Journal of Petroleum & Environmental Biotechnology

Abstract

Molecular Cloning and Expression of Cellulase and Polygalacturonase Genes in E. coli as a Promising Application for Biofuel Production

Eman Ibrahim, Kim D. Jones, Ebtesam N. Hosseny and Jean Escudero

0Lignocellulosic biomass has potential for bioethanol, a renewable fuel. A limitation is that bioconversion of the complex lignocellulosic material to simple sugars and then to bioethanol is a challenging process. Recent work has focused on the genetic engineering of a biocatalyst that may play a critical role in biofuel production. Escherichia coli have been considered a convenient host for biocatalysts in biofuel production for its fermentation of glucose into a wide range of short-chain alcohols and production of highly deoxygenated hydrocarbon. The bacterium Pectobacterium carotovorum subsp. carotovorum (P. carotovorum) is notorious for its maceration of the plant cell wall causing soft rot. The ability to destroy plants is due to the expression and secretion of a wide range of hydrolytic enzymes that include cellulases and polygalacturonases. P. carotovorum ATCC™ no. 15359 was used as a source of DNA for the amplification of celB, celC and peh. These genes encode 2 cellulases and a polygalacturonase, respectively. Primers were designed based on published gene sequences and used to amplify the open reading frames from the genomic DNA of P. carotovorum. The individual PCR products were cloned into the pTAC-MAT-2 expression vector and transformed into Escherichia coli. The deduced amino acid sequences of the cloned genes have been analyzed for their catalytically active domains. Estimation of the molecular weights of the expressed proteins was performed using SDS-PAGE analysis and celB, celC and peh products were approximately 29.5 kDa, 40 kDa 41.5 kDa, respectively. Qualitative determination of the cellulase and polygalacturonase activities of the cloned genes was carried out using agar diffusion assays.