Screening And Enzymology Properties Of Cellulase-producing Strains

The cellulose resources is extremely rich in Natural,if these cellulose resources could be used,not only protect the environment,and it is realize the resource recycling,so the cellulase research has always been a hot research topic in domestic and overseas.This study screening a cellulase-producing Actinomycetes from soil,through the single factor experiment and orthogonal experiment to determine the optimal fermentation medium.The strain was identified by morphology.molecular biology,physiological and biochemical analysis.And the enzymatic properties of the strain was studied.At last,the research purification of cellulase.This research paper was provided a theoretical basis for characteristic of cellulase of Actinomycetes,and has guiding significance for development and utilization of cellulase.1.The study was obtained eighteen strains has ability of degradation cellulose,including five strains of transparent cir-cle diameter ratio greater than three.Through the determine of enzyme activity of endo-?-glucanase,exo-?-glucanase,?-glucanase and FPase of these strains,it is screened strain NO.11,eventually.The enzyme activity of endo-(3-glucanase,exo-?-glucanase,(3-glucanase and FPase of srain NO.11 in the liquid fermentation was 30.06 U mL-1,10.40 U mL-1,8.05 U·mL-1 and 13.88 U mL-1,respectively.2.The study was determined the morphology,molecular biology,physiological and biochemical of strain NO.11.The result showed that the strain NO.11 is Streptomyces canus.3.The study was optimized the liquid fermentation medium of strain NO.11,In optimization experiment,the optimum nitrogen source was 0.2%beef extract;the optimal carbon sources for 2%rice straw powder,the optimal initial pH is pH 7.5,the optimal KH2PO4 adding amount is 0.3%,the optimal inoculum size is 6%,the optimal fermentation temperature is 32? and the fermentation time is five days.Under the this culture condition,the CMCase of strain NO.11 is 55.28 U·mL-1,the CMCase has improved significantly.4.In order to obtain a higher purity of cellulose enzyme,the study separated and purified cellulase used method of ammonium sulfate salting out,dialysis,G-100 column chromatography.After the purification,the CMCase is 53.09 U mL-1,the specific activity is 211.22 U mg-1,the purification multiple is 1.90.Through the SDS-PAGE showed that the molecular weight of endo-?-glucanase is obout 65 kDa,and it is achieved electrophoresis pure by ammonium sulfate salting out,dialysis and Sephadex G-100.5.In the study of enzymatic properties,the optimum pH of endo-P-glucanase,exo-?-glucanase,?-glucanase and FPase is pH 4.5,the optimum reaction temperature of exo-?-glucanase,?-glucanase and FPase is 50 ?,and the endo-?-glucanas is 55 ?,the relative enzyme activity of endo-?-glucanase can keep in over 70%in pH 4.0-6.5 and 30?-60 ?;the Mg2+ Ca2+,Mn2+ have active effect on cellulase.the Zn2+ have active effect to exo-?-glucanase but have negative effect to endo-?-glucanase and ?-glucanase,the Fe3+ have negative effect to ?-glucanase.