Purification and Substrate Specificity of endo-Type Inulinase from Penicillium purpurogenum

Abstract

An inulinase was highly purified from the culture broth of Penicillium purpurogenum by chromatographies on DEAE-Sepharose CL-6B, Toyopearl HW-65, and Bio-Gel P-100. The enzyme was homogeneous by disc electrophoretic analysis. The molecular weight was 6.4 × 104 by SDS-disc electrophoresis and gel filtration on Bio-Gel P-150. The isoelectric point was pH 3.6 by isoelectric focusing. The enzyme hydrolyzed inulin rapidly, but did not affect sucrose. By paper chromatography analysis, the major products from inulin were tri-, tetra-, penta-, and hexasaccharides. The substrate specificity of the enzyme on hydrolyses of fructo-oligosaccharides[1F(1-β-D-fructofuranosyl)n sucrose (n = 1 to 6 and [Formula omitted] (average of polymerization degree) = 8)] were examined. The Km values and relative maximum velocities for the hydrolyses of inulin and fructo-oligosaccharides (GFn, n = 2 to 7 and [Formula omitted]= 9) were as follows: inulin, ([Formula omitted]= 35) 0.21 mM and 100; GF9, 0.24 mM and 86.5; GF7, 0.33 mM and 132; GF6, 0.85 mM and 71.2; GF5, 3.8 HIM and 25.4; GF4, 2.8 mM and 28.8; GF3, (nystose) 16 mM and 0.8; GF2 (1-kestose), 8.4 mM and 0.2. The molecular activities for the hydrolyses of fructo-oligosaccharides (GFn, n = 2 to 6) were increased depending on the degree of polymerization of fructosyl residues, and were nearly constant if the polymerization degree was over seven. These results strongly suggested that the endo-type inulinase from Penicillium purpurogenum had a subsite structure consisting of at least seven subsites. © 1988, Japan Society for Bioscience, Biotechnology, and Agrochemistry. All rights reserved.