Screening Of A Thermostable Cellulase Producing Strain, Coloning And Expression Of The Cellulase Gene

Cellulose is the most abundant renewable resources on Earth, mainly synthesized by green plants through photosynthesis. For the lack of cellulose in gastrointestinal Tract of Monogastric animals, Feed nutrients can not make fully use by animals, which results in a waste of feed source and serious environmental pollution. Cellulase, referring to a group of enzymes that degrade cellulose to glucose,The enzymes degrades cellulose to cellobiose and oligosaccharides,and ultimately these intermediates will be degraded to monosaccharides.the present cellulose produced by microorganism can’t reach the industrial production requests because of the low enzyme activity.High temperature caused great damage to the cellulase (?) while feed pelletization.Therefort as a practical approach,to screen a thermostable cellulose gene and make it express efficiently using gene modification techniques has attracted great attention.Bacillus subtilis as a widely used expression system was made further study.The system has a clear genetic background and can secret extracell proteins directly.Compared to traditional expressing system,E coli,it has no pathogenicity.a high efficiency signal peptide screending and expressing system was construced in Bacillus subtilis in our lab,filter expression system,which can be used to produce feed enzymes.1. In this study, a hyperthermophilic cellulase-producing strain CY15-10was isolated from decayed wood and compost through the Congo red medium plate method.Based on morphology,biochemical and physiological characterization,16S rRNA sequence analysis and construction of phylogenetic tree, the homology99%same with the16S rDNA sequence of Paenibacillus polymyxa. In conclusion, strain CY15-10was identified as Paenibacillus polymyxa.2. A cellulose gene was amplified by specific primers designed according to the cellulose gene of Paenibacillus polymyxa published on NCBI,the total genome DNA of CY15-10as the template.The fragment was inserted into pMD19-T and sequenced,thehomology between the gene sequence and reported sequence reached99%through BLAST online.The gene had1482bp and an interal ORF,coding493amino acids. The signal peptide prediction results showed that the first36amino acids was the signal peptide,the cellulose protein was approximately51kDa tested by SDS-PAGE and zymogram analysis.3. In order to determine the optimal bacterial growth and enzyme production conditions,5unattached single factor experiments were at temperature37℃,pH6.0and36h cultivation;the optimum carbon source w.as CMC-Na,while the nitrogen source was beef cream.The best enzyme activity conditions were at temperature55℃,pH6.0; The enzyme performed good thermal stability:it can tolerant to75℃for10min,70%of enzyme activity remained.4. Two resitriction enzyme sites Bam H I and Sac I were introduced in the primers C2up/down,the cellulose gene was ligated to expressing vector pPS,which was digested by the same restriction enzymes,the integrating vector was transfonned into Bacillus subtilis WB700and the gene was induced expression extracellularly by3%maltose,the supernatant enzyme activity was2.4U/mL and was increased by double compared to its wild type.