Cloning And Expression Of Endoglucanase Gene In Bacillus Subtilis WB600

Cellulose was the most renewable resources, which can be transformed into energy, food and chemical materials through degradation, it had great and real significance to solve the food shortage and energy crisis for people. Cellulose was decomposed and convered by cellulose produced by microbiology was a effective way. Cellulase is a complex enzyme system component of the endo-glucanase, exo-glucanase, β-glucosidase, which can degrade cellulose into glucose through synergy. In the process of cellulose degradation, EG priority in the role of amorphous cellulose, cutting beta1,4primary key, exoglucanase and B-glucosidase complete thoroughly degradation. Nowadays, cloning and expression of cellulose gene was the study hot of cellulose molecular biology. In this paper, based on Rhizopus stolonifer var.reflexus TP-02with high cellulase activity which was screened from the rotten wood from the ecological forests of Huangshan, the gene engineering bacteria of endoglucanase was constructed in order to incrrease endoglucanase activity. The main results are as follows:Library of cDNA expression was constructed successfully, and screen recombinants from the library, which can produce endoglucanase. Two different sequences were obtained, both of which are novel endoglucanase coding sequences. The result of SDS-PAGE electrophoresis of the recombinant endoglucanases purified by G100showed that the molecular weight of the two recombinant proteins is44kDa and46kDa, both of which have only one band. The recombined Bacillus subtilis WB600were cultured in the CMC-Na medium, the results showed that the maximum activity of recombined endoglucanase were gotten at36h,54h earlier than that of the original strain Rhizoupus stolonifer var. reflexus TP-02, and were1.034IU/ml and1.174IU/ml.Single factor and Response Surface Methodology was used to optimize fermentation conditions included packing volume, initial pH, inoculum concentration, rotational speed of the cradle and yeast extract in this study. The optimum fermentation conditions were:packing volume55mL/250mL, initial pH7.2,incolum concentration10%,rotational speed of the cradle170r/min and yeast extract0.4%.The endoglucanase yield was promoted by33.9%from1.174IU/mL to1.573IU/mL under optimum conditions.According to eg2gene of higher endoglucanase activity screened from the cDNA library of Rhizopus stolonifer var.reflexus TP-02. The primers were designed as follows:P1:5’-GAATTCAAACGACGGCCAGTGAAT-3’(the words underlined is EcoR Ⅰ restriction site), P2:5’-GAATTCACCATGATTACGCCAAGC-3 ’(the words underlined is EcoR I restriction site). PCR amplified bgll gene with the recombinant plasmid pMA5-eg2as a template. The eg2was linked with the double digested pHY-p43, and it was successfully transformed into the Bacillus subtilis WB600. The recombined Bacillus subtilis WB600were cultured in the CMC-Na medium, the results showed that the maximum activity of recombined endoglucanase was2.013IU/mL at36h.