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.