title: Efficient purification, characterization and partial amino acid sequencing of two alpha-1,4-glucan lyases from fungi.
abstract: alpha-1,4-Glucan lyases from the fungi Morchella costata and M. vulgaris were purified by affinity chromatography on beta-cyclodextrin-sepharose, followed by ion exchange and gel filtration. The purified enzymes produced 1,5-anhydro-D-fructose from glucose oligomers and polymers with alpha-1,4-glucosidic linkages, such as maltose, maltosaccharides, amylopectin, and glycogen. The lyases were basically inactive towards glucans linked through alpha-1,1, alpha-1,3 or alpha-1,6 linkages. For both enzymes the molecular mass was around 121,000 Da as determined by matrix-assisted laser desorption mass spectrometry. The pI for the lyases from M. costata and M. vulgaris was 4.5 and 4.4, respectively. The lyases exhibited an optimal pH range of pH 5.5 to pH 7.5 with maximal activity at pH 6.5. Optimal temperature was between 37 degrees C and 48 degrees C for the two lyases, depending on the substrates. The lyases were examined with 12 inhibitors to starch hydrolases and it was found that they were inhibited by the -SH group blocking agent PCMB and the following sugars and their analogues: glucose, maltitol, maltose, 1-deoxynojirimycin and acarbose. Partial amino acid sequences accounting for about 35% of the lyase polypeptides were determined. In the overlapping region of the sequences, the two lyases showed 91% identity. The two lyases also cross-reacted immunologically.
authors.affiliation: Danisco Biotechnology, Danisco A/S, Langebrogade 1, Copenhagen K, Denmark. g7sy@danisco.dk
authors.forename: S
authors.initials: S
authors.lastname: Yu
authors.forename: T M
authors.initials: TM
authors.lastname: Christensen
authors.forename: K M
authors.initials: KM
authors.lastname: Kragh
authors.forename: K
authors.initials: K
authors.lastname: Bojsen
authors.forename: J
authors.initials: J
authors.lastname: Marcussen
mesh_terms: D000595:Amino Acid Sequence; D005658:Fungi / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D011133:Polysaccharide-Lyases; C080474:alpha-1,4-glucan lyase