Screening Of A Strain Producing Cellulase, Its Enzymatic Properties And Clone And Expression Of The Cellulase Gene

Cellulose is the most abundant renewable resource on earth, and the straw occupies a very large proportion of cellulose, thus how to improve the utilization rate of crude fiber of ruminants become an important direction of our research. One effective approache is to search the bacterium which can degrade the cellulose and then produce enough amount of cellulase. Lactobacillus is generally recognized as a security microorganism (GRAS), the recombinant genetic engineering Lactobacillus can set prebiotic and the dual function of exogenous gene expression in one, has a certain application prospect. This research was designed to screen and identify the strain producing thermostable cellulase from the excretion of Mulberry longicorn and identified by morphologic and biologic observation, its enzymatic properties and degradation rate of the crude fiber in the feed (bran, wheat straw, oat straw) were studied as well; meanwhile, artificial recombinant lactobacillus is constructed by connecting the cellulase gene into lactobacillus expression vector. The main results of this research are:1. A cellulase-producing bacterial strain named HY3was screened by Congo red staining method and DNS method, identified by morphologic and biologicobservation, the strain was identified as Bacillus licheniformis.2. The appropriate pH and incubation time of growth and enzyme production of HY3are6.5-7and24h respectively. The optimum temperature of the reaction with substrate is70℃, belonging to hot-resistant cellulase:at75℃for1h,65%of cellulase activities still remained; the optimum pH is6.5, and the enzyme has high reactivity in a wide range of pH (pH=5-9),but weak acid-base tolerance, it has good stability under neutral condition.3. The cellulase activity inthe medium supplemented with oat straw was the highest (5.01U/mL) and the degradation rate to crude fiber of bran was thehighest (7.41%), and the degradation rate in the medium added wheat straw was the lowest, thedegradation rate of crude fiber had no significant change over time.4. Cellulase gene Cell2A was cloned from bacillus licheniformis genomic DNA by homology primers. Cellulase gene of Bacillus licheniformis is786bp, and encodes231amino acids. The gene fragment was inserted into pET-28(a+) vector to construct prokaryotic expression plasmid pET-28-cel which then transformed into E.coli BL21(DE3).Induced by IPTG, Cell2Acan be successly expressed endoglucanase gene protein in E. coli, the cellulase protein was about30kD tested by using SDS-PAGE.5. The recombinant genetic engineering Lactobacillus can produce cellulase is successfully constructed by connecting cellulase gene Cell2A into the lactobacillus expression vector pLEM415, CMCase activity assay of0.489U/mL, has higherenzyme activity.