Engineering of Alicyclobacillus hesperidum L-arabinose isomerase for improved catalytic activity and reduced pH optimum using random and site-directed mutagenesis.
A mutation, D478N, was obtained by an error-prone polymerase chain reaction using the L-arabinose isomerase (L-AI) gene from Alicyclobacillus hesperidum URH17-3-68 as the template. The mutated isomerase showed higher activity for D-galactose isomerization. The mutation site obtained from random mutagenesis was then introduced as a single-site mutation using site-directed mutagenesis. Single-site variants, D478N, D478Q, D478A, D478K, and D478R, were constructed. The optimum temperatures were all higher than 60 °C. D478A, D478N, and D478Q retained more than 80 % of the maximum relative activity of the wild-type L-AI at 75 °C. With the exception of the D478A variant, all variants showed decreased optimum pH values in the acidic range (6.0-6.5). All of the variant L-AIs could be significantly activated by the addition of Co(2+) and Mn(2+). D478N and D478Q showed higher catalytic efficiencies (k cat/K m) toward D-galactose than that of wild-type L-AI. In addition, the D478N and D478Q variants exhibited a much higher conversion ratio of D-galactose to D-tagatose at 6.0 than the wild-type L-AI. According to the molecular model, residue D478 was located on the surface of the enzyme and distant from the active site. It was supposed that the charged state of residue 478 may influence the optimum pH for substrate binding or isomerization.
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, China.
Chen
C
Fan
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, China.
Wei
W
Xu
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, China.|Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, 214122, China.
Tao
T
Zhang
Roquette America, Keokuk, IA, 52632, USA.
Leon
L
Zhou
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, China.|Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, 214122, China.
Bo
B
Jiang
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, China. wmmu@jiangnan.edu.cn.|Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, 214122, China. wmmu@jiangnan.edu.cn.
Wanmeng
W
Mu
D019747:Aldose-Ketose Isomerases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D056450:Alicyclobacillus / Q000201:enzymology* / Q000235:genetics*; D055162:Biocatalysis; D020134:Catalytic Domain; D006601:Hexoses / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D013696:Temperature
D006601:Hexoses; D008670:Metals; D019747:Aldose-Ketose Isomerases; C019715:L-arabinose isomerase; C030192:tagatose
D-tagatose; Error-prone PCR; L-Arabinose isomerase; Random mutagenesis; Site-directed mutagenesis