Intracellular Alginolytic Enzymes of the Marine Bacterium Pseudoalteromonas citrea KMM 3297

S. A. Alekseeva^*, I. Yu. Bakunina, O. I. Nedashkovskaya, V. V. Isakov, V. V. Mikhailov, and T. N. Zvyagintseva

Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences, pr. 100-letiya Vladivostoka 159, Vladivostok 690022, Russia; fax: (4232) 31-4050; E-mail: mik@piboc.dvo.ru

^* To whom correspondence should be addressed.

Received December 24, 2002; Revision received April 15, 2003

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The marine bacterium Pseudoalteromonas citrea KMM 3297 is an associate of the holothurian Apostichopus japonicus. When grown in a medium containing glucose, the strain produces two intracellular alginolytic enzymes, AlI and AlII. Fucoidan from the brown alga Fucus evanescens induces synthesis of one more alginolytic enzyme, AlIII. These enzymes were separated using anion-exchange chromatography. The alginate lyase AlI completely retains its activity at 35°C, AlII and AlIII being stable at 45°C. The alginate lyases exhibit maximal activities in the range of pH 7-8. The molecular weights of AlI, AlII, and AlIII determined by gel filtration are 25, 79, and 61 kD, respectively. All the investigated enzymes are endo-type alginate lyases. They catalyze degradation of polyguluronate (poly-G) and polymannuronate (poly-M) yielding oligosaccharides of the polymerization degree of 5 >= n >= 3 with the unsaturated bond between the C4 and C5 atoms of the non-reducing terminus. A mixture of these three enzymes exhibits synergism while acting on the polymeric substrate. The K_m values of the alginate lyase AlI for poly-G and poly-M are 24 and 34 µg/ml, respectively. Alginate lyase AlIII exhibits less affinity to poly-M (K_m = 130.0 µg/ml) than to poly-G (K_m = 40.0 µg/ml). NaCl (0.2 M), MgCl₂ and MgSO₄ (0.01 M) activate all three enzymes more than twofold. The presence of several alginolytic enzymes of different specificity provides efficient destruction of alginic acids of brown algae by the strain P. citrea KMM 3297.

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KEY WORDS: alginate lyase, marine bacterium, Pseudoalteromonas citrea, alginic acid, brown algae

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