Screening Cellulase-Producing Bacillus Amyoliquofaciens Tx26 And Expression Of Its β-Endoglucanase Gene In Escherichia Coli

One strain of cellulase-producing Bacillus which can degrade sodium carboxymethylcellulos (CMC-Na) was isolated on the selected medium using CMC-Na as only carbon source from rotten rice straw and leaves. It was identified as Bacillus amyoliquefaciens by 16S rDNA sequences homology analysis.The production of enzyme by microorganism needs some kind of inducing factors. Various culture conditions for Tx26 strain were studied. The results showed that the optimal harvesting period of enzyme was 48h, the optimal fermenting temperature was 37℃, pH7 was the optimal fermenting pH condition, and the best carbon source was the corn-cob powder, while the best nitrogen source was yeast extract with peptone in our experiment condition.The endoglucanase gene of B. amyloliquefaciens Tx26 was amplified by PCR with primers based on DNA sequences of Bacillus cellulase genes cloned previously which downloaded from GenBank. The amplified PCR fragment was ligated to the E. coli plasmid pGEX-6p-l. The recombinant plasmid was transformed into the E.coli DH5αstrain. And the recombinant plasmid (named pGEX-Tx26) was transformed into E.coli BL21 (DE3) and induced to express. The transformants on the selective CMY plate (100μg/mL Amp added) were detected for CMCase by the Congo red method. The transformants were cultured in LB (100μg/mL Amp added) and induced by 1mmol/L IPTG for 12-16h. The CMCase activity of the recombinant was 341.86 IU/mL, which was about 4.4 times of the initial strain. SDS-PAGE showed that the molecular mass of recombinant protein was about 82 kD.The character of recombinant endoglucanase was studied in this experimentation. The results showed that the optimum reaction pH was pH5.5; the optimum reaction temperature was 55℃; Enzyme stability experiment showed that the residual activity was 70% after over night treatment at pH5-pH9, and 60% after treatment at 30℃-50℃for 1h, respectively. The result of the effect of metal ions on enzyme activity showed that Hg²⁺, Zn²⁺ and Cu²⁺ strongly inhibited the CMCase activity of the enzyme, while other metal ions showed no activation nor inhibition on the activity of the enzyme. The recombinant endoglucanase showed consistent character of the initial strain. Sequecing analysis showed that the sequence ofβ-endoglucanase gene fragment of B. amyoliquofaciens Tx26 exhibited the nucleotide homology was 99% with the endoglucanase gene of Bacillus subtilis isolate JA18, and the amino acid homology was 97%; and with eglC of Bacillus amyloliquefaciens FZB42, the nucleotide homology was 97%, and the amino acid homology was 97%.