CLONING OF A XYLANASE GENE FROM THE RUMINAL FUNGUS NEOCALLIMASTIX-PATRICIARUM-27 AND ITS EXPRESSION IN ESCHERICHIA-COLI

An endo-beta-1,4-xylanase gene was cloned from Neocallimastix patriciarum 27 in the bacteriophage vector lambdagtWESlambdaB and was subcloned into the plasmid vectors pUC18 and pUC19 in which xylanase activity was expressed in both orientations. The xylanase was located in the periplasmic space of the host, Escherichia coli HB101. The pH and temperature optima for periplasmic xylanase activity were 6.2 and 40-degrees-C, respectively, and the K(m) for oat spelt xylan hydrolysis was 0.89 mg . mL-1. It also exhibited hydrolytic activity on carboxymethyl cellulose that was equivalent to 28% of the activity exhibited by the enzyme on xylan. It bound to crystalline cellulose, but lacked hydrolytic activity on amorphous cellulose. SDS-PAGE followed by zymogram analysis showed active bands of 68, 58, and 51 kDa. Isoelectric focusing in gels combined with zymogram analysis showed one band of xylanase activity with a pI of 3.6.