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Expression Of The Paecilomyces Sp.FLH 30 Endo ?-1,3(4) Glucanase Gene And Selenomonas Ruminantium Xylosidase Gene In Pichia Pastoris

Posted on:2017-07-28Degree:MasterType:Thesis
Country:ChinaCandidate:W HuFull Text:PDF
GTID:2310330488469798Subject:Food Science
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From Pecilomyces sp. FLH 30 ?-1,3(4)-glucanase has an effect on the cell wall of plants, it could efficiently hydrolysis plant cell wall.Selenomonas ruminantium is a dominant bacteria in the rumen of the rumen, The production of Selenomonas ruminantium plays a key role in the process of complete degradation of xylanase.This experiment takes Paecilomyces sp.FLH30 endo ?-1,3(4) glucanase gene and Selenomonas ruminantium xylosidase gene as the research object. The results are as follows:Optimization of codon sequence of Paecilomyces sp.FLH30 endo ?-1,3(4) glucanase, build a new carrier, using Sac I enzyme digestion DNA, by 1500 V of the current stimulation, transduction into the strain GS115, and the level of test tube, shake flask and fermentation tank were expressed respectively, the enzyme activity increased from the original 11.26 IU/mL to 259.73 IU/mL. The results of recombinant enzyme GLUnm measured in the experiment showed: The change of temperature in the temperature of 37-50? is small, and the change is not remarkable in the category of pH 5.0-8.0. GLUnm could hydrolysis of Lichenim and Barley ?-glucan, it has no hydrolysis effect on Oat spelt xylan, CMC-Na and pNPX.Optimize the synthesis of xylosidase gene, get a new xylosidase gene XSAnm, construction of recombinant vector pPIC9K-XSAnm, using Bgl II enzyme digestion DNA, by 1500 V of the current stimulation, transduction into the strain GS115, It is expressed in three different expression environments, in the test tube, in shake flask, in 10 L fermentation tank to express, the recombinant enzyme activity was increased from 17.54 IU/mL to 287.61 IU/mL. The enzymatic property experiments showed: The recombinant xylosidase enzyme was stable in pH 5.0-6.0 and the temperature was 40-55?, The recombinant xylosidase can degrade pNPX, Oat spelt xylan, the recombinant xylosidase had no hydrolysis effect on CMC-Na, Lichenim and Barley ?-glucan, SDS and EDTA will reduce the recombinant xylosidase XSAnm activity.When GLUnm malt juice recombinant enzyme reaction, add the recombinant enzyme GLUnm filtration rate, reaction rate obviously was added to the control group of malt juice improve the 44.04%; adding recombinant xylosidase reaction rate was added filtration rate of exogenous enzymes increased 25.31%.
Keywords/Search Tags:Paecilomyces, ?-1,3(4)-glucanase, Selenomonas ruminantium, xylosidase, Pichia pastoris, expression, enzymatic properties
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