Purification and characterization of two different agarose-degrading enzymes.

Agarase was concentrated and purified from culture filtrates of an agar-degrading Pseudomonas-like bacteria by affinity chromatography on divinyl sulphone cross-linked Sepharose 4B. By fractionation on Sephadex G-200 three fractions were obtained two of which, agarases I and II, had agarase activity. Agarase II was further purified by isoelectric focusing, and the main peak, agarase IIb, was isoelectric at pH 5.1. Molecular weight determinations indicated agarase I to be a dimer with Mr approximately 210 000. The Mr of agarase IIb was 63 000 as determined by analytical ultra-centrifugation, polyacrylamide gel electrophoresis in sodium dodecyl sulphate and molecular sieve chromatography on Sepharose 4B in 6M Gdn-HCl. The amino acid compositions of the two proteins were very similar and both were found to be glycoproteins. The pH optimum of the enzymes was 6.7 and the optimal temperature was 38 degree C for agarase I and 43 degree C for agarase IIb. Melted agarose and agarose gel were used as substrates for the enzymes. The ratio of the activities towards the different substrates was 4.3 for agarase I and 1.0 for agarase IIb. Agarase I hydrolyzed the beta-linkages in neoagarooctaose so as to produce two moles of neoagarotetraose or one mole of neoagarohexaose and one mole of neoagarobiose. Agarase IIb hydrolyzed only the central beta-linkage to form two moles of neoagarotetraose.