PURIFICATION AND CHARACTERIZATION OFAXYLANASE FROM ASPERGILLUS NIGER DB 106

Abstract

When growing in solid state fermentation with com cob as carbon source, strain Aspergillus niger DB 106 produced various xylanases. The predominant endoxylanase (signed as xylanase P) was purified to homogeneity by a four-step procedure including strong anion exchanger column, Econo Pac P6 (BioRAD) Gel filtration column, anion exchanger chromatography Econo Pac High Q and Sephadex G-50 gel filtration. Some physico-chemical properties of the xylanase P were characterized: The purified xylanase P showed a single protein band with a molecular mass of 32 kDa as estimated by SDS-PAGE and zymogram analysis with oat spelts xylan (OSX). It had an optimum activity at pH and temperature of 5.5 and 55^oC, respectively. This enzyme was stable at a wide range pH (2.5 - 8.0), however it was not thermostable (lost activity at 60^oC). Vmax, and Km values were 20000 units.mg^-1 and 38 mg.ml^-1, respectively, as determined in the enzyme reaction with insoluble OSX as substrate at 55°C and pH 5.5. The enzyme was slightly inhibited by Zn+2 , Mn+2 , Fe+2 and Hg+2 but not by Na+, K+, Ca2+, Cu2+, Co2+, Mg2+, and EDTA. The xylanase P could not degrade carboxylmethyl cellulose (CMC), p-nitrophenyl galactopyranoside and p-nitrophenyl xylopyranoside. All these data showed that xylanase P was a true xylanase, with no cellulase, p-galactosidase, amylase and beta-xylodase activities. Hydrolysis products of the purified enzyme action on OSX substrate were analyzed on TLC showing that xylanase P was an endoxylanase that completely degraded xylan to mainly short xylooligosaccharides and minor amount of xylose