STUDIES ON THE FUSION OF LIGNINOLYTIC ENZYME cDNAS AND THEIR EXPRESSION

Jun Xie, Lei Feng, Ning Xu, Jun Yang, Xiaoli Xu, Shiyu Fu

Abstract


Manganese peroxidase (MnP) and lignin peroxidase (LiP) are two major peroxidases involved in lignin biodegradation. The cDNA mnp1 encoding a kind of MnPs, and cDNA clg5 encoding a kind of LiPs were fused to one cDNA mnp1- clg5 (rmc15) by over-lap PCR technology in this research. Then the recombinant cDNA rmc15 was cloned into a vector pTrcHisB to construct its efficient expression plasmid pTHmc15 in Escherichia coli. The E. coli transformed by pTHmc15 was induced by isopropyl--D-thiogalactoside. The expressed protein was analyzed by SDS-PAGE, and a new one was observed with a molecular weight of about 77KD. Enzyme activities of MnP and LiP could not be observed in the unfolded fused protein. However, the enzyme activity of MnP was detected in the recombinant protein after it was refolded and activated by Ca2+ and heme, while the activity of LiP was not detected. These results show that the enzyme activity of the protein at N-terminal was not affected, but at C-terminal it was affected in the fusion protein of ligninolytic enzymes. Therefore, it is unfeasible to construct the gene of bifunctional ligninolytic enzyme with the fusion of the cDNA mnp1 encoding MnP and cDNA clg5 encoding LiP.

Keywords


Ligninolytic Enzymes; Over-lap PCR technology; Gene Expression; Folding and Activating of proteins

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