q000737:chemistry / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D013696:Temperature
chemical_list: D008084:Lipoxygenase
keywords: Computer-aided rational design; Lipoxygenase; Site-directed mutagenesis; Thermostability