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United States Department of Agriculture

Agricultural Research Service

Research Project: BIOLOGICAL AND BIORATIONAL CONTROL OF THE FORMOSAN SUBTERRANEAN TERMITE Title: Differential cellulolytic activity of native-form and C-terminal tagged-form cellulase derived from coptotermes formosanus and expressed in E. coli

Authors
item Zhang, Dunhua
item Lax, Alan
item Raina, Ashok
item Bland, John

Submitted to: Insect Biochemistry and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 25, 2009
Publication Date: July 22, 2009
Repository URL: http://hdl.handle.net/10113/34952
Citation: Zhang, D., Lax, A.R., Raina, A.K., Bland, J.M. 2009. Differential cellulolytic activity of native-form and C-terminal tagged-form cellulase derived from coptotermes formosanus and expressed in E. coli. Insect Biochemistry and Molecular Biology 39:516–522

Interpretive Summary: Formosan subterranean termites are natural wood-eating insects. The success in using and digesting of the wood/cellulosic materials depends on the cellulolytic enzymes present in the termite gut system. One of the key enzymes is cellulase, which breaks internal bonds to disrupt the crystalline structure of cellulose and expose individual cellulose polysaccharide chains. In this study, the gene encoding for the cellulase was isolated and cloned into bacterial (E. coli) expression vector, which could produce the gene-encoding protein. For the first time, we demonstrated that we could produce the recombinant cellulase in E. coli cells in large quantity with high biochemical activity. This allowed us to characterize the properties of the cellulase in vitro. The success of this study has dual impacts on future research: screening of cellulase-inhibiting chemicals/molecules which could be developed into (bio-) pesticides and applying for degrading of agricultural by-products (cellulosic materials) for sugar based biofuel production.

Technical Abstract: The endogenous cellulase gene (CfEG3a) of Coptotermes formosanus, an economically important pest termite, was cloned and overexpressed in both native form (nCfEG) and C-terminal His-tagged form (tCfEG) in E.coli. Both forms of recombinant cellulases showed hydrolytic activity on cellulosic substrates. While the tCfEG could be purified to near homogeneity with a simple procedure, the nCfEG was more active and stable than tCfEG. The differential activities of nCfEG and tCfEG were also evidenced by hydrolytic products they produced on different substrates. On CMC, both acted as endoglucanase, randomly hydrolyzing internal ß-1,4-glycosidic bonds and resulting in smear polymers with different lengths, though cellobiose, cellotriose, and cellotetraose equivalents were noticeable. The hydrolytic products of tCfEG were one unit sugar less than those produced by nCfEG. Using filter paper as substrate, however, the major hydrolytic products of nCfEG were cellobiose, cellotriose and trace of glucose; those of tCfEG were cellobiose, cellotriose and trace of cellotetraose, indicating a property similar to that of cellobiohydrolase, an exoglucanase. The results represented in this report for the first time will have impact on further investigation on designing cellulase-inhibitor based pesticides and applying the cellulases for sugar based biofuel production.

Last Modified: 7/24/2014
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