Screening,Identification Of Mongolian Horse Source Cellulose-decomposing Strains And Cloning And Expression Of Cellulase Gene

Mongolian horse is lived in Plateau with excellent characteristics of a strong adaptability and high tolerance,adapting to graze on all-year-round pasture and mainly feed on grass.To improve the utilization efficiency of Mongolian horse for fodder grass,this study Mongolian horse cecum contents as experiment material,designed to screen out the degradation ability strong cellulolytic strains,to identify,clone and express with cellulose enzyme activity of exogenous proteins,for horse source cellulose decomposition microbes and the theoretical basis for the industrial production and utilization of cellulase.The results were as belows:(1)In this study,we isolated a strain with high cellulose decomposition ability with Carboxymethylcellulose agar plates(CMC-Na)and shake-flask cultures.It was identified based on Gram staining?Physiological and biochemical characterization and 16 S rDNA partial sequence analysis,and named it as Bacillus amyloliquefaciens H3.(2)It is found that,Bacillus amyloliquefaciens H3 grew fast,its growth curve entered the logarithmic phase after 8-32 h and the stable growth phase between 32-52 h,reproduction went into recession gradually after 52 h.Optimal reaction conditions of cellulase was pH4.8 and 65°C with a reaction time of 5min,and the cellulase has strong acid.In addition,the optimum carbon source of Bacillus amyloliquefaciens H3 for fermentation was 1% corn flour,while 2% peptone and yeast powder mixture were the best nitrogen sources.The initial pH during fermentation was 4.8,with 4% inoculum,resulting in a highest and maintained a certain degree of stability for enzyme in 18-42 h.(3)Cellulase gene(endo-beta-1,4-glucanase(egl),GenBank: KT800419.1)was cloned from the Bacillus amyloliquefaciens H3 of Mongolian horse source.The length was 1500 bp,and was able to encoding 499 amino acids.It was belonged to glycoside hydrolase family 5,which consists of the glucoside hydrolase family 5 of cellulose and CMB-3 domain.(4)In this study,we successfully constructed lactic acid engineering bacteria with cellulose degradation capacity,and the enzyme activity was 0.91U/mL,which was higher than that of the original strain(0.60U/mL).