Screening Of Producing Cellulase Bacteria And Ultraviolet Mutagenesis

With Energy crisis and environment pollution, biomass ethanol, one of the renewable clean energy, further research and development is imminent. Although for fermentation technology and metabolic engineering research at home and abroad is relatively broad, but the progress is still larger deficiencies. On the one hand, the known cellulase activity is low, and cannot be directly effective degradation natural crystallization of lignocellulose. Nature, on the other hand, a large number of microorganisms can direct the rapid use of natural wood cellulose to rapid reproduction. Screening and access highly active cellulase and its bacteria, it has great significance for cellulose ethanol research.This first step of experiment is got the346single colony strains of bacteria through bacterial culture, which is isolated from the nature of the environment in the degradation of cellulose samples of the material. Preliminary screening is the halo of Congo red method. We got143bacteria strains with cellulase activity. And in the beginning of screening process, furthermore, we found that the cellulaseactivity with the size of halo diameter of CMC degradation has no direct relation. At the beginning screen we got143strains that have the cellulase activity. To do further more accurate second screening, through the enzyme production after training, we determined of reducing sugars by DNS method. After second screening, we choose the6strains of bacteria whose cellulose degradation enzyme activity are highest, and these6strains of bacteria strains for the next experiment.Through ultraviolet mutagenesis, enzyme activity is highest in second scree ning, the6strains of bacteria by mutagenic treatment. Get a lot of strain after mutation, and respectively determination of enzyme activity after enzyme prod uction cultivation, through the way of DNS method. Filter the improved of enz yme activity after a mutation and the genetic stability of strain, named strain E140’, which enzyme activity is0.90IU/mL, increased by32.35%than the en zyme activity (0.68IU/mL) of starting strain. Using DNA sequence homology for the sources and species of bacterial strains E140’. PCR clone with bacteria highly conserved coding sequence of16S ribosomal RNA (rRNA). After a high-fidelity enzyme PCR cloning, connected pMD(?)18-T Simple Vector, transformation competence, sequencing by the BGI. After MEGA5.05software by the phylogenetic tree construction analysis, E140in the same evolutionary branches with Bacillus amyloliquefaciens. While, gram staining proved E140for gram-negative bacteria, and the morphology of E140is rod-shape.For strain E140’ conditions of enzyme production, carries on the single factor optimization experiment, we got a single enzyme production optimization of cultivation conditions:bran as the best carbon source, the initial pH6, temperature of37℃, with fluid amount to30mL, enzyme production cultivation time is4days, and add a little of Ca2+and Mg2+can increase the crude enzyme activity. According to the optimization of enzyme production culture conditions for enzyme production fermentation experiment, get strain E140’of the enzyme activity of1.47IU/mL, than the original enzyme production conditions (enzyme activity is0.9IU/mL) enzyme activity increased by63.33%, while increased by116.18%than the original strains E140enzyme activity (0.68IU/mL).By the optimization of multi-factor experiment, we got the further optimization of enzyme production conditions:3.3d training time, liquid volume of58mL, of188rpm, bran content is85g/L, then strain E140’producing enzyme enzyme activity reached1.74IU/mL. It is1.93times than the enzyme activity before optimization (0.90IU/mL), and is2.56times than the enzyme activity of strain E140, the original strain, before optimization (enzyme activity0.68IU/mL).