Cloning And Expression Of Novel β-Glucosidase Genes From Rhizopus Stolonifer Var. Reflexus

The plants on the earth produces a large number of cellulosic biomass resources by the photosynthesis, most of which can't be developed and utilized very well. The development and utilization of cellulose resources is still a world technical problem. The cellulase Produced by microbial degrades the cellulose to produce the clean new energy potential ethanol in order to solve the problem of shortage of energy. Cellulase is a complex enzyme system component of the endo-glucanase, exo-glucanase,β-glucosidase, which can degrade cellulose into glucose effectively. In the process of cellulose degradation, because the activity ofβ-glucosidase is low and the lowest, this inhibits the activity of cellulase. Therefore, in the proportion of three enzymes. The low activity ofβ-glucosidase activity is the bottleneck of improving the activity of cellulaseIn this paper, based on Rhizopus stolonifer var.reflexus TP-02 with high cellulase activity which was screened from the rotten wood from the ecological forests of Huangshan, the gene engineering bacteria of β-glucosidase was constructed in order to incrrease P-glucosidase activity. The main results are as follows:According to the principle that esculin sesqinhydrate can be hydrolyzed into glucose and esculetin, and esculetin can react with ammonium iron critrate to produce the black substance 6,7-dihydroxy-2H-chromen-2-one, the reconbinants could be screened easily by means of the black circle produced by the reconbinants growing on the esculin sesqinhydrate plate. Two different sequences were obtained, both of which are novel P-glucosidase coding sequences, and have been submitted to GenBank under accession no. HQ222598 and HQ222599. The result of SDS-PAGE electrophoresis of the recombinantβ-glucosidases purified by G100 showed that the molecular weight of the two recombinant proteins is 46 kDa and 47 kDa, both of which have only one band.Two novelβ-glucosidase genes were expressed in Pichia pastrois successfully and correctly. Under the condition of 1% methanol, the activity of recombinantβ-glucosidases was 1.0-fold and 1.41-fold higher than that of the original strain Rhizoupus stolonifer var. Reflexus TP-02 and reached the peak 12 h earlier. Pichia pastoris expression system is used to separately express P-glucosidases.According to bgl1 gene of higherβ-glucosidase activity screened from the cDNA library of Rhizopus stolonifer var.reflexus TP-02. The primers were designed as follows:P1:5'-GCGAAGCTTATGTGTCC CGAGGACTAT-3'(the words underlined is EcoR I restriction site), P2: 5'-TAGAATTCTCACGCCGCCTCAATCCAG-3'(the words underlined is HindⅢrestriction site). PCR amplified bgll gene with the recombinant plasmid GS115/pPIC9K-bgl1 as a template. The bgll was linked with the double digested pMA5, and it was successfully transformed into the Bacillus subtilis WB600.After the fermentation conditions of recombinant Bacillus subtilis was optimized, it was determined that in the fermentation medium, the optimal concentration of carbon cellobiose concentration was 0.8%, the best organic nitrogen source was peptone and the optimal concentration of it was 1.5%, the optimal concentration ofvpotassium dihydrogen phosphate was 1%. In the final medium,β-glucosidase activity of the recombinant strain WB600/pMA5-bgll reached 18.05 IU/mL fermentation in 34 h. Final medium composition were:0.8% cellobiose, 1.5% peptone,1% potassium dihydrogen phosphate,0.07% magnesium sulfate,0.1% yeast extract,0.5% sodium chloride,40ug/ml ampicillin.