Purification and Characterisation of a Novel Agarase Secreted by a Marine Bacterium, Pseudoalteromonas sp. Strain CKT

Abstract

A marine bacterium which grows on agar as its sole carbon and energy source shows an agar liquefying ability, was isolated from sea mud and identified as Pseudoalteromonas sp. An extracellular agarase was purified to homogeneity through a four-step process: ultra-filtration (10 kDa < enzyme < 0.2 μm), two series of DEAE-Sephacel chromatography, and Sephacryl S-300 gel filtration. The enzyme was estimated to have a molecular mass of 56 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and gel filtration, and be composed of a 37 kDa protein and 19 kDa of sugar, namely a glycoprotein. The optimum conditions for its action were 30°C, pH of 6.5-7.5, and 0 mM NaCl. Km to agarose was determined as 6.28 mM. The enzyme was active at 15-65°C, pH 4.0-9.0 and 0-4,000 mM salt concentration (NaCl). It was found to be an endo-type agarase which hydrolyses agar and agarose to yield galactose, neoagarobiose and a trace amount of neoagarohexaose. In addition, the enzyme had both α- and β-galactosidase activities. © 2000, Japanese Society of Microbial Ecology – The Japanese Society of Soil Microbiology. All rights reserved.