Directed Evolution Of Xylanase Using P. Pastoris Expression System And The Xylo-oligosaccharides Production

Xylanase as a semi-cellulose degrading enzyme, can be applied to interrupt β-1,4-glycosidic bond from xylan and longer wood oligosaccharide chain. It is one ofthe promising industrial enzymes, widely used in the area of food, textile, medince,pulp and paper. Espically in the industrial production of xylooligosaccharides,pretreatmenthe of raw material is alkaline conditions; A thermostable andalkali-tolerant xylanase is needed to meet the requirements of the industrialprocesses.This paper mainly studied the directed evolution of Trichoderma reeseixylanase using the P. pastoris system. About the optimization of fermentation andpreparation of xylo-oligosaccharides in the application.Replacing AOX promoter with GAP promoter, a constitutive expression vectorpPGAPZαB-xynII was constructed and an engineering strain H-B was obtained.which could constitutively secreted3.82IU/mL recombinant xylanase. Thus,theconstitutive expression of the recombinant xylanase not only significantly increasedsecurity in food and feed industry application, but also solve screening problemduring the induced expression.Based on the random mutagenesis technique, a mutant library of P. pastorissystem was constructed. Using Remazol Brilliant Blue R-D-Xylan as a chromogenicsubstrate for high throughput screening methods, a series of good mutations wereobtained. Enzyme characterization analysis showed mutant A10had obviouslyimproved thermostability and pH tolerance. Enzyme activity increased21.5%,relative activity increased from50.5%to77.8%at pH7, increased by54%.Thus, A10is more suitable for industrial environment.According to the experimental results in Shake flask, the optimum medium wasconstructed: glucose3.54%, yeast extract3.65%, bran6.4%.The optimumfermentation conditions were:30°C, inoculation10%pH6.0,220rpm. Afteroptimization, the highest xylanase activity of11.53IU/mL was achieved after48hfermentation, increased by2.4times.NBS Bioflo1103L fermentation for high density fermentation were furtherstudied.These results showed GYB is superior to BSM. Glucose is a better carbonsource than glycerol for fed-batch. More conducive to cell growth and secretion of enzyme production, manual feeding of glucose cell growth is rapider, but dissolvedoxygen linkage fermentation enzyme activity is higher.The crude recombinant xylanase was used for preparation ofxylooligosaccharides.When the mountain beech xylan was used as raw materials,30g/L, enzymatic hydrolysis rate reached29.7%at4h hydrolysis with recombinantxylanase of20IU/g and PEG6000of1.5g/L. Xylooligosaccharides yield increased32.37%and xylooligosaccharides products distribution (%)was: X4.93; X229.31;X347.17; X412.34; X54.88; X61.36.