Isolation And Identification Of A New Strain Producing Celluase And Optimization Of Its Fermentation Condition

Cellulose is the major part of plant cell walls and the most abundant renewal resource in the world. But this resource is always wasted because it’s hard to digest and absorb by animals. Under the condition of shortage of feed source, it’ll be the hot pot in the field of feed industry and animal nutrition to utilize the cellulose effectively.Cellulases are glycoside hydrolases which can make cellulose into oligosaccharides, even into glucose. It’s an effective pathway to utilize the cellulose by degrading cellulose using cellulase. There are some limiting factors to restrict wide application of cellulase, as follows:(i) low activity of cellulase (ii) poor tolerance for heat, acid and alkali environment (iii) microorganism producing cellulase is pathogenic.In this study, we used Congo-method to screen one strain, which can degrade cellulose effectively, from dead wood and decaying vegetation collected from the surrounding area of Yangling. By morphology and16S rDNA identification, this strain is one member of Bacillus subtilis and named Bacillus subtilis NP29. Bacillus subtilis is a generally recognized safety microorganisms (Generally Recognized As Safe, GRAS). The highest cellulase activity of strain NP29was1.8U/mL when grown at37℃, pH7.0for36h, the generation of this cellulase was closely relatively to its growth rate.We cloned one fragment of DNA, which is1446bp length, by PCR method. Bioinformatics analysis showed that this fragment contained complete open reading frame (ORF). It coded481amio acids. Sequence alignment of the protein showed that the protein is one of Glucanase family and have a high degree of similarity with a variety of heat-resistant cellulase. The result indicated that the cellulase produced by NP29strain has good heat resistance.Enzymatic properties analysis indicated that the cellulase has good tolerance to heat, acids, alkalis. The optimum temperature and pH value is65℃and4.5respectively. Heat resistance tests showed that the activity of cellulase can maintain more than85%after120seconds under65℃,70℃treatment and more than60%after120seconds under75℃ treatment. pH value tolerance test showed that the activity of cellulase can maintain more than91%after120minutes incubation in pH4.5and pH5.0environment.In order to improve the production, we used response surface methodology (RSM) to optimize the fermentation conditions of Bacillus subtilis NP29. Firstly, we used Plackett-Burman design to screen out several key factors from9fermentation factors. Then the path of steepest ascent was taken to approach the optimal region of fermentation conditions. Finally, the optimal combined conditions for maximum enzyme activity were further optimized by Box-Behnken surface response design methodology.The results showed that the best fermentation condition is as follows:starch21.44g/L, Inoculation ratio1.88%, temperature41.07℃. When strain NP29was cultured under the optimized condition for36h, CMCase activity increased from2.021U/ml to4.278U/ml, which is211.68%higher than preliminary culture.