Purification and Properties of Alternanase, a Novel Endo‐α‐1,3‐α‐1,6‐d‐Glucanase

• Published in: European journal of biochemistry Vol. 226; no. 2; pp. 633 - 639
• Main Authors: Biely, Peter, Côté, Gregory L., Burgess‐Cassler, Anthony
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.12.1994
• Subjects: Bacillus; Bacillus - enzymology; Calcium - pharmacology; Carbohydrate Conformation; Cations; Edetic Acid - pharmacology; Enzyme Stability; Glucans - chemistry; Glucans - metabolism; Glycoside Hydrolases - chemistry; Glycoside Hydrolases - isolation & purification; Glycoside Hydrolases - metabolism; Hydrogen-Ion Concentration; Isoelectric Point; Kinetics; Molecular Weight; Substrate Specificity; Temperature
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1994.tb20090.x

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 α‐1,3‐α‐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°C. The enzyme is stable up to 50°C but loses activity rapidly at 60°C. Its action is inhibited by EDTA and stimulated by Ca2+. The enzyme requires, for its action, d‐glucan chains in which α‐1,3‐linkages alternate with α‐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 [Côté, G. L. & Biely, P. (1994) Eur. J. Biochem. 226, 641–648] the reason for this finding is that the main hydrolytic products are non‐reducing, novel types of cyclic oligosaccharides.