Cloning and expression of Erwinia caratovara cellulase in Escherichia coli.

摘要

The conversion of citrus waste into fuel ethanol is an attractive alternative renewable source of energy. The enzymes required for the hydrolysis of polysaccharides in citrus waste are cellulase, pectinase, and beta-galacturonase. Erwinia caratovara is a wood-rotting bacterium known for its ability to rapidly degrade cellulosic materials using these very enzymes. The long-term goal of our project is to engineer a bacterium that will be able to hydrolyze the waste as well as use the fermentable sugars to produce ethanol.;To this end, we have cloned the open reading frame (ORF) encoding the Erwinia caratovara cellulase protein and expressed it in the bacterium Escherichia coli. A PCR product of the expected size (1121 bp) was produced and cloned into pGEMT-EASY (Promega). Following sequence confirmation, the cellulase ORF was cloned into the E. coli expression vector pTACMAT (Sigma). The expression of the enzyme has been optimized and digestion of cellulose examined.