Purification and Characterization of Agarase from Marine Bacteria Acinetobacter sp. PS12B and Its Use for Preparing Bioactive Hydrolysate from Agarophyte Red Seaweed Gracilaria verrucosa
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Acinetobacter strain PS12B was isolated from marine sediment and was found to be a good candidate to degrade agar and produce agarase enzyme. The extracellular agarase enzyme from strain PS12B was purified by ammonium sulfate precipitation followed by DEAE-cellulose ion-exchange chromatography. The specific activity of the crude enzyme which was 1.52 U increased to 45.76 U, after two-stage purification, with an enzyme yield of 9.76%. Purified enzyme had a molecular mass of 24 kDa. The optimum pH and temperature for activity of purified agarase were found to be 8.0 and 40 °C, respectively. The Km and Vmax values for agarase were 4.69 mg/ml and 0.5 μmol/min, respectively. Treatment with EDTA reduced the agarase activity by 58% at 5 mM concentration. The enzyme activity was stimulated by the presence of Fe2+, Mn2+, and Ca2+ ions while reducing reagents (β-mercaptoethanol and dithiothreitol, DTT) enhanced its activity by 30–40%. The purified agarase exhibited tolerance to both detergents and organic solvents. Major hydrolysis products of agar were DP4 and also a mixture of longer oligosaccharides DP6 and DP7. The enzyme hydrolysed seaweed (Gracilaria verrucosa) exhibited strong antioxidant activity in vitro. Successful hydrolysis of seaweed indicates the potential use of the enzyme to produce seaweed hydrolysate having health benefits as well as the industrial application like the production of biofuels.
KeywordsAcinetobacter Agarase Degree of polymerization (DP) Gracilaria verrucosa Oligosaccharide Antioxidant activity
The authors wish to thank the Director, CSIR-CFTRI for encouragement, and the facilities provided. First author thanks UGC, Govt. of India (Grant No. 1269/NET-June 2011) for the support in the form of fellowship.
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no conflict of interest.
- 1.Chi W.J., Y.K. Chang, Y.K. and Hong, S.K. (2012) Agar degradation by microorganisms and agar-degrading enzymes. Applied Microbiology and Biotechnology 94, 917–930, 4.Google Scholar
- 4.Chen, H. M., Zheng, L., & Yan, X. J. (2005). The preparation and bioactivity research of agarooligosaccharides. Food. Technol. Biotechnol., 43, 29–36.Google Scholar
- 5.Kobayashi, R., M. Takisada, Suzuki T., Kirimura K. and S. Usami S. (1997) Neoagarobiose as a novel moisturizer with whitening effect. Biosci. Biotechnol. Biochem. 61, 162–163, 1.Google Scholar
- 8.Li, M., Li, G., Zhu, L., Yin, Y., Zhao, X., Xiang, C., Yu, G., & Wang, X. (2014). Isolation and characterization of an agaro-oligosaccharide (AO)-hydrolyzing bacterium from the gut microflora of Chinese individuals. PLoS One, 9, 1–9.Google Scholar
- 10.Higashimura, Y., Naito, Y., Takagi, T., Uchiyama, K., Mizushima, K., Ushiroda, C., Ohnogi, H., Kudo, Y., Yasui, M., Inui, S., Hisada, T., Honda, A., Matsuzaki, Y., & Yoshikawa, T. (2016). Protective effect of agaro-oligo-saccharides on gut dysbiosis and colon tumorigenesis in high-fat diet-fed mice. American Journal of Physiology. Gastrointestinal and Liver Physiology, 310, 367–375.CrossRefGoogle Scholar
- 14.Lee, Y.H., Jun, S.E. and Shin, H.D. (2005) Low molecular weight agarose-specific alpha-agarase from agarolytic marine microorganism Pseudoalteromonas sp. BL-3 which hydrolyzes alpha-1, 3-glycoside bond of agar or agarose to produce agarobiose and agarotetraose. Patent KR2005079035.Google Scholar
- 17.Leema Roseline, T., & Sachindra, N. M. (2016). Characterization of extracellular agarase production by Acinetobacter junii PS12B, isolated from marine sediment. Biocatal. Agri. Biotechnol 6, 219–226.
- 18.Nitin, T., Ravi, S., John, B., Vishal, G., Reddy, C.R.K., Arvind, M. and Lali, Bhavanath, J. (2016) An integrated process for the extraction of fuel and chemicals from marine macroalgal biomass. Sci. Rep. 6, 30728.
- 21.Lowry, O. H., Rosebrough, A. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. The Journal of Biological Chemistry, 193, 265–273.Google Scholar
- 23.Merril, C. R., & Washart, K. M. (1998). Protein detection methods. In B. D. Hames (Ed.), Gel electrophoresis of proteins, a practical approach (pp. 53–92). Oxford: Oxford University Press.Google Scholar
- 27.Wada, M., Kido, H., Ohyama, K., Ichibangase, T., Kishikawa, N., & Ohba, Y. (2007). Chemiluminescent screening of quenching effects of natural colorants against reactive oxygen species, evaluation of grape seed, monascus, gardenia and red radish extracts as multi-functional food additives. Food Chemistry, 101(3), 980–986.CrossRefGoogle Scholar
- 29.StatSoft. (1999) STATISTICA for windows. StatSoft, Inc., Tulsa.Google Scholar
- 34.Ohta, Y., Hatada, Y., Nogi, Y., Miyazaki, M., Li, Z., Akita, M., Hidaka, Y., Goda, S., Ito, S., & Horikoshi, K. (2004). Enzymatic properties and nucleotide and amino acid sequences of a thermostable β-agarase from a novel species of deep-sea Microbulbifer. Applied Microbiology and Biotechnology, 64(4), 505–514.CrossRefGoogle Scholar
- 35.Ohta, Y., Nogi, Y., Miyazaki, M., Li, M., Hatada, Y., Ito, S., & Horikoshi, K. (2004). Enzymatic properties and nucleotide and amino acid sequences of a thermostable β-agarase from the novel marine isolate, JAMB-A94. Bioscience, Biotechnology, and Biochemistry, 68(5), 1073–1081.CrossRefGoogle Scholar
- 39.Ha, J.C., Kim, G.T., .Kim, S.K., Oh, T.K., Yu, J.H. and Kong, I.S. (1997) β-agarase from Pseudomonas sp. W7, purification of the recombinant enzyme from Escherichia coli and the effects of salt on its activity. Biotechnology and Applied Biochemistry 26, 1–6.Google Scholar
- 40.Sugano, Y., Terada, I., Arita, M., Noma, M., & Matsumoto, T. (1993). Purification and characterization of a new agarase from a marine bacterium, Vibrio sp. strain JT0107. Applied and Environmental Microbiology, 93, 1549–1554.Google Scholar
- 41.Fu, X. T., Cheol-Ho, P., Hong, L., & Sang, M. K. (2009). Gene cloning, expression, and characterization of a β-Agarase, AgaB34, from Agarivorans albus YKW-34. Journal of Microbiology and Biotechnology, 19(3), 257–264.Google Scholar
- 43.Gupta, V., Trivedi, N., Kumar, M., Reddy, C. R. K., & Jha, B. (2013). Purification and characterization of exo-b-agarase from an endophytic marine bacterium and its catalytic potential in bioconversion of red algal cell wall polysaccharides into galactans. Biomass and Bioenergy, 49, 290–298.CrossRefGoogle Scholar
- 51.Kim, J. H., Yun, E. J., Seo, N., Yu, S., Kim, D. H., Cho, K. M., An, H. J., Kim, J. H., Choi, I. G., & Kim, K. H. (2016). Enzymatic liquefaction of agarose above the sol–gel transition temperature using a thermostable endo-type β-agarase, Aga16B. Applied Microbiology and Biotechnology, 101, 1111–1120.Google Scholar
- 53.Araki, T. Lu, Z. and Morishita, T. (1998) Optimization of parameters for isolation of protoplasts from Gracilaria verrucosa (Rhodophyta). Journal of Marine Biotechnology 6, 193–197, 3.Google Scholar
- 59.Ruth, J., & Adhikary, S. P. (2004). Effect of alkali treatment on the yield and quality of agar from red algae Gracilaria verrucosa occurring at different salinity gradient of Chilika lake. Ind. Journal of Marine Science, 33, 202–205.Google Scholar