beta-agarases I and II from Pseudomonas atlantica. Purifications and some properties.
The agarose-degrading system of Pseudomonas atlantica has been re-examined. In addition to the previously reported extracellular endo-beta-agarase [Yaphe, W. (1966) in Proceedings 5th International Seaweed Symposium, pp. 333-335] a second, membrane-bound endo-enzyme activity, beta-agarase II has been discovered. These two enzymes act in concert to degrade agarose to neoagarobiose [3,6-anhydro-alpha-L-galactopyranosyl-(1 leads to 3)-D-galactose] and also to degrade partially 6-O-methylated agarose to neoagarobiose and 6(1)-O-methyl-neoagarbiose. Novel assays were devised for beta-agarase II and the associated disaccharidase, neoagarobiose hydrolase. These allowed the critical purification of beta-agarase I and II. beta-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 beta-agarase II. Dodecylsulphate/polyacrylamide gel electrophoresis indicated a homogeneous protein and a molecular weight of 32000. 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 degrees C but only for up to 2 h at 40 degrees C. beta-Agarase II was partially purified (5-fold) from the soluble fraction of disrupted cells by chromatography on Sephadex G-100, hydroxyapatite and DEAE-Sepharose CL-6B. This preparation was free of beta-agarase I and disaccharidase. beta-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.
D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D004186:Disaccharidases / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology*
D004186:Disaccharidases; D006026:Glycoside Hydrolases; C039308:neoagarobiose hydrolase; C019624:agarase