New Insights Into The Thermostability By Deleting Two-terminal Amino Acid Residus Of The Xylanase XynⅢ From Aspergillus Niger

Microbial xylanases have attracted considerable research interest in recent years because their potential application in the animal feed , food , paper and pulp industries. and have become one of the hot spots of modern enzymology.However, it doesn't work well in the extreme environments such as highly acidic or highly alkalic.So it's all-important to reconstruct the enzyme to fulfill the industrial needs,This study chose the gene, xynⅢwhich encoding the mature protein of an endo-β-1,4-xylanase obtained from Aspergillus niger A-25 as materials to reconstrust the mutated xylanases XYNⅢ-2aa and XYNⅢ-5aa and then overexpressed in Escherichia coli to investigate the characterizations of the mutated proteins.There were three parts in this study:1) Mutated site: Using bioinformatics, three-dimensional structure of the wild-type xylanase XynⅢfrom Aspergillus niger A-25 was modeled by the software. Guided by the modeling analysis, we recognize that the enzyme exists in the form of free amino acid residues in its two terminus segment:N-terminus free residue is made up of five amino acid residus and C-terminus free residue is made up of two Ser amino acid residue. and the two fresidues is free, they don't take part in secondary structure of enzyme.2)Construction of the mutated genes: By PCR Technology, we successully deleted the wild-type genes XynⅢfrom Aspergillus niger A-25 in the N-terminus free residue and C-terminus free residue.3) The XynⅢ,XynⅢ-5aa和XynⅢ-2aa were expressed in Escherichia coli : The xynⅢ,xynⅢ-5aa and xynⅢ-2aa were inserted into the expression vector of pET20b(+) separately and transformed into E.coli BL21(DE3) which were successfully expressed in Escherichia coli by inducing with IPTG. But wasn't secreted. The mutated proteins and wild-typed protein were purified by Ni-NTA sepharose 6BFF.4) The properties of the mutated enzymes were determined:The results revealed that the optimal temperature of the mutated enzyme XynⅢ-2aa(deleting C-terminus segment) was at 52℃, It is 4℃higher than the parental enzyme;and the optimal pH(3.8) was the same as parental enzyme. Which has been obviously improved compared with those of the parental enzyme.The thermostability of mutated enzyme XynⅢ-2aa is 79.4%,73.8% after incubating the properly diluted enzyme solutions at 50℃for 40min and 52℃for 20min, respectively. While the parental enzyme is 12.6% and 2.2% respectively. While the mutated enzyme optimal pH is the same as parental enzyme. However, the deletion of N-terminus free amino acid residue has an effect on the optimal temperature. But the thermostability at 42℃for 1h and 45℃for 1h is slightly improved compared with wild-type protein.