Full Length Research Paper
Abstract
The aim of this study was to purify and analyse a Phanerochaete chrysosporiumcbhI.1 gene-product expressed as an inducible, secreted, heterologous protein from anEscerichia coli pGEXcbhI.1 clone. Using glutathione Sepharose 4B affinity chromatography, the expressed protein was purified from the supernatant of an inducedE. 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.
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