ISSN 2736-1624
Frontiers of Agriculture and Food Technology Vol. 1 (1), pp. 001-004, December, 2011. © International Scholars Journals
Full Length Research Paper
Characterisation of a chimeric Phanerochaete chrysosporium cellobiohydrolase expressed from Escherichia coli
R.L. Howard1*, P. Masoko1, M.B. Mowa1, E. Abotsi2 and Howard S3
1Microbiology, School of Molecular and Life Sciences, University of the North, P/Bag X1106, Sovenga, 0727, South Africa.
2Biochemistry, School of Molecular and Life Sciences, University of the North, P/Bag X1106, Sovenga, 0727, South Africa.
3Nutrition, School of Health Sciences, University of the North, P/Bag X1106, Sovenga, 0727, South Africa.
Accepted 09 September, 2011
Abstract
The aim of this study was to purify and analyse a Phanerochaete chrysosporium cbhI.1 gene-product expressed as an inducible, secreted, heterologous protein from an Escerichia coli pGEXcbh I.1 clone. Using glutathione Sepharose 4B affinity chromatography, the expressed protein was purified from the supernatant of an induced E. coli transformed with pGEXcbhI.1 and ran as a single band on a Sodium dodecyl sulphate-polyacrylamide gel. The glutathione S-transferase (GST) fused CBHI.1 was approx-imately 80 kDa in size, approximately 2.2 kDa smaller than the theoretically predicted size. The purified protein exhibited time dependent hydrolytic reaction against carboxy-methyl-cellulose (CMC) and Avicel. On CMC the highest hydrolytic reaction occurred at 120 min. whereas for Avicel it was at 150 min. Optimum pH and temperature for activity of the protein against these cellulose substrates were pH 6 and 55oC, respectively, and the protein remained stable under these optimum conditions for 24 h.
Key words: Phanerochaete chrysosporium, cellobiohydrolase purification, heterologus expression.