Expression Of Fibrobacter Succinogenes1,3-1,4-β-glucanase Gene In Pichia Pastoris

1,3-1,4-β-glucanase(EC3.2.1.73), as an important industrial enzyme, has been widely used in thebrewing, animal feed and textile industry. Ruminant rumen is considered to be the highest conversionefficiency of NSP bioreactor. Among them, β-glucan enzymes of Fibrobacter succinogenes play animportant role in the degradation of plant cell walls. This experiment is increased the expression with geneoptimization and phenotype. The recombinant enzymatic properties were studied.According to the codon usage frequency of highly expressed genes in Pichia pastoris,1,3-1,4-β-glucanase gene derived from rumen bacterium Fibrobacter succinogenes was optimized, and193nucleotides were changed. The G+C content was adjusted from54%to44.2%, and AT-rich stretches wereeliminated to avoid premature termination. The original gene and modified gene were chemical synthesizedand constructed two yeast expression vectors pPIC9K-GLU and pPIC9K-GLUm. Then, the optimized andwild β-glucanase gene was expressed in Pichiapastoris GS115. Results showed, by comparison with thewild β-glucanase gene, the activity of the optimized glucanase increased25.1%at the flask level, and theactivity of the optimized glucanase increased above2times in the density level.In order to study the effect of different phenotype, the recombinant vectors pPIC9K-GLUmrespectively after being linearized with Sal I and Bgl II are transformed into Pichia pastoris GS115withelectronic pulse, then the recombinant yeasts Mut+å'ŒMutsare obtained through culturing on nutritiondeficient medium.. The expression of Mut+is higher than Mutsby the test of Congo red. The expression ofMut+is53times than Mutsin the density level.The properties of recombinant yeast176express of specific activity reach to10397U/mg afterpurification. The MW of recombinant enzyme is between44.3KD and29KD, it is approximately87.2%of the total secreted protein. Enzymatic properties analysis showed that, the optimum pH and reactiontemperature of the purification recombinant glucanase GLUm was5.0and37℃. Substrate specificityanalysis showed that, GLUm could hydrolysis barley β-glucan and lichenan, but not oat spelt xylan, birchxylan and sodium carboxymethyl cellulose. The optimum reaction time and enzyme concentration of thepurification recombinant glucanase GLUm was3h and40U/g by digestion outside. And the best digestionwas barely among three feeds. In the action of pepsin and trypsin after60min, the GLUm still retained50.5%of the maximal activity. Therefore, this β-glucanase had potential application as a feed enzyme.