Screening And Mutation Breeding Of A Cellulase Producing Strain And Studying On Its Conditions Of Enzyme Production

Cellulose is an inexhaustible renewable resource in nature, its rational utilization has received widespread attention. China is a large agricultural country, the annual output of crop stalks can reach700million tons. The main material of these crop stalks is cellulose. And these crop stalks are mostly not been fully rational exploitation and utilization, they have been treated by incineration or landfill disposal. It would cause environmental pollution. Cellulose can be degraded to glucose by cellulases. On the one hand, the glucose can be converted to the protein and fat by fermentation for feed. On the other hand, it can produce alcohol, butyl alcohol, citric acid, glycerol as chemicals. Cellulose conversion is accomplished by the concerted action of a group of enzymes, collectively termed cellulases. Cellulase can be widely used in the fields of food, textiles, paper making, feed, energy and so on, which can bring huge economic benefits to human.Bacillus subtilis M7which was isolated from alpine meadow soil in Gannan Tibetan Autonomous Prefecture can produce cellulase. Use ultraviolet radiation and EB mutation methods for mutagenesis breeding. The optimal liquid shaking flask fermentation and solid-state fermentation for cellulase were determined. The results are as follows:A mutant strain EB-5was obtained through treatment of ultraviolet radiation and EB mutation. After each treatment, the optimum of mutagenic conditions was researched. Two methods were used in the process of screening. The primary is congo red culture and filter liquid culture, the second is measurement of cellulose activity. The enzyme activity of EB-5was higher than the initial strain. The result of higher were:CMCase was6.11times, FPA was6.32times. CMCase was30.86U/mL, FPA was43.11U/mL.Mutant strain EB-5was fermented in liquid media and fermentation conditions were optimized. The highest level of cellulase activity was induced in the medium containing bran, with1%(NH4)2SO4as the best nitrogen source. The rotational speed, cultural original pH, cultural temperature and time of production of EB-5are160r/min,4.83,30℃and72h respectively. The age of the strain was24h, the addition amount of Tween-80was0.6%.On the base of single factors experiments, response surface methodology (RSM) was employed to optimize the conditions of EB-5producing CMCase and FPA, which3factors3levels test was designed by Box-Behnken, CMCase and FPA activity as response value. After the model prediction and verification test, the results of CMCase showed that the suitable temperature was31℃, initial pH the rotational speed was160r/min, cultural time was81h, the results of FPA showed that the suitable temperature was30℃, initial pH the rotational speed was160 r/min, cultural time was80h.The ratio of wheat straw to bran is3:2for cellulase production. The activity of cellulase appear highest when nitrogen source is (NH4)2SO4The ratio of stuff to water is1:1.The optimal pHis5.83, the optimal temperature is30℃,the optimal time of fermentation is72h.