β‐Agarases I and II from Pseudomonas atlantica. Purifications and some properties

Abstract

The agarose‐degrading system of Pseudomonas atlantica has been re‐examined. In addition to the previously reported extracellular endo‐β‐agarase [Yaphe, W. (1966) in Proceedings 5th International Seaweed Symposium, pp. 333–335] a second, membrane‐bound endo‐enzyme activity, β‐agarase II has been discovered. These two enzymes act in concert to degrade agarose to neoagarobiose [3,6‐anhydro‐α‐l‐galactopyranosyl‐(1→3)‐d‐galactose] and also to degrade partially 6‐O‐methylated agarose to neoagarobiose and 61‐O‐methyl‐neoagarobiose. Novel assays were devised for β‐agarase II and the associated disaccharidase, neoagarobiose hydrolase. These allowed the critical purification of β‐agarase I and II. β‐Agarase I was purified 670‐fold from the bacterial medium by a new method using ammonium sulphate precipitation and gel filtration on Sephadex G‐100. The enzyme was resolved from the small amount of extracellular β‐agarase II. Dodecylsulphate/polyacrylamide gel electrophoresis indicated a homogeneous protein and a molecular weight of 32 000. Activity was observed against agar over the pH range 3.0–9.0 and optimally at pH 7.0. The enzyme could be used indefinitely at 30°C but only for up to 2 h at 40°C. β‐Agarase II was partially purified (5‐fold) from the soluble fraction of disrupted cells by chromatogm raphy on Sephadex G‐100, hydroxyapatite and DEAE‐Sepharose CL‐6B. This preparation was free of β‐agarase I and disaccharidase. β‐Agarase II was stimulated by NaCl, optimally in the range 0.10–0.20 mol dm−3 (2.4‐fold the activity at 0.010 mol dm−3 NaCl). Alkali earth metal (0.002 mol dm−3 CaCl2 or 0.005 mol dm−3 MgCl2) gave 1.2‐fold the normal activity. Optimum activity was over pH 6.5–7.5. Copyright © 1983, Wiley Blackwell. All rights reserved