A simple procedure for the large-scale purification of β- d-xylanase from Trichoderma viride

Abstract

A simple procedure is described for the purification of gram quantities of β- d-xylanase from a commercially available Trichoderma viride culture filtrate. Chromatography of the crude extract on CM-Sepharose CL-6B gave two partially separated peaks of β- d-xylanase activity, and for convenience these have been termed xylanases I and II. Each has cellulase activity. The cellulase and xylanase activities were not separated by further purification on Ultrogel AcA 54 or Phenyl Sepharose CL-4B. Each of these xylanases was purified essentially to homogeneity (by the criterion of isoelectric focusing) and was free of protease, amylase and glycosidase activities. Physical and kinetic properties of xylanases I and II were identical, indicating that the separation of CM-Sepharose CL-6B may simply have been an artefact of chromatography. However, this pattern was reproducible, being obtained on several occasions. Each enzyme separated into two protein bands on isoelectric focusing. The major band had a pI of 8·45 and a very minor component had a pI of 7·3. Optimal activity was at pH 4·5 and 50°C and the enzymes were stable over a pH range of 3·4–7·9 and at temperatures below 55°C. Apparent K m s were 3·33 mg ml −1 on rye flour arabinoxylan and 1·33 mg ml −1 on larch wood xylan. The enzymes partially hydrolysed larch wood xylan to oligosaccharides with two or three d-xylosyl residues. Rye flour arabinoxylan was hydrolysed to high molecular weight oligosaccharides which were not fractionated on Bio-Gel P-2.