Cloning Of Exocellobiohydrolase Gene Form Bacillus Pumilus And Expression In Pichia Methanolica

Exocellobiohydrolase (CBH) is the important components of cellulase. It is also the rate-limiting enzyme in the process of cellulose enzymatic hydrolysis. In this paper, Bacillus pumilus AC-4 which can produce CBH was screened. The Cbh gene of the strain was expressed in Pichia methanolic. The main research content is as follows:Firstly, a strain produced CBH was isolated and identified. The bacteria strain AC-4 was screened, which CBH activity is 0.625±0.003U/mL. The optimal pH of the crude enzyme is 9.5. And the optimal temperature is 50℃. Based on the morphological characteristics and the 16S rDNA sequence analysis, the strain AC-4 was identified as Bacillus pumilus.Secondly, the Cbh gene of Bacillus pumilus AC-4 was cloned by PCR amplification. The length of the Cbh gene is 2106bp, encoding 701 amino acids. The similarity of the Cbh gene sequence between Bacillus pumilus AC-4 and Bacillus pumilus SAFR-032 is 94%.Finally, the Cbh gene of Bacillus pumilus AC-4 was expressed in Pichia methanolic. The Cbh gene was connected with secretory expression vector pMETaA. The recombinant expression plasmid pMETaA-Cbh was constructed to integrate the Cbh gene into the genome of Pichia methanolic. SDS-PAGE protein profiles showed the Cbh gene has been successfully expressed in Pichia methanolic PMAD16. The molecular weight of the heterologous protein, CBH, is 78kDa. After small-scale expression induced by methanol, the CBH activity produced by the recombination strain NM-Cbh is 5.4U/mL.