Department of Biochemistry, Gulbarga University, Gulbarga 585106, Karnataka, India
PROCESS BIOCHEMISTRY (Impact Factor: 2.52). 09/2009; 44(9):999-1003. DOI: 10.1016/j.procbio.2009.04.025
The extracellular β-agarase LSL-1 produced by an agar-liquefying, soil bacterium Acinetobacter sp., AG LSL-1 was purified to homogeneity by combination of ion-exchange and size exclusion chromatography with final yield of 44%. The enzyme has a specific activity of 397 U mg−1 protein and with a molecular mass of 100 kDa. The agarase was active in the pH range of 5.0–9.0, optimally at pH 6.0 and temperature between 25 °C and 55 °C and optimal at 40 °C. The enzyme retained 63% of native activity at 50 °C suggesting it is a thermostable. The activity of the agarase was completely inhibited by metal ions, Hg2+, Ag+ and Cu2+, whereas 25–40% of native activity was retained in the presence of Zn2+, Sn2+ and SDS. Neoagarobiose was the final product of hydrolysis of both agarose and neoagarohexaose by the purified agarase LSL-1. Based on the molecular mass and final products of agarose hydrolysis, the β-agarase LSL-1 may be further grouped under group III β-agarases and may be a member of GH-50 family. This is the first report on the purification and biochemical characterization of β-agarase from an agar-liquefying Acinetobacter species.
"SY37-12 (Wang et al. 2006), Agarivorans albus YKW-34 (Fu et al. 2008), and Acinetobacter sp. Ag LSL-1 (Lakshmikanth et al. 2009) retained activities after incubation as follows; 20 % after 1 min at 70 °C, 10 % after 60 min at 70 °C, and none after 60 min at 60 °C, respectively. "
[Show abstract][Hide abstract] ABSTRACT: An agar-degrading archaeon Halococcus sp. 197A was isolated from a solar salt sample. The agarase was purified by hydrophobic column chromatography using a column of TOYOPEARL Phenyl-650 M. The molecular mass of the purified enzyme, designated as Aga-HC, was ~55 kDa on both SDS-PAGE and gel-filtration chromatography. Aga-HC released degradation products in the order of neoagarohexose, neoagarotetraose and small quantity of neoagarobiose, indicating that Aga-HC was a β-type agarase. Aga-HC showed a salt requirement for both stability and activity, being active from 0.3 M NaCl, with maximal activity at 3.5 M NaCl. KCl supported similar activities as NaCl up to 3.5 M, and LiCl up to 2.5 M. These monovalent salts could not be substituted by 3.5 M divalent cations, CaCl2 or MgCl2. The optimal pH was 6.0. Aga-HC was thermophilic, with optimum temperature of 70 °C. Aga-HC retained approximately 90 % of the initial activity after incubation for 1 hour at 65–80 °C, and retained 50 % activity after 1 hour at 95 °C. In the presence of additional 10 mM CaCl2, approximately 17 % remaining activity was detected after 30 min at 100 °C. This is the first report on agarase purified from Archaea.
Electronic supplementary material
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"It is remarkable that the agarase from Acinetobacter sp. AGLSL-1 show a specific activity of 397 U/mg , which is the highest among the native agarases have been reported up to date. There are no special properties as comparing the above four agarases with marine derived agarases in pH and temperature properties. "
[Show abstract][Hide abstract] ABSTRACT: Agarases are the enzymes which catalyze the hydrolysis of agar. They are classified into alpha-agarase (E.C. 220.127.116.11) and beta-agarase (E.C. 18.104.22.168) according to the cleavage pattern. Several agarases have been isolated from different genera of bacteria found in seawater and marine sediments, as well as engineered microorganisms. Agarases have wide applications in food industry, cosmetics, and medical fields because they produce oligosaccharides with remarkable activities. They are also used as a tool enzyme for biological, physiological, and cytological studies. The paper reviews the category, source, purification method, major characteristics, and application fields of these native and gene cloned agarases in the past, present, and future.
[Show abstract][Hide abstract] ABSTRACT: Microbulbifer strain CMC-5 was isolated from decomposing seaweeds, and was found to degrade agar, alginate, carboxymethyl cellulose, carrageenan,
xylan, and chitin. The extracellular agarase enzyme from strain CMC-5 was purified 103-fold by ultrafiltration, ion-exchange
chromatography, using diethylaminoethyl sepharose FF, and gel filtration, using sephacryl S-300HR, with a yield of 6.7%. Zymogram
and protein staining of the purified agarase on a SDS-polyacrylamide gel revealed a single band, with an apparent molecular
weight of 59 kDa. The purified enzyme was endo-type β-agarase, as it was able to hydrolyze the β-1, 4 glycosidic linkages
of agarose, releasing neoagarotetraose and neoagarohexaose as the end products. The optimum pH and temperature of agarase
were 7 and 50°C, respectively. Thermal stability studies indicated that the agarase retained 62% of its activity after incubating
at 50°C for 30 min. Treatment with EDTA reduced the agarase activity by 54%. The agarase activity was stimulated by the presence
of Ca2+ and Mg2+ ions; whereas, Zn2+, Hg2+, Cu2+, Fe2+, and Co2+ abolished the activity. Further, the presence of NaCl at concentrations lower than 100 mM caused a decrease in the agarase
activity; whereas, the activity was enhanced up to a concentration of 500 mM.
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