Screening Of Strains For High β-Glucosidase Producing From The Giant Panda Intestine And Cloning, Expression Of Itsβ-Glucosidase Gene

The giant panda is a rare animal endemic to China, whose staple food is rich in high fiber like bamboo, but its ability to digest cellulose is poor, mainly depends on the intestinal microbial decomposition of cellulose, β-glucosidase is an important rate limiting enzyme in the process of microbial degradation of cellulose, the different sources of β-glucosidase and its gene are different in the size, structure and physicochemical characteristics. The study include isolation and screening of cellulose decomposing bacteria from giant panda intestine, further study of the B-glucosidase activity and the influencing factors, construction of engineering bacteria of B-glucosidase gene expression, is of great significance to study the giant panda gut micro flora.Isolation, screening from Ya’an Bifengxia zoo 15 giant pandas in fecal samples of 5 strains with cellulose decomposing ability of bacteria, physiological and biochemical, morphological identification of 5 strains of bacteria,5 strains of bacteria were inferred as bacillus,5 strains and related species 16SrDNA sequence phylogenetic tree construction with the Bacillus, are found in sp. (HM17927.1) in the same branch of evolution, the shortest distance, the results are consistent with the BLAST results. Combined with the 16SrDNA sequence analysis,5 strains of bacteria were determined for the Bacillus of Firmicutes.The results of β- glucosidase activity of strains QY, HH, YH, YY, AA were measured by salicin respectively were 3.65 U/mL,2.43 U/mL,2.51 U/mL,1.36 U/mL,7.32 U/mL. Select a higher enzyme activity of strain QY, YH, AA, through inoculation, change the culture time, temperature, initial pH of medium, carbon source, nitrogen source to optimized condition of enzyme production, eventually found the enzyme activity of 3 strains were both increased, reaching 21.83 U/,19.05 U/mL mL 20.77 U/mL respectively.Select strain QY bacteria as the β-glucosidase gene provider, cloning of B-glucosidase gene bgl H, which size is 1321 bp, the bgl H gene was transformat and expression successfully by the expression vector pET-22b (+)in Escherichia coli BL21, we found the enzyme activity of induction of supernatant of BL21/pET-bglH have reached 36.3 U/mL.Research the enzymatic properties of 13-glucosidase of expression of the crude product, we found that the obtained β-glucosidase by expression, its optimum temperature is 36℃, the optimum pH is 5.5, its have a good thermal stability at 30℃, the enzyme activity can still maintain the original enzyme activity in 60% which deal with at 40℃ for 20 min, when the environmental temperature over 50 ℃, the enzyme activity decreased rapidly, the higher the temperature, the enzyme activity decreased more quickly. In the pH in the range of 5-6.5, the relative enzyme activity is higher, when pH exceeds this range, the enzyme activity decreased rapidly. In the alkaline environment, the enzyme activity decreased faster, showing the stability is worse, when pH exceeds 8, the enzyme activity almost disappear.The results of this study show that, cellulose decomposing bacteria exist in panda intestine, through the optimization of the fermentation conditions of bacteria, can improve the activity of cellulose decomposing bacteria. At the same time, the cloning of β-glucosidase gene and construction of engineering bacteria, managed to get high level expression of β-glucosidase, provides the bacteria source reference for giant panda intestinal probiotics.