Purification and characterization of β-agarase from agar-liquefying soil bacterium, Acinetobacter sp., AG LSL-1

Department of Biochemistry, Gulbarga University, Gulbarga 585106, Karnataka, India; Department of Biotechnology, MVJ College of Engineering, Bangalore 560067, Karnataka, India
Process Biochemistry 01/2009; DOI: 10.1016/j.procbio.2009.04.025

ABSTRACT 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.

  • [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.
    Biotechnology and Bioprocess Engineering 01/2011; 16(3):513-519. · 1.28 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Agar is a major gelling agent used both in food and pharmaceutical applications. Traditional purification of agar is generally performed by sequential time consuming chemical and/or physical steps, leading to both poor recovery yields and low productivities. As a consequence, only 30% of the amount of agar produced is actually available under purified form to feed the world market. The current limiting factor for purification is the presence of sulphated compounds such as sulphated-agaropectin, which strongly affect the technological properties of the agar gel such as gel strength, melting and fusion temperatures and electroendosmosis. In this context, this communication aims at discussing about the development of a biorefining agar purification approach which allows overcoming the current limitations associated with traditional purification methods. More specifically, this article focuses on the potential role of arylsulphatases in agar purification processes to reduce the number of purification steps and to improve recovery yields. This review first presents the global gelling agents market before focusing on agar characteristics and production processes. Then, after a brief reminder of the sulphur metabolism, the roles, classes and properties of the different arylsulphatases are described to draw perspectives on their integration in current or new agar production processes.
    Process Biochemistry 09/2013; 48(12):1861-1871. · 2.44 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Agar is a cell wall component of macro red algae that can be hydrolyzed by agarase. Agarases are classified into -agarase (E.C. and -agarase (E.C., in accordance with their cleavage pattern, and can be grouped in the glycoside hydrolase (GH)-16, -58, -86, -96, and -118 family according to the amino acid sequences of the proteins. Many agarases and/or their genes have been detected, isolated, and recombinantly expressed from bacteria, and metagenomes have their origins in sea and terrestrial environments. Products of agarases, agarooligosaccharides and neoagarooligosaccharides, represent wide functions such as antitumor, immune stimulation, antioxidation, prebiotic, hepa-protective, antibacterial, whitening, and moisturizing effects; hence, broad applications would be possible in the food industry, cosmetics, and medical fields. In addition, agarases are also used as a tool enzyme for research. This paper reviews the sources, purifications and detection methods, and application fields of agarases. The role of agarases in agar metabolism and the function of their enzymatic products are also surveyed.
    Journal of Life Science. 01/2012; 22(2).