PURIFICATION AND PHYSICOCHEMICAL PROPERTIES OF AN ENDO-1,5-ALPHA-L-ARABINANASE (EC-3.2.1.99) ISOLATED FROM AN ASPERGILLUS-NIGER PECTINASE PREPARATION

Using low and medium (FPLC) pressure column chromatographic separation methods, an endo-1,5-alpha-L-arabinanase (EC 3.2.1.99) was purified f rom an Aspergillus niger pectinase preparation. The purification proce dure combined different anion exchange and hydrophobic interaction chr omatographies followed by gel filtration, yielding a highly pure and s pecific enzyme. The isolated endo-1,5-alpha-L-arabinanase was most act ive on linear 1,5-alpha-L-arabinan (90.2 U/mg), whereas branched arabi nans from sugar beets were degraded to a lesser extent (similar to 14 U/ mg). No other significant carbohydrate degrading activities could b e detected. The enzyme was shown to be electrophoretically pure after silver staining and its identity was confirmed through specific bindin g to an antiserum directed against endo-1,5-alpha-L-arabinanase. The m ajor physico-chemical characteristics of the enzyme were the following : M(r) 42 500 (SDS-PAGE), M(r) 33 900 (MALDI-TOF-MS), pI less than or equal to 3.0, pH optimum 4.8, temperature optimum 55 degrees C, pH sta bility 3.5-8.0, temperature stability less than or equal to 45 degrees C, K-m = 0. 205 mg/ml, V-max = 1.77 x 10(-4) mu mol/ min, E(A) = 12.2 0 kcal/mol; Zn2+ and Hg2+ showed potent inhibitory effects.