The Stusy On Plant Cell Wall Degrading Fungi And B Their Enzyme

We screened50fungi strains of which isolated from the Qinghai–Tibet Plateau andstored in Qin-Hai University Lab. Among them,4strains had higher capacity of degradingplant cell wall were identified. Based on the different characteristics of these strains,xylanase characteristics,xylanase gene cloning,bioinformatics analysis, synergisticfunction were studied in this paper.We screened fungal isolates of Q7-31for higher,more stable,and more comprehensiveenzyme activity toward powder from rice cell walls. Degrading enzyme was induced byculturing each strain for3d in a liquid medium supplemented with0.3%powder as thesole carbon source. The culture supernatant as crude enzyme was immediately used forassaying cell wall-degrading activity via the dinitrosalicylic acid method. Among ourscreened fungal strains,the crude enzyme of Fusarium sp. Q7-31hydrolyzed the ricepowder to reducing sugars at a rate of265g min^-1mL^-1. To compare their stability, crudeenzymes of Fusarium sp. Q7-21and Q7-31strains were stored at25or40oC. Regardlessof temperature, the latter strain remained stable, with at least75%of its initial activity,for3d.Furthermore, this Q7-31enzyme showed even higher degradabilities toward othercereal plant powders, including local varieties of barley, wheat, corn, oats, and rye, thantoward rice. These results suggest that Fusarium sp. Q7-31is capable of producinghighly stable enzymes to degrade the polysaccharides within cereal cell walls. Thus,it hasgreat potential for cost-efficient bioethanol production from such feedstocks.The rice mutants of T-DNA were screened by using the crude Enzyme of Fusariumsp. Q7-31to degrading the cell wall of rice mutants, it showed that:four rice mutantstrains were selected based on the highest reducing sugar production. The geneticmutation of the strains were identified.1B-0G636:xyloglucan endotransglucosylase/hydrolase protein1precursor;1B-08331:cellulose synthase;2D-00494:CESA5-cellulosesynthase;3A-50357:xyloglucanase inhibitor. After degrading, the capacity of the mutantincreased3-5time compared with the wild type of the rice. Through single factor analysisand orthogonal experiments, effects of carbon source, nitrogen source and other factorson xylanase production from Fusarium sp. Q7-31were studied. The optimal mediumcomposition for the xylanase production was: corn steep0.5%, NaNO30.6%, K2HPO4 0.1%, MgSO4·7H2O0.05%, KCl0.05%, FeSO4·7H2O0.001%, and the pH was5.0. Afterscreening, the xylanase activity increased by3.73times from3.145U/mL to11.72U/mL.The characteristics of culture, xylanase producing condition and characteristics ofenzyme of Ref1were studied. The result showed that Ref1was a psychrophile, and thebest growing conditions were pH6,20℃and yeast extract as nitrogen sourcewhile thebest xylanase producing conditions were pH3-7,15℃and yeast extract as nitrogensource. Xylanase activity of optimized Ref1reached118.7u/mL and soluble protein60ug/mL; the specific activity of the crude enzyme was1250U/mg protein. Thexylanase has an optimal activity at50℃and pH5.0and the relative enzyme activity ofxylanase remains80%while keeping at the temperatures of15℃-40℃. Mg²⁺, Na+and8mmol/L Fe²⁺, Cu²⁺, Zn²⁺were strong inhibitors, while Ca²⁺,4mmol/L Fe²⁺, Cu²⁺, Zn²⁺and8mmol/L Mn²⁺were stimulators of the enzyme. The maximum velocity of reactionand the Kmof the xylanase were163.38mmol/mg/min and0.75mg/mL, respectively.Non-mixed and mixed enzymes produced by liquid state fermentation of fourdifferent fungi which are come from Fusarium sp., Trichoderma sp., Aspergillus sp. andPestacotiopsis sp. were used to degrading rice straw. The degrading effect which isexpressed by amounts of reduced sugar (μg.min^-1.mL^-1) was tested by DNS method.Meanwhile CMCase and Xylanase of the non-mixed and mixed enzymes weredetermined respectively. The results showed that more or less synergistic effects areubiquity in mixed enzymes on digesting rice straw, CMC and Xylan. For example, thedegrading effects of mixed enzymes of Trichoderma sp..and Aspergillus sp.improved85.60%on digesting Rice straw. To be mentioned, in these strains, CMCase and Xylanaseof enzyme of Pestacotiopsis sp. are the highest, which is up to0.3149U/mL and38.4817U/mL respectively. It’s digesting effect on Rice straw is also not bad which is28.9490μg/mL.The xlanase gene of xyn8which was cloned by our laboratory was cloned fromcDNA of Fusarium sp. Q7-31. Xyn8encodes a xylanase with a calculated molecularweight of25.7KD. The theoretical pI is6.9. The protein has a signal peptide with thesequence MVSFKFLLVAASAITGALA. The xylanase Xyl8was belonging to G11xylanase family. Construct the recombinant expression vector of pGEX_5x-1-xyn8. Thexylanase gene xyn8was expressed in Ecoli.BL21（DE3）. The production of recombine xylanase was identified by SDS-PAGE and Western blot. Recombine xylanase waspurified by affinity chromatography. The characteristics of expressed Xyl8were studied.The study of xylanase of deferent fungi has very important role in utilization of plantcell wall.