PURIFICATION AND CHARACTERIZATION OF A NEW XYLANASE (APX-II) FROM THEFUNGUS AUREOBASIDIUM-PULLULANS Y-2311-1

Aureobasidium pullulans Y-2311-1 produced four major xylanases (EC 3.2 .1.8) with pI values of 4.0, 7.3, 7.9, and 9.4 as revealed by isoelect ric focusing and zymogram analysis when grown for 4 days on 1.0% oat s pelt xylan. The enzyme with a pI of 9.4 was purified by ammonium sulfa te precipitation, chromatography on a DEAE-Sephadex A-50 column, and g el filtration with a Sephadex G-75 column. The enzyme had a mass of ab out 25 kDa as determined by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography. The purified e nzyme had a K(m) of 7.6 mg . ml-1 and a V(max) of 2,650 mumol . min-1 . mg-1 for birchwood xylan at 28-degrees-C and pH 4.5. It lacked activ ity towards carboxymethylcellulose, cellobiose, starch, mannan, p-nitr ophenyl (pNP)-beta-D-xylopyranoside, pNP-beta-D-glucopyranoside, pNP-a lpha-D-glucopyranoside, pNP-beta-D-cellobioside, pNP-beta-D-fucopyrano side, or pNP-alpha-D-galactopyranoside. The predominant end products o f birchwood xylan or xylohexaose hydrolysis were xylobiose and xylose. The enzyme had the highest activity at pH 4.8 and 54-degrees-C. Sixty percent of the activity remained after the enzyme had been incubated at 55-degrees-C and pH 4.5 for 30 min. The sequence of the first 68 am ino acid residues at the amino terminus showed homology to those of se veral other xylanases. Immunoblot analysis with antiserum raised again st the purified xylanase revealed that two immunologically related pol ypeptides of 25 and 22 kDa were produced in A. pullulans cultures cont aining oat spelt xylan or xylose as carbon sources but not in cultures containing glycerol or glucose.