Recombinant construction,optimization and enzymatic properties analysis of cellobiohydrolase 1

In this study,the c DNA sequence was cloned from the total RNA of the strain Rhizopus stolonifer TP-02.Sequence analysis showed that this gene encoded a polypeptide with 526 amino acids. Homology modeling was established by DS2.5 software to study cbh1 catalytic hydrolysis and the active site of CBHI was a tunnel structure of “barrel. The cbh1 c DNA sequence of the gene was inserted into expression vector p ET22b(+) and expressed in E.coli BL21(DE3). Using two-step chromatography column to purification of CBHI. Enzymatic properties of the recombinant CBHI were also investigated. Results showed that the recombinant CBHI activity could reach 3.57 IU/mg at the IPTG concentration of 0.5 mmol/L at 20 ℃ for induction 14 h. The optimum reaction temperature and p H were 55 ℃ and 5.5,and it was stable over a range of p H 4.5~6.5. Fe3+and Co2+were promoted to CBHI activity and the Kmwas 6.1 mg·m L-1with avicel.