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Efficient Expression Of Aspergillus Niger Xylanase Gene In Pichia Pastoris And Study On Enzymatic Properties

Posted on:2010-05-19Degree:MasterType:Thesis
Country:ChinaCandidate:S Q XuFull Text:PDF
GTID:2120360302455189Subject:Microbiology
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Xylan which is mainly found in plant cell wall is the most abundant regenerate resource secondary to cellulose. For complicated structure of xylan , the degradation of it needs many kinds of hydrolases. Xylanase is the key hydrolases for degradation of xylan. Xylanase can catalyze the hydrolysis ofβ-1,4 xylose linkage of xylan into xylooligosaccharides and xylose. With this catalyze procedure, micromolecul carbohydrate which can be reused is produced. For exploitation of xylan resource, we must get xylanase with good property.In this dissertation, native xylanase xylB and mutational xylanase ST7(Ser33 and Thr186 were substituted with Cys. In order to introduce a disulfide bridge into catalytic domain. Ser39,Ser54,Thr101,Thr105 and Thr151 were substituted with Arg.) were our research objects. xylB and ST7 gene were expressed in Pichia pastoris GS115. Native xylanase and mutational xylanase were purified. Characterization and thermostability of recombinant xylanase were researched. Inducing condition of mutational xylanase recombined Pichia pastoris was optimized, and the results showed that:1. Expression of native xylanase xylB and mutational xylanase ST7 in Pichia pastorisxylB and ST7 were cloned into Pichia pastoris expression vector pPIC9K, then transferd them into Pichia pastoris GS115. Xylanase were successfully expressed after inducing with methanol. Xylanase were secreted into medium. We got two recombinants with high xylanase activity, xylanase activity can reach 82.9 U/ml and 390.2 U/ml.2. Purification of native xylanase and mutational xylanaseWith the knowledge that some xylanase can bind xylan specific, Aspergillus niger xylanase were purified with xylan. Single protein band were found in the SDS-PAGE , demonstrated that xylanase were purified. 3. Characterization of native xylanase and mutational xylanaseCompared with native xylanase, optimal reaction temperature of mutational xylanase increased from 45℃to 50℃. Half-life of the mutational xylanase increased from lower than 10 min to 120 min. Accumulation amplitude of enzyme react product increased from 36.9% to 276.3%. These dates confirmed that thermostability of mutational xylanase was greatly improved. Specific activity of xylanase increased from 2127.9±83.9 U/mg to 3330.9±173.4 U/mg. Km of xylanase decreased from 56.9 mg/ml to 37.2 mg/ml, affinity of xylanase was greatly improved. Vmax of xylanase decresed from 82.9 mmol/ml.min.mg to 27.0 mmol/ml.min.mg.4. Optimization the induce condition of the mutational xylanase Pichia pastorisInfluence of methanol quantity,initial cell density,pH and inducing time to enzyme production were reseached with four factors and three levels Orthogonal design. The importance of these factors for enzyme production were : methanol quantity>inducing time>pH>initial cell density. The best inducing condition were : BYPN medium(pH8.0), added 1% methanol every 12 h, initial cell density was OD600=3, induced 7 d. Activity of mutational xylanase can reached 1850.6±25.2 U/ml under the best inducing condition.With the purpose of improving the yield of xylanase, we are reseaching on the inducing condition of mutational xylanase production with bio-fermenter.
Keywords/Search Tags:Aspergillus niger, xylanase, site-directed mutagenesis, thermostability, Pichia pastoris
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