Applied Biochemistry and Biotechnology

, Volume 186, Issue 1, pp 66–84 | Cite as

Purification and Characterization of Agarase from Marine Bacteria Acinetobacter sp. PS12B and Its Use for Preparing Bioactive Hydrolysate from Agarophyte Red Seaweed Gracilaria verrucosa

  • T Leema Roseline
  • NM SachindraEmail author


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.


Acinetobacter 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. 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
  2. 2.
    Fernandes, P. (2014). Marine enzymes and food industry: insight on existing and potential interactions. Frontiers in Marine Science, 1, 1–18.CrossRefGoogle Scholar
  3. 3.
    Fu, X. T., & Kim, S. M. (2010). Agarase: review of major sources, categories, purification method, enzyme characteristics and applications. Marine Drugs, 8(12), 200–218.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 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. 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
  6. 6.
    Lee, D. G., Jang, M. K., Lee, O. H., Kim, N. Y., Ju, S. A., & Lee, S. H. (2008). Over-production of a glycoside hydrolase family 50 beta-agarase from Agarivorans sp. JA-1 in Bacillus subtilis and the whitening effect of its product. Biotechnology Letters, 30(5), 911–918.CrossRefPubMedGoogle Scholar
  7. 7.
    Hu, B., Gong, Q., Wang, Y., Ma, Y., & Li, J. (2006). Prebiotic effects of neoagaro-oligosaccharides prepared by enzymatic hydrolysis of agarose. Anaerobe, 12(5-6), 260–266.CrossRefPubMedGoogle Scholar
  8. 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
  9. 9.
    Enoki, T., Tominaga, T., Takashima, F., Ohnogi, H., & Sagawa, H. (2012). Anti-tumor-promoting activities of agaro-oligosaccharides on two-stage mouse skin carcinogenesis. Biological & Pharmaceutical Bulletin, 35(7), 1145–1149.CrossRefGoogle Scholar
  10. 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
  11. 11.
    Higashimura, Y., Naito, Y., & Takagi, T. (2013). Oligosaccharides from agar inhibits murine intestinal inflammation through the induction of heme oxygenase-1 expression. Journal of Gastroenterology, 48(8), 897–909.CrossRefPubMedGoogle Scholar
  12. 12.
    Potin, P., Richard, C., Rochas, C., & Kloareg, B. (1993). Purification and characterization of the α-agarase from Alteromonas agarlyticus (Cataldi) comb. nov., strain GJ1B. European Journal of Biochemistry, 214(2), 599–607.CrossRefPubMedGoogle Scholar
  13. 13.
    Ohta, Y., Hatada, Y., Miyazaki, M., Nogi, Y., Ito, S., & Horikoshi, K. (2005). Purification and characterization of a novel α-agarase from a Thalassomonas sp. Current Microbiology, 50(4), 212–216.CrossRefPubMedGoogle Scholar
  14. 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
  15. 15.
    Lakshmikanth, M., Manohar, S., Souche, Y., & Lalitha, J. (2006). Extracellular β-agarase LSL-1 producing neoagarobiose from a newly isolated agar-liquefying soil bacterium, Acinetobacter sp., AG LSL-1. World Journal of Microbiology and Biotechnology, 22(10), 1087–1094.CrossRefGoogle Scholar
  16. 16.
    Lakshmikanth, M., Manohar, S., & Lalitha, J. (2009). Purification and characterization of β-agarase from agar-liquefying soil bacterium, Acinetobacter sp., AG LSL-1. Process Biochemistry, 44(9), 999–1003.CrossRefGoogle Scholar
  17. 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. 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.
  19. 19.
    Wu, F., Wu, J., Liao, Y., Wang, M., & Shih, I. (2014). Sequential acid and enzymatic hydrolysis in situ and bioethanol production from Gracilaria biomass. Bioresource Technology, 156, 123–131.CrossRefPubMedGoogle Scholar
  20. 20.
    Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31(3), 426–428.CrossRefGoogle Scholar
  21. 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.PubMedGoogle Scholar
  22. 22.
    Laemmeli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 277, 680–685.CrossRefGoogle Scholar
  23. 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
  24. 24.
    Duan, X. J., Zhang, W. W., Li, X. M., & Wang, B. G. (2006). Evaluation of antioxidant property of extract and fractions obtained from a red alga, Polysiphonia urceolata. Food Chemistry, 95(1), 37–43.CrossRefGoogle Scholar
  25. 25.
    Sowmya, R., & Sachindra, N. M. (2012). Evaluation of antioxidant activity of carotenoid extract from shrimp processing by products by in vitro assays and in membrane model system. Food Chemistry, 134(1), 308–314.CrossRefGoogle Scholar
  26. 26.
    Tsai, P. J., Tsai, T. H., Yu, C. H., & Ho, S. C. (2007). Comparison of NO-scavenging and NO-suppressing activities of different herbal teas with those of green tea. Food Chemistry, 103(1), 181–187.CrossRefGoogle Scholar
  27. 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
  28. 28.
    Dinis, T. C. P., Madeira, V. M. C., & Almeida, L. M. (1994). Action of phenolic derivates acetoami nophen, salycilate and 5-aminosalycilate as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers. Archives of Biochemistry and Biophysics, 315(1), 161–169.CrossRefPubMedGoogle Scholar
  29. 29.
    StatSoft. (1999) STATISTICA for windows. StatSoft, Inc., Tulsa.Google Scholar
  30. 30.
    Dong, J., Tamaru, Y., & Araki, T. (2007). A unique β-agarase a from a marine bacterium, Vibrio sp. strain PO-303. Applied Microbiology and Biotechnology, 74(6), 1248–1255.CrossRefPubMedGoogle Scholar
  31. 31.
    Jonghee, K., & Hong, S. K. (2012). Isolation and characterization of an agarase-producing bacterial strain, Alteromonas sp. GNUM-1, from the West Sea, Korea. Journal of Microbiology and Biotechnology, 22, 1621–1628.CrossRefGoogle Scholar
  32. 32.
    Kang, N. Y., Choi, Y. L., Choi, Y. S., Kim, B. K., Jeon, B. S., Cha, J. Y., Kim, C. H., & Lee, Y. C. (2003). Cloning, expression and characterization of a β-agarase gene from a marine bacterium, Pseudomonas sp. SK38. Biotechnology Letters, 25(14), 1165–1170.CrossRefPubMedGoogle Scholar
  33. 33.
    Fu, X. T., Lin, H., & Kim, S. M. (2008). Purification and characterization of a novel β-agarase AgaA34, from Agarivorans albus YKW-34. Applied Microbiology and Biotechnology, 78(2), 265–273.CrossRefPubMedGoogle Scholar
  34. 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.CrossRefPubMedGoogle Scholar
  35. 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.CrossRefPubMedGoogle Scholar
  36. 36.
    Long, M., Ziniu, Y., & Xun, X. (2010). A novel β-agarase with high pH stability from marine Agarivorans sp. LQ48. Marine Biotechnology, 12(1), 62–69.CrossRefPubMedGoogle Scholar
  37. 37.
    Fu, W., Baoqin, H., Delin, D., Liu, W., & Wang, C. (2008). Purification and characterization of agarase from a marine bacterium Vibrio sp. F-6. Journal of Industrial Microbiology & Biotechnology, 35(8), 915–922.CrossRefGoogle Scholar
  38. 38.
    Zhang, W., & Sun, L. (2007). Cloning, characterization, and molecular application of a β-agarase gene from Vibrio sp. strain V134. Applied and Environmental Microbiology, 73(9), 2825–2831.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 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.PubMedGoogle Scholar
  40. 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. 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.PubMedGoogle Scholar
  42. 42.
    Zhu, Y., Zhao, R., Xiao, A., Li, L., Jiang, Z., Chen, F., & Ni, H. (2016). Characterization of an alkaline β-agarase from Stenotrophomonas sp. NTa and the enzymatic hydrolysates. Int. J. Biol. Macromolecules., 86, 525–534.CrossRefGoogle Scholar
  43. 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
  44. 44.
    Jonnadula, R., & Ghadi, C. S. (2011). Purification and characterization of β-agarase from seaweed decomposing bacterium Microbulbifer sp. strain CMC-5. Biotechnology and Bioprocess Engineering, 16(3), 513–519.CrossRefGoogle Scholar
  45. 45.
    Van der Meulen, H. J., & Harder, W. (1975). Production and characterization of the agarase of Cytoplaga flevensis. Antonie Van Leeuwenhoek, 41(1), 431–447.CrossRefPubMedGoogle Scholar
  46. 46.
    Lin, B., Lu, G., Zheng, Y., Xie, W., Li, S., & Hu, Z. (2012). Gene cloning, expression and characterization of a neoagarotetraose-producing β-agarase from the marine bacterium Agarivorans sp. HZ105. World Journal of Microbiology and Biotechnology, 28(4), 1691–1697.CrossRefPubMedGoogle Scholar
  47. 47.
    Xie, W., Lin, B., Zhou, Z., Lu, G., Lun, J., Xia, C., Li, S., & Hu, Z. (2013). Characterization of a novel β-agarase from an agar-degrading bacterium Catenovulum sp. X3. Applied Microbiology and Biotechnology, 97(11), 4907–4915.CrossRefPubMedGoogle Scholar
  48. 48.
    Suzuki, H., Sawai, Y., Suzuki, T., & Kawai, K. (2003). Purification and characterization of an extracellular β-agarase from Bacillus sp. MK03. Journal of Bioscience and Bioengineering, 93, 456–463.CrossRefGoogle Scholar
  49. 49.
    Shi, Y. L., Lu, X. Z., & Yu, W. G. (2008). A new β-agarase from marine bacterium Janthinobacterium sp. SY12. World Journal of Microbiology and Biotechnology, 24(11), 2659–2664.CrossRefGoogle Scholar
  50. 50.
    Ma, C., Lu, X., Shi, C., Li, J., Gu, Y., Ma, Y., Chu, Y., Han, F., Gong, Q., & Yu, W. (2007). Molecular cloning and characterization of a novel β-agarase, AgaB, from marine Pseudoalteromonas sp. CY24. The Journal of Biological Chemistry, 282(6), 3747–3754.CrossRefPubMedGoogle Scholar
  51. 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
  52. 52.
    Liang, Y., Ma, X., Zhang, L., Li, F., Liu, Z., & Mao, X. (2017). Biochemical characterization and substrate degradation mode of a novel exo-type β-agarase from Agarivorans gilvus WH0801. J. Agri. Food Chem., 65(36), 7982–7988.CrossRefGoogle Scholar
  53. 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
  54. 54.
    Michel, G., Nyval-Collen, P., Barbeyron, T., Czjzek, M., & Helbert, W. (2006). Bioconversion of red seaweed galactans, a focus on bacterial agarases and carrageenases. Applied Microbiology and Biotechnology, 71(1), 23–33.CrossRefPubMedGoogle Scholar
  55. 55.
    Goh, C. H., & Lee, K. T. (2010). A visionary and conceptual macroalgae based third-generation bioethanol (TGB) biorefinery in Sabah, Malaysia as an underlay for renewable and sustainable development. Renewable and Sustainable Energy Reviews, 14(2), 842–848.CrossRefGoogle Scholar
  56. 56.
    Kim, N. J., Li, H., Jung, K., Chang, H. N., & Lee, P. C. (2011). Ethanol production from marine algal hydrolysates using Escherichia coli KO11. Bioresource Technology, 102(16), 7466–7469.CrossRefPubMedGoogle Scholar
  57. 57.
    Wu, S. C., & Pan, C. L. (2004). Preparation of algal-oligosaccharide mixtures by bacterial agarases and their antioxidative properties. Fisheries Science, 70(6), 1164–1173.CrossRefGoogle Scholar
  58. 58.
    Ahola, S., Turon, X., Österberg, M., Laine, J., & Rojas, O. J. (2008). Enzymatic hydrolysis of native cellulose nanofibrils and other cellulose model films, effect of surface structure. Langmuir, 24(20), 11592–11599.CrossRefPubMedGoogle Scholar
  59. 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
  60. 60.
    Khambhaty, Y., Mody, K., Gandhi, M. R., Thampy, S., Maiti, P., Brahmbhatt, H., Eswaran, K., & Ghosh, P. K. (2012). Kappaphycus alvarezii as a source of bioethanol. Bioresource Technology, 103(1), 180–185.CrossRefPubMedGoogle Scholar
  61. 61.
    Yanagisawa, M., Nakamura, K., Ariga, O., & Nakasaki, K. (2011). Production of high concentrations of bioethanol from seaweeds that contain easily hydrolyzable polysaccharides. Process Biochemistry, 46(11), 2111–2116.CrossRefGoogle Scholar
  62. 62.
    Tsao, R. (2010). Chemistry and biochemistry of dietary polyphenols. Nutrients., 2(12), 1231–1246.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Meat and Marine SciencesCSIR-Central Food Technological Research InstituteMysoreIndia

Personalised recommendations