The disruption of two salt bridges of the cold-active xylanase XynGR40 results in an increase in activity, but a decrease in thermostability.
Cold-active xylanases are of great interest due to their large potential for application in the food industry. In this study, salt bridges of the eight glycoside hydrolase (GH) family 10 cold-active xylanases reported to date were predicted and the salt bridges specific to the cold-active xylanase XynGR40 were identified. Seven mutants were constructed to disrupt salt bridges specific to XynGR40. The results suggested that five mutants lost their xylanase activity, while the other two mutants, D30N and D83N, displayed different properties when compared with the wild-type XynGR40. First, both mutations showed an obvious decrease in thermostability, with the T1/2of D30N and D83N at 50 °C being about one half and one sixth of the wild-type, respectively. Second, both D30N and D83N had a higher specific activity than the wild-type, with activities about 13 and 163% higher, respectively. Third, both D30N and D83N had high kcatand Kmvalues, which resulted in a higher catalytic efficiency of the mutant D83N, but a lower catalytic efficiency of the mutant D30N compared to the wild-type. Our results suggested that salt bridges play important roles in both the activity and thermostability of the cold-active xylanase XynGR40. The mutant D83N had a higher kcatand higher relative activity at low temperatures than the wild-type, and is a good candidate for application in the food industry.
Fujian Key Laboratory of Marine Enzyme Engineering, Fuzhou University, Fuzhou, 350116, PR China.
Guozeng
G
Wang
Fujian Key Laboratory of Marine Enzyme Engineering, Fuzhou University, Fuzhou, 350116, PR China.
Jingjing
J
Wu
Fujian Key Laboratory of Marine Enzyme Engineering, Fuzhou University, Fuzhou, 350116, PR China.
Juan
J
Lin
Fujian Key Laboratory of Marine Enzyme Engineering, Fuzhou University, Fuzhou, 350116, PR China. Electronic address: xiuyunye@fzu.edu.cn.
Xiuyun
X
Ye
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, PR China. Electronic address: binyao@caas.cn.
Bin
B
Yao
D019943:Amino Acid Substitution; D001665:Binding Sites; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000648:ultrastructure*; D004789:Enzyme Activation; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000648:ultrastructure*; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013696:Temperature
D006026:Glycoside Hydrolases; D043364:Endo-1,4-beta Xylanases
Cold-active; Mutation; Salt bridge; Thermostability; Xylanase