An effective method for isolating alginate lyase-producing Bacillus sp. ATB-1015 strain and purification and characterization of the lyase.
A new alginate lyase-producing micro-organism, designated as Bacillus sp. strain ATB-1015, was effectively isolated from soil samples pretreated for 3 months with a substrate of the enzyme, sodium alginate. Alginate lyase activity was assayed by the degrading activity of biofilm of Teflon sheet discs, which was formed by a mucoid strain of Pseudomonas aeruginosa PAM3 selected from clinical isolates. The extracellular alginate lyase was precipitated with ammonium sulphate from the culture broth, and purified by gel filtration and anion exchange chromatography. The molecular weight of the lyase was estimated to be 41 kDa by SDS polyacrylamide gel electrophoresis and Sephacryl S-200 HR column chromatography. The optimum pH and temperature for the enzyme activity were around 7.5 and 37 degrees C, respectively, and the Km value was 0.17% with the substrate, sodium alginate. The lyase activity was completely inhibited by treatment with 1 mmol l-1 of EDTA and the decreased activity was almost completely recovered by the addition of 2 mmol l-1 of CaCl2. The activity was not affected by treatment with the protein denaturants, 0.01 mol l-1 of SDS or 1 mmol l-1 of urea. The lyase had substrate specificity for both the poly-guluronate and poly-mannuronate units in the alginate molecule.
Nakagawa
A
A
Department of Biosciences, Teikyo University, Utsunomiya, Japan.
Ozaki
T
T
Chubachi
K
K
Hosoyama
T
T
Okubo
T
T
Iyobe
S
S
Suzuki
T
T
D000464:Alginates / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology*; D018441:Biofilms; D020723:Glucuronic Acid; D006603:Hexuronic Acids; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D011133:Polysaccharide-Lyases / Q000302:isolation & purification* / Q000378:metabolism; D011489:Protein Denaturation; D011550:Pseudomonas aeruginosa / Q000166:cytology; D012988:Soil Microbiology; D013379:Substrate Specificity; D013696:Temperature
D000464:Alginates; D006603:Hexuronic Acids; D008670:Metals; D020723:Glucuronic Acid; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase