Purification and characterization of agarases from a marine bacterium, Vibrio sp. PO-303.

Abstract

A marine bacterium, Vibrio sp. PO-303, produced three kinds of extracellular agarases. These enzymes were purified to homogeneity by ammonium sulfate precipitation and successive column chromatographies. The molecular masses of agarase-a, -b, and -c were estimated to be 87.5, 115, and 57 kDa by SDS-PAGE with isoelectric point of 6.6, 3.4, and 8.4, respectively. These enzymes had maximal activity at pH 6.5-7.5 and at around 38-55 degreesC. They differed in their sequences at the amino termini of the protein chains. All enzymes were inhibited completely by Hg2+. Ag+, Cu2+, and Zn2+ strongly inhibited agarase-a and -c compared with agarase-b, and the activity of agarase-c fell wide by Al3+, Fe3+, and EDTA. Agarase-a hydrolyzed agarose to give neoagarotetraose and -hexaose as predominant products, but could not cleave neoagarotetraose. The main hydrolysis products of agarase-b were neoagarobiose from agarose and neoagarooligosaccharides more than dimer. Agarase-c could not cleave neoagarohexaose.