Study On Induction And Action Mechanism Of Cellualase In Two Bacillus

Cellulose is the most abundant renewable polysaccharide in nature. Its biodegradation by microorganisms is one of the major steps of the carbon cycle on Earth. The efficient utilization of this process could make a significant contribution to the solving of present environmental and ecological problems.Two strains of bacteria producing higer cellulase activities were selected by screening plate stained with Congo red and liquid culture from soil samples and preserved strains by our labrotory. X18 was purched by our labrotory, and named as Bacillus licheniformis. X10-1-2 was identified as Bacillus cereus according to classification basis. Cellulases produced by two Bacillus sp. were exocellular enzyme. Adsorption ability of produced cellulases by two Bacillus sp. on crystalline cellulose was very poor. It could be concluded, these cellulases were lack of cellulose-binding domains.Optimal culture conditions for production of cellulases using Bacillus sp. X18 and Bacillus sp. X10-1-2 were investigated. The highest level of cellulase activities were induced in the medium containing wheat bran. Optimal mass concentration for Bacillus sp. X18 and Bacillus sp. X10-1-2 were 2.5% and 3%, respectively. Bean cake was the best nitrogen source. Optimal mass concentration for Bacillus sp. X18 and Bacillus sp. X10-1-2 were 4% and 1%, respectively. Optimal culture temperature of cellulase production of Bacillus sp. X18 and Bacillus sp. X10-1-2 was 30℃. Optimal culture pH of cellulase production of Bacillus sp. X18 and Bacillus sp. X10-1-2 were 6⁸ and 5, respectively. Optimal time of cellulase production of Bacillus sp. X18 and Bacillus sp. X10-1-2 were 20h and 60h, respectively.The induction of cellulase production in two Bacillus sp. was studied, by means of measuring cellulase activities under the condition of different carbon sources. The results indicated that cellulases could not be induced by pure cellulose material as a sole carbon source. Instead, they could be induced by monosaccharide or disaccharide with reducing group. Moreover, the expression of cellulase components was synergistic. When cell wall/ envelope enzyme and endoenzyme from two Bacillus sp. acted on these inducers, analysis of reaction products by HPLC revealed that cell wall/envelope enzyme and endoenzyme from two Bacillus sp. were inactive on these inducers.Wheat bran and straw were employed for degradation by Bacillus sp. X18 and Bacillus sp. X10-1-2. During the research of fermentation, concentration of reducing sugar and soluble total sugar, activities of cellulase and hemicellulase were assayed, while weight loss, crystallinity, FT-IR and surface structure of residue were analyzed. The results showed that the process of biodegradation of lignocellulose was actually simultaneous saccharification and fermentation. Cellulose, hemicellulose and lignin in wheat bran and straw were biodegraded to diffirent tent after 5d cultivation. Surface structure of wheat bran and straw had changed evidently after biodegradation.CMCase produced by Bacillus sp. X10-1-2 and Bacillus sp. X18 were purified to homogeneity from culture supernatant. The enzyme from Bacillus sp. X10-1-2 and Bacillus sp. X18 had molecular mass of 51.3kDa and 37.2kDa, respectively.The optimum temperature of CMCase from Bacillus sp. X10-1-2 and Bacillus sp. X18 was 55℃and 50℃, respectively. The CMCase from Bacillus sp. X10-1-2 was stable below 55℃, but the CMCase from Bacillus sp. X18 had bad thermostability. The cellulases from Bacillus sp. X10-1-2 and Bacillus sp. X18 were optimally active in the pH8 and pH9, respectively, and they were stable at 50℃in a pH range 7.0⁹.0. The activity of the enzyme from Bacillus sp. X10-1-2 was increased by Mn²⁺, Fe²⁺, and EDTA. Ba²⁺, Cu²⁺, Mg²⁺, and C2O42- caused loss in enzyme activity to a lower extent. In contrast, the activity of the enzyme from Bacillus sp. X18 was increased by Fe²⁺, Mn²⁺, Mg²⁺, Cu²⁺, Co²⁺, and K+. Zn²⁺, urea, SDS, Ba²⁺, and Na+ caused loss in enzyme activity.The cellulases from Bacillus sp. X10-1-2 and Bacillus sp. X18 showed highest activity with carboxymethylcellulose. Slight activities were also observed with filter paper and xylan as substrate. For carboxymethycellulose the CMCase from Bacillus sp. X10-1-2 had a Km of 2.12mg/mL and Vmax of 5.37μg/mL·min, while CMCase from Bacillus sp. X18 had a Km of 9.84mg/mL and Vmax of 11.19μg/mL·min. The FT-IR spectrum of two CMCases showed characteristic cellulase peaks. Infrared spectrum of the amide I and II bands of CMCase showed that secondary structure of two CMCases mainly consist ofα-helix structures in solid phase. According to the proton magnetic resonance spectrum of two CMCases, we concluded that primary structure of two CMCase contain less aromatic amino acid and more alkalinity amino acid.