An effective method for isolating alginate lyase-producing Bacillus sp. ATB-1015 strain and purification and characterization of the lyase

Abstract

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 on 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 °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.

Screening of Alginate Lyase-Producing Bacteria and Optimization of Media Compositions for Extracellular Alginate Lyase Production

“…In this context, Hansen and coworkers [16] identified strain B. circulans, which specifically degraded polyM alginate. Nakagawa et al [19] also identified Bacillus sp. strain ATB-1015, which had substrate specificity for both the polyG and polyM blocks in the alginate.…”

“…Interestingly, no difference was observed in the diversity of the isolated bacteria from sprayed soil samples with or without sodium alginate solution. However, Nakagawa et al [19] have reported that treatment with sodium alginate solution is an effective method for the isolation of lyase-producing microorganisms. Most of the colonies were creamcolored, pale yellow, and wrinkled but some of them were flat.…”

“…To isolate alginate-degrading bacteria, 1.0% sodium alginate solution was poured over specified 100 cm 2 areas of soil once a week for three months [19] . In addition, another area of the soil was sampled directly (without alginate solution).…”

“…algL has also a potential application for enhancing the bacteriocidal effects of antibiotics against mucoid P. aeruginosa in biofilms [18] . Considering the enzymatic treatment of infections caused by biofilm-forming bacteria, a search was carried out for a new algL from soil bacteria [19] . Moreover, genetic studies on algL-producing microorganisms have revealed that the algL genes are clustered with other alginate biosynthetic gene loci [20] .…”

Alginate Lyase from Indonesian Bacillus megaterium S245 Shows Activities Toward Polymannuronate and Polyguluronate

“…This research was the first finding of a new alginate lyase producing Bacillus megaterium S245. Another work that reported bacteria producing alginate lyase from genus Bacillus were Bacillus sp ATB-1015 (Nakagawa, Ozaki, Chubachi, Hosoyama & Okubo, 1998) and Bacillus subtilis (El Ahwani & Elborai, 2012). Figure 3.…”