Purification and properties of a beta-mannanase from alfalfa seeds

Abstract

A beta-mannanase (EC 3.2.1.25) has been purified from germinating Alfalfa seeds by successive chromatography steps; on hydroxyapatite, DEAE-cellulose and ECTEOLA-cellulose. The enzyme preparations were homogeneous as judged by gel electrophoresis. A 5000-fold increase in specific activity (from the crude extract) was obtained. The purified enzyme has a molecular weight of 40 000. Several of its properties were determined: pH optimum 5.2 and optimal temperature of activity 50 degrees C. The hydrolysis of galacto- and gluco-mannans (with various ratio of mannose to galactose and glucose) as well as that of mannooligosaccharides was studied in detail. A prefered point of attack at the third position from the non-reducing end was shown. Comparative results from the hydrolysis of intact galactomannans, of galactomannans previously hydrolysed by galactosidase, suggest that galactose hinders the accessibility of the mannan backbone to the enzyme.