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

Abstract

Lignocellulosic 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.

Metabolic engineering of Bacillus subtilis with an endopolygalacturonase gene isolated from Pectobacterium. carotovorum; a plant pathogenic bacterial strain

“…Previously, expression of a polygalacturonase encoding PehA gene from Pectobacterium carotovorum in E . coli was analysed by Ibrahim et al [ 41 ] who reported the molecular mass of 41.5 kDa which is lower than to that found by us. Previously, a p ehA gene encoding endopolygalacturonase expression in Pichia pastoris was presented by Liu et al [ 24 ] which exhibited a molecular weight of 40 kDa.…”

Recombinant E. coli Cellulases, β-Glucosidase, and Polygalacturonase Convert a Citrus Processing Waste into Biofuel Precursors

“…carotovorum (Pcc) expressed by Escherichia coli (E. coli) and examination of their potential for lignocellulosic biomass conversion and/or biofuel production was conducted in our earlier study for cellulases, cel8C and cel12B, and polygalacturonase, peh28. 20 The characteristics of the enzymes, cel8C, cel12B, and peh28, in terms of protein secondary or tertiary structure as well as their biochemical properties on cellulose, in conjunction with another β-glucosidase from E. coli for cel8C/ cel12B, or on galacturonic acid, for peh28, have also been reported. 21 Characterization of these enzymes and evaluation of their ability to degrade recalcitrant products found in citrus waste are the main objectives of this study.…”

“…carotovorum ( Pcc ) expressed by Escherichia coli (E. coli) and examination of their potential for lignocellulosic biomass conversion and/or biofuel production was conducted in our earlier study for cellulases, cel8C and cel12B , and polygalacturonase, peh28 . The characteristics of the enzymes, cel8C, cel12B, and peh28, in terms of protein secondary or tertiary structure as well as their biochemical properties on cellulose, in conjunction with another β-glucosidase from E.…”