Introduction Of Disulfide Bridge Enhances The Thermostability Of The GH11Xylanase Xyn â…¢ From Aspergillus Niger

The xylanases had widely application foreground in the paper industry, the feedindustry as well as in the foodstuff industry. Our lab had screened aspergillus A-25which was ahigh yield strain in xylanase. The β-1,4-endoxylanse from A-25was belong to G/11familiy withoptimum temperature at46℃which was not suitable for high-temperature enviroment. Inprophase of this study we introduced three extra disulfide bonds:33(S130C/N174C)，F61(G30C/D47C) and G16(V128C/F178C)；then cut two free amino acid from mutant33’s carbonterminal to generated the mutant S4(33C-2S). through sequencing we discovered that all of thefour mutants had20-30abnormal amino acids to their N terminal. So we only measuredthe properties of crude enzymes.Making the mutant obtained at prophase of this study as template to cut off the abnormalamino acids from N terminal by PCR. Then linked it to PET-20b(+) and expressed in E. coliBL21(DE3) to get four mutants:33(S130C/N174C)，F61(G30C/D47C)，G16(V128C/F178C)，S4(33C—2S) with correct sequence. Analysis these character ofter expression and purification.。After measuring these pured xylanase we known:(1)The wild type xylanase23’s optimum pH was3.8, and mutant S4, G16and33were all at3.6comparatively. By contrasting with wild type they decreased0.2and0.4respectively. So thosemutants’ acid-resistant had enforced.(2) Those mutants’ optimum temperature were at48℃and improved2℃by contrast to thewild type.(3) The wild type’s half-life was27min at the50℃. The mutant S4, F61,33, G16’s half-lifewere366,175,68and55min and extended by366,175,68and1times respectively. The mutant S4was the best obviously increased.(4) The mutant S4’s Km was scant below the wild type, so it was appetency had beenenhanced. The Vmax of those mutant S4, F61,33and G16had been increased by3.2,12.4,7and2.1times respectively. The Kcat of F61and33had been improved by29.6and7.7times.All of those result had approved that the thermalstability of xylanase had enhanced, and thecharacter of those xylanases had improved.