Abstract
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.
Highlights
The massive usage of petroleum and petroleum products in the last decade, with the consequent reverse effect on minimizing consumption of these unsustainable resources, has increased the demand for the development of renewable sources [1,2]
Based on the available conserved domain sequences in the National Center for Biotechnology information (NCBI) website, the deduced amino acid sequences of the 3 cloned open reading frames (ORFs) were found to belong to Glycoside hydrolases (GHs)-12, 8, 28 for the cloned celB, celC and peh, respectively [45,46]
The enzymes belonging to GH-28 family are classified into several categories in accordance with their catalytic hydrolysis mechanism and are given specific E.C. numbers based on the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology
Summary
The massive usage of petroleum and petroleum products in the last decade, with the consequent reverse effect on minimizing consumption of these unsustainable resources, has increased the demand for the development of renewable sources [1,2]. The bioconversion of lignocellulosic materials is a challenging process which requires two steps. The lignin and the hemicellulosic parts are first degraded into simpler sugars and/or organic acids, followed by a deoxygenating step to produce a liquid fuel [4]. Design of a genetically modified microorganism for direct lignocellulosic biomass conversion purposes has recently been taken into consideration [5]. The production of several types of fuel through direct lignocellulosic biomass conversions has been demonstrated by various studies [6,7]. A genetically engineered E. coli capable of degrading pectin-rich lignocellulosic biomass by cellulolytic and pectinolytic activities has been developed [8]. The ability to ferment several pentoses and hexoses makes E. coli an ideal ethanologen for biofuel production [5,13]
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