Isolation, Identification, Mutagenesis, Fermentation Conditions And Enzymatic Properties Of Cellulose-Degrading Bacterium Bacillus Licheniformis CMC-4

Cellulase is an enzyme that hydrolyzes the β-1,4-glycosidic bonds in cellulose to release glucose units. The cellulases contain exoglucanase(EC.3.2.1.91), endoglucanase (EC.3.2.1.4) and β-glucosidase (EC.3.2.1.21). Applications of cellulases is somewhat limited by both their low catalytic efficiency and stability. Researches about the cellulose degradation microbes have been widely involved bacterias due to their higher growth rate, more complex glycoside hydrolases providing synergy with higher potency because of organismal diversity of extreme niches. For the industrial application requirements, discovering new special cellulase is becoming the research focus. Among bacteria, Bacillus spp. has received widespread attention for their growth rate, extracellular secretion and biosafety capacity, etc. In recent years, many researchers have reported about purification and characterization of cellulases from isolated Bacillus spp., such as B.brevis, B.pumilus, B.amyoliquefaciens and Bacillus subtilis, but few was about Bacillus licheniformis.In present study, a strain with high efficient to degrade cellulose was screened from straw returning soil, termed CMC-4. CMC-4 was identified as Bacillus licheniform is by 16s rDNA identification and physiological and biochemical identification. Through sodium nitrite mutagenesis, a mutant strain termed CMC-4-3 with high stable cellulase production was obtained. The comparative analysis of enzymatic properties and enzyme producing conditions of CMC-4 and CMC-4-3 were performed. Through determining the amount of reducing sugar released by dinitrosalicylic acid (DNS) method, the cellulase activity of CMC-4 and CMC-4-3 were 95.89 U·mL-1 and 160.29 U·mL-1 respectively. The enzyme activity of the mutant strain is up roughly 67.5% from the original isolate.The production of extracellular cellulase in microorganisms is significantly influenced by a number of factors such as temperature, pH, aeration, and medium constituents. The optimal enzyme production temperatures for CMC-4 and CMC-4-3 were all 37℃, and higher or lower temperature would inhibit the strain’s growth or the production of cellulase. As with the temperature, pH is also a vital factor for cellulase production. The optimal enzyme production pH in CMC-4 was 7.0, while 6.0 in CMC-4-3. The optimal source of carbon were maltose and glucose respectively, which were better than CMC-Na, and this was inconsistent with previous studies. And this phenotypic modulation could be traced to the decrease of ability to hydrolyze maltose caused by mutagenesis. Moreover, the optimal nitrogen source were all peptones, and the results from other workers also demonstrated that peptone had significant effect on cellulase production. The fluid quantity 60 mL/250 mL and inoculation size 2.0% were optimum conditions for CMC-4 and CMC-4-3. It was found that by comparison with CMC-4, some optimal enzyme production conditions of CMC-4-3, such as pH and source of carbon, have changed. This could be due to the mutation in cellulases or the effect of mutagenesis on other enzymes.Factors affecting the efficient of cellulose degradation by cellulase includes reaction pH, temperature, activators and inhibitors. Cellulase from CMC-4 and CMC-4-3 showed activity over a broad range of temperature (20～80℃) with the optimal activity at 50℃. Sufficient activity (more than 60%) was present at 30～70℃ for 2 h. More than 70% of the maximal activity was obtained at 80℃ for CMC-4-3, while a significant reduction in CMC-4 (approximately 40%). Mutagenesis made the scope of temperature expanded. And the optimal temperature of the cellulase from CMC-4-3 was slightly changed, while the ability of CMC-4-3 to maintain the relative enzyme activity and to keep stability was more stronger than CMC-4. This kind of stability might comply with the industrial process requirements because of its’prolonged stability under high temperature. The reaction pH have greater influences on cellulase activity. The both cellulases hydrolyzed CMC in the pH range of 3.0～9.0, and exhibited highest activity at pH 6.0. However, high activity were also recorded on either side of this point which indicated the cellulases have characteristically broad range of pH activity. Also, the two cellulases had a broad range of pH (4.0～7.4) stability where it retained more than 60% for 24h at 4℃, though maximum stability was found at pH 5.0. Among the useful properties of the Bacillus cellulases are that many are active and stable over a wide range of pH and temperature, and from this perspective, the organism CMC-4-3 has the potential to produce thermostable cellulase with broad range of pH and temperature stability which could have potential applications for wide range of industries. In our research, Fe2+, Mg2+, Co2+ and K+ can activate cellulases, while other metal ions have some inhibiting effects on enzyme activity. Among inhibitory ions, the inhibition levels of Cu2+ and Ca2+ were strongest, with a 50% decrease in enzyme activity, making up the strongest inhibitory factors of the cellulase.The enzymatic properties and enzyme producing conditions changed partly, and the mutant strain showed more wide scope of environment adaption. On these grounds, a strain of Bacillus licheniformis termed CMC-4-3 with high stable cellulase production and wide adaption scope was obtained.