Characterisation of a chimeric Phanerochaete chrysosporium cellobiohydrolase expressed from Escherichia coli

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.