PURIFICATION AND PROPERTIES OF ALTERNANASE, A NOVEL ENDO-ALPHA-1,3-ALPHA-1,6-D-GLUCANASE

A newly isolated soil bacterium strain NRRL B-21195, tentatively identified as a Bacillus species, was found to be a constitutive producer of a novel type of glycanase that hydrolyses in an endo-fashion the polysaccharide alternan, an alpha-1,3-alpha-1,6-D-glucan, referred to in the literature as B-1355 dextran (fraction S), synthesized from sucrose by alternansucrase of Leuconostoc mesenteroides. The glycanase, named alternanase, has been purified to homogeneity from a cell-free culture fluid of the bacillus grown in a liquid medium containing D-glucose, and has been characterized. The enzyme has a molecular mass of 110000 Da (SDS/PAGE) and an isoelectric point of approximately 4.0. Optimum activity occurs at pH 7 and at a temperature of 40 degrees C. The enzyme is stable up to 50 degrees C but loses activity rapidly at 60 degrees C. Its action is inhibited by EDTA and stimulated by Ca2+. The enzyme requires, for its action, D-glucan chains in which alpha-1,3-linkages alternate with alpha-1,6-linkages; i.e., it is specific for alternan. Monitoring of alternan hydrolysis by determination of liberated reducing sugars pointed to an unusually low extent of hydrolysis and a low specific activity of the enzyme. As shown in the accompanying paper [Cote, G. L. and Biely, P. (1994) Eur. J. Biochem. 226, 641-648] the reason for this finding is that the main hydrolytic products are nonreducing, novel types of cyclic oligosaccharides.