ALPHA-1,4-GLUCAN LYASE, A NEW CLASS OF STARCH GLYCOGEN DEGRADING ENZYME .1. EFFICIENT PURIFICATION AND CHARACTERIZATION FROM RED SEAWEEDS

This study presents the first purification and characterization of an alpha-1,4-glucan lyase. The enzyme degraded alpha-1,4-glucan to produce 1,5-anhydrofructose. A simple and efficient purification procedure has been developed and the enzyme has been purified to homogeneity from two red seaweeds Gracilariopsis lemanciformis and Gracilaria verrucosa. Alpha-1,4-Glucan lyase was apparently a single polypeptide as a molecular weight of 111 000 was observed in SDS-gel electrophoresis, and 98 000 by gel filtration chromatography on Sephacryl S-200. Amino acid composition analysis of the enzyme showed high amounts of Asp/Asn, Gly and Glu/Gln. The isoelectric point of the enzyme was 3.9, as revealed by isoelectrofocusing. The concentrations of maltotriose, maltose and amylopectin that yield half of the maximum activity were 798 mug ml-1 (1.58 mM), 1,418 mug ml-1 (4.14 mM) and 1,600 mug ml-1, respectively. Alpha-1,4-Glucan lyase exhibited a wide pH optimum range from pH 2.5 to 7.0 for maltose and from pH 3.5 to 7.5 for amylopectin. The optimal temperature for activity of the algal lyase was 50-degrees-C when maltose or amylopectin was used as a substrate under the assay conditions. The Arrhenius activation energies were 45.8 and 44.0 kJ mol-1 for maltose and amylopectin as substrate, respectively. Only one form of alpha-1,4-glucan lyase was found in cell-free extracts of the two red seaweeds.