PRODUCTION, PURIFICATION, AND CHARACTERIZATION OF XYLANASE FROM A HYPERXYLANOLYTIC MUTANT OF ASPERGILLUS-OCHRACEUS

Enhancement of the productivity of xylanase and β‐xy‐losidase of Aspergillus ochraceus was investigated by multistep mutagenesis. The spores of the wild strain were subjected to UV and N‐methyl‐N‐nitro‐N‐nitro‐soguanidine (NTG). The hyperxylanolytic mutant (NG‐13), which showed good clearing on the surface of the xylan‐agar plate, secretes xylanase and β‐xylosidase at high levels during growth on commercial xylan and on agricultural wastes. Both liquid and solid state cultures were employed in the study for enzyme production. The xylanase from NG‐13 was purified to homogeneity by ammonium sulfate precipitation and gel filtration. This purified enzyme showed a pH optimum of 6.0 and was stable in the range of pH 5 to 10. Prolonged stability of the enzyme was observed at 45°C though its activity was maximal at 50°C. The molecular weight of the enzyme was estimated to be 4.3 × 104 by SDS‐polyacrylamide gel electrophoresis and 5 × 104 by gel filtration on Sephadex G‐75. The kinetic data showed that the Km and Vmax values for xylan were 1 × 10−3M and 19.6 μmol/ min/mg protein, respectively. The enzyme was both more active and thermostable in the presence of K+and was inactivated by thiol reagents such as Hg2+, p‐hydroxymercuribenzoate (PHMB), 3′, 5′‐dithiobis (2′‐nitrobenzoic acid) (DTNB), and N‐ethylmaleimide (NEM). Copyright © 1990 John Wiley & Sons, Inc.