Studies On The Strain Screening And Biochemical Characteristics Of A Cellulase-producing Strain From The Larval Gut Of Bombyx Mori

Cellulose existing widely in nature is the most cheap and the most abundant renewableresources in the word. Cellulase produced by microorganism can transform cellulose intoenergy material. That not only can reduce pollution on the environment caused by fossilenergy, still can alleviate or solve the energy crisis.12cellulase-producing strains were isolated from the larval gut of Bombyx mori by theselective culture medium. They were fermented to produce cellulase and CMCase specificactivity of the cellulase was detected. CMCase specific activity of cellulase produced by thestrain BMC-2was the highest and was12.742U/mg.The results of morphological observation showed that the colony of strain BMC-2suborbicular, edges neat; surface appeared white villous; mycelium was close and branching.Sporangiophore solitary, was without the diaphragm, grew from aerial hyphae directly; itscolour and lustre were as aerial hyphae. Sporangium was spherical or pyriform. Spore wascolorless, round or oval. Combining above morphological observation and “Manual ofFungal Identification”, strain BMC-2was maybe Mucor sp. Through the analyses of18SrRNA gene sequences, the strain BMC-2was identified as Mucor sp. And its accessionnumber of GenBank was KC009579.Response Surface Methodology （RSM） was used to optimize components of culturemedium for optimizing cultural conditions of the strain BMC-2. The results showedCMC-Na, peptone and CaCl₂had influence on cellulase-producing significantly. Theaffecting order was CMC-Na, CaCl₂and peptone, in turn. The optimized components ofculture medium were as follows: CMC-Na20.4g/L, peptone10.0g/L, CaCl₂6.0g/L. Underthe optimal conditions, the CMCase specific activity was16.209U/mg.Fermentation conditions for the cellulase-producing, in which CMCase specificactivity was taken as response value, were optimized with Plackett-Burman design, steepestascent test and Box-Benhken design. The results showed that fermentation time,fermentation temperature, initial pH of medium and rotational speed had influence oncellulase-producing significantly. The affecting order was fermentation time, initial pH ofmedium, fermentation temperature and rotational speed, in turn. The optimized fermentationconditions were as follows: fermentation time94.35h, fermentation temperature30.3oC,initial pH of medium7.01and rotational speed179r/min. Under the optimal conditions, the CMCase specific activity was25.526U/mg that was increased by1times compared withthe initial fermentation condition.The characteristics of the cellulase produced by the strain BMC-2were researched. Thecellulase showed high activity and satisfactory stability at pH6.0⁹.0and below55oC. Theoptimal conditions for the enzymatic reaction were55oC and pH8.0. The metal ions,including Ca²⁺, Mn²⁺, Fe²⁺and K⁺, showed activated functions on cellulase, while the ions,including Cu²⁺, Na+, Zn²⁺, Mg²⁺and Ba²⁺, inhibited the cellulase activity. The inhibitingorder was Ba²⁺, Mg²⁺, Zn²⁺, Na+and Cu²⁺, in turn.