Agarase Production by Marine Pseudoalteromonas sp. MHS: Optimization, and Purification

Source:
By:The author(s)

DOI: https://doi.org/10.30564/jms.v4i1.3875

Abstract:

Agar is an essential polysaccharide that has been utilized in numerous fields. Many kinds of literature have been published regarding agarolytic microorganisms’ isolation and agarases biochemical studies. In this search, a local marine agarolytic bacterium associated with marine alga Ulva lactuca surface was isolated and identified as Pseudoalteromonas sp. MHS. The agarase production was parallel to the growth of Pseudoalteromonas sp. MHS as cells displayed a lag phase (2 h), subsequently an exponential growth that prolonged till 10 h where maximum growth (OD550nm = 3.9) was achieved. The enzyme activity increased rapidly as cells increased exponentially where the maximum activity of 0.22 U/mL was achieved after 8h and remained constant till 12 h during the stationary phase of growth. Agarase production was optimized using Plackett-Burman statistical design by measuring enzyme activity as a response and the design was validated using a verification experiment; the activity of the enzyme increased from 0.22 U/mL to 0.29 U/mL. Pseudoalteromonas sp. MHS agarase was partially purified and its molecular weight (MW) was determined by SDSPAGE (15-25 kDa). Agarase showed approximately 94% of its activity at 40 °C. The enzyme stability decreased as the temperature increased; the enzyme could retain about 98, 90, 80, 75, and 60% of its activity at 20, 30, 40, 50, and 60 °C, respectively. Biomass of the red alga Pterocladia capillacea proved to be a suitable substrate for agarase production using Pseudoalteromonas sp. MHS; the enzyme activity recorded after 24 h of incubation was 0.35 U/mL compared to 0.29 U/mL from the optimized medium.

References:

[1]Comba-González, N.B., Ruiz-Toquica, J.S., López-Kleine, L., Montoya-Castaño, D., 2016. Epiphytic bacteria of macroalgae of the Genus Ulva and their potential in producing enzymes having biotechnological interest. Mar Biol Oceanogr. 5, 1-9. [2]Song, T., Zhang, W., Wei, C., Jiang, T., Xu, H., Cao, Y., Cao, Y., Qiao, D., 2015. Isolation and characterization of agar-degrading endophytic bacteria from plants. Curr. Microbiol. 70, 275-281. [3]Seok, J.H., Kim, H.S., Hatada, Y., Nam, S.W., Kim, Y.H., 2012. Construction of an expression system for the secretory production of recombinant α-agarase in yeast. Biotechnol. Lett. 34, 1041-1049. [4]Popper, Z.A., Michel, G., Hervé, C., Domozych, D.S., Willats, W.G., Tuohy, M.G., Kloareg, B., Stengel, D.B., 2011. Evolution and diversity of plant cell walls: from algae to flowering plants. Annu. Rev. Plant Biol. 62, 567-590. [5]Martin, M., Barbeyron, T., Martin, R., Portetelle, D., Michel, G., Vandenbol, M., 2015. The cultivable surface microbiota of the brown alga Ascophyllum nodosum is enriched in macroalgal-polysaccharide-degrading bacteria. Front Microbiol. 6, 1487. [6]Martin, M., Portetelle, D., Michel, G., Vandenbol, M., 2014. Microorganisms living on macroalgae: diversity, interactions, and biotechnological applications. Appl. Microbiol. Biotechnol. 98, 2917-2935. [7]Trincone, A., 2011. Marine Biocatalysts: Enzymatic features and applications. Mar. Drugs. 9, 478-499. [8]Chi, W.J., Chang, Y.K., Hong, S.K., 2012. Agar degradation by microorganisms and agar-degrading enzymes. Appl. Microbiol. Biotechnol. 94, 917-930. [9]Saraswathi, S., Vasanthabharathi, V., Kalaiselvi, V., Jayalakshmi, S., 2011. Characterization and optimization of agarase from an estuarine Bacillus subtilis. Microbiol. Res. 5, 2960-2968. [10]Ziayoddin, M., Lalitha, J., Shinde, M., 2014. Optimization of agarase production by alkaline Pseudomonas aeruginosa ZSL-2 using Taguchi experimental design. ILNS. 12, 180-193. [11]Chi, W., Park, J., Kang, D., Hong, S., 2014. Production and characterization of a novel thermostable extracellular agarase from Pseudoalteromonas hodoensis newly isolated from the west sea of South Korea. Appl. Biochem. Biotechnol. 173, 1703-1716. [12]Choi, H.J., Hong, J.B., Park, J.J., Chi, W.J., Kim, M.C., Chang, Y.K., Hong, S.K., 2011. Production of agarase from a novel Micrococcus sp. GNUM-08124 strain isolated from the East Sea of Korea. Biotechnol. Bioprocess Eng. 16, 81. [13]Temuujin, U., Chi, W.J., Chang, Y.K., Hong, S.K., 2012. Identification and biochemical characterization of Sco3487 from Streptomyces coelicolor A3(2), an exo and endo-type β-agarase-producing neoagarobiose. Bacteriol. 194, 142-149. [14]Wu, H., Chen, G., Bian, Y., Zeng, W., Sun, B., Liang, Z., 2017. Identification and characterization of a new agar-degrading strain with the novel properties of saccharides inhibition and nitrogen fixation. Microbiol. 55, 475-482. [15]Abouelkheir, S.S., Abdelghany, E.A., Ghozlan, H.A., Sabry, S.A., 2020. Characterization of Biofilm Forming Marine Pseudoalteromonas spp. J. Mar. Sci. 2(1), 31-37. [16]Abouelkheir, S.S., Abdelghany, E.A., Sabry, S.A., Ghozlan, H.A., 2021. Biofilm Formation by Marine Cobetia marina alex and Pseudoalteromonas spp: Development and Detection of Quorum Sensing N-Acyl Homoserine Lactones (AHLs) Molecules. Mar. Sci. 3(3), 1-12. DOI: https://doi.org/10.30564/jms.v3i3.3397. [17]Fu, X.T., Lin, H., Kim, S.M., 2009. Optimization of medium composition and culture conditions for agarase production by Agarivorans albus YKW-34. Process Biochem. 44, 1158-1163. [18]Gu, W.X., Chen, Y.L., Niu, H.N., Lu, X., Mao, X.Z., Du, Z.J., Liu, X.L., 2012. Enhanced Activity of Intracellular Agarase from a Novel Marine Strain Agarivorans gilvus WH0801. In Adv. Mater. Res. Trans Tech Publications, Ltd. 554-556, 1227-1232. DOI: https://doi.org/10.4028/www.scientific.net/ amr.554-556.1227 [19]Jung, C., Kwon, H., Park, C., Lee, J., 2012. Optimization of Pseudoalteromonas sp. JYBCL 1 culture conditions, medium composition and extracellular β-agarase activity. Biotechnol. Bioprocess Eng. 17, 937-945. [20]Saqib, A.A., Whitney, P.J., 2011. Differential behaviour of the dinitrosalicylic acid (DNS) reagent towards mono- and di-saccharide sugars. Biomass and Bioenergy. 35(2011), 4748-4750. [21]Calissendorff, J., Falhammar, H., 2017. Lugol’s solution and other iodide preparations: perspectives and research directions in Graves’ disease. Endocrine. 58, 467-473. DOI: https://doi.org/10.1007/s12020-017-1461-8 [22]https://www.bio-rad.com/webroot/web/pdf/ lsr/literature/Bulletin_6040.pdf. [23]Faturrahman, F., Meryandini, A., Junior, M.Z., Rusmana, I., 2011. Isolation and identification of an agar-liquefying marine bacterium and some properties of its extracellular agarases. Biodiversitas. 12, 192-197. [24] Feng, Z., Li, M., 2013. Purification and characterization of agarase from Rhodococcus sp. Q5, a novel agarolytic bacterium isolated from printing and dyeing wastewater. Aquaculture. 372, 74-79. [25] Cui, F., Dong, S., Shi, X., Zhao, X., Zhang, X., 2014. Overexpression and characterization of a novel thermostable β-Agarase YM01-3, from marine bacterium Catenovulum agarivorans YM01T. Mar. Drugs. 12, 2731-2747. [26] Furusawa, G., Lau, N.S., Suganthi, A., Amirul, A.A.A., 2017. Agarolytic bacterium Persicobacter sp. CCBQB2 exhibited a diauxic growth involving galactose utilization pathway. Microbiologyopen. 6, 1-11. [27] Moyes, R.B., Reynolds, J., Breakwell, D.P., 2009. Differential staining of bacteria: negative stain. Curr. Protoc. Microbiol. A-3F. John Wiley & Sons, Inc. [28] Duguid, J.P., 1951. The demonstration of bacterial capsules and slime. Pathol. 63, 673-685. [29] Buck, J.D., 1982. Nonstaining (KOH) method for determination of gram reactions of marine bacteria. Appl. Environ. Microbiol. 44, 992-993. [30] Phanse, N., Chincholikar, A., Patel, B., Rathore, P., Vyas, P., Patel, M., 2011. Screening of PHA (poly hydroxyalkanoate) producing bacteria from diverse sources. Biosci. 1, 27-32. [31] Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular cloning: a laboratory manual (No. Ed. 2). Cold spring harbor laboratory press. [32] Saravanan, D., Kumar, V.K., Radhakrishnan, M., 2015. Isolation and optimization of agarase producing bacteria from marine sediments. Chem. Tech. Res. 8, 1701-1705. [33] Deshavath, N.N., Mukherjee, G., Goud, V.V., Veeranki, V.D., Sastri, C.V., 2020. Pitfalls in the 3, 5-dinitrosalicylic acid (DNS) assay for the reducing sugars: Interference of furfural and 5-hydroxymethylfurfural. Int. J. Biol. Macromolecules, 156 (2020) xxx. https:// www.researchgate.net/deref/https%3A%2F%2Fdoi. org%2F10.1016%2Fj.ijbiomac.2020.04.045. [34] Yang, M., Mao, X., Liu, N., Qiu, Y., Xue, C., 2014. Purification and characterization of two agarases from Agarivoransalbus OAY02. Process Biochem. 49, 905-912. [35] Plackett, R.L., Burman, J.P., 1946. The design of optimum multifactorial experiments. Biometrika. 33, 305-325. [36] Li, J., Hu, Q., Li, Y., Xu, Y., 2015. Purification and characterization of cold-adapted beta-agarase from an Antarctic psychrophilic strain. Braz J Microbiol. 46, 683-690. [37] Liu, Y., Tian, X., Peng, C., Du, Z., 2019. Isolation and Characterization of an Eosinophilic GH 16 β-Agarase (AgaDL6) from an Agar-Degrading Marine Bacterium Flammeovirga sp. HQM9. Microbiol. Biotechnol. 29(2), 235-243. DOI: https://doi.org/10.4014/jmb.1810.09065 [38] Rhee, Y.J., Han, C.R., Kim, W.C., Jun, D.Y., Rhee, I.K., Kim, Y.H., 2010. Isolation of a novel freshwater agarolytic Cellvibrio sp. KY-YJ-3 and characterization of its extracellular beta-agarase. Microbiol. Biotechnol. 20, 1378-1385. [39] Oh, Y.H., Jung, C., Lee, J., 2011. Isolation and characterization of a novel agarase- producing Pseudoalteromonas spp. bacterium from the guts of spiny turban shells. Microbiol. Biotechnol. 21, 818-821. [40] Kim, J., Hong, S.K., 2012. Isolation and characterization of an agarase-producing bacterial strain, Alteromonas sp. GNUM-1, from the West Sea, Korea. Microbiol. Biotechnol. 22, 1621-1628. [41] Egan, S., Holmström, C., Kjelleberg, S., 2001. Pseudoalteromonas ulvae sp. nov., a bacterium with antifouling activities isolated from the surface of a marine alga. Syst. Evol. Microbiol. 51, 1499-1504. [42] Song, T., Cao, Y., Xu, H., Zhang, W., Fei, B., Qiao, D., Cao, Y., 2014. Purification and characterization of a novel β-agarase of Paenibacillus sp. SSG-1 isolated from soil. Biosci. Bioeng. 118, 125-129. [43] Li, J., Sha, Y., Seswita-Zilda, D., Hu, Q., He, P., 2014. Purification and characterization of thermostable Agarase from Bacillus sp. BI-3, a thermophilic bacterium isolated from Hot Spring. Microbiol. Biotechnol. 24, 19-25. [44] Lee, D.G., Jeon, M.J., Lee, S.H., 2012. Cloning, expression, and characterization of a glycoside hydrolase family 118 beta-agarase from Agarivorans sp. JA-1. Microbiol. Biotechnol. 22, 1692-1697. [45] Tang, J., Wang, M., Zhou, Q., Nagata, S., 2011. Improved composting of Undaria pinnatifida seaweed by inoculation with Halomonas and Gracilibacillus sp. isolated from marine environments. Bioresour. Technol. 102, 2925-2930. [46] Kang, S., Kim, J.K., 2015. Reuse of red seaweed waste by a novel bacterium, Bacillus sp. SYR4 isolated from a sandbar. Microbiol. Biotechnol. 31, 209-217. [47] Abouelkheir, S.S., Kamara, M.S., Atia, S.M. et al., 2020. Novel research on nanocellulose production by a marine Bacillus velezensis strain SMR: a comparative study. Sci Rep. 10, 14202. DOI: https://doi.org/10.1038/s41598-020-70857-7 [48] Silva, L.M.C.M., Lima, V., Holanda, M.L., Pinheiro, P.G., Rodrigues, J.A.G., Lima, M.E.P., Benevides, N.M.B., 2010. Antinociceptive and Anti-inflammatory Activities of Lectin from Marine Red Alga Pterocladiella capillacea. Biol. Pharm. Bull. 33, 830-835. [49] Sebaaly, C., Karaki, N., Chahine, N., Evidente, A., Yassine, A., Habib, J., Kanaan, H., 2012. Polysaccharides of the red algae ―Pterocladia”growing on the Lebanese coast: Isolation, structural features with antioxidant and anticoagulant activities. J App Pharm Sci. 2, 1-10. [50] Chauhan, P.S., Saxena, A., 2016. Bacterial carrageenases: an overview of production and biotechnological applications. 3 Biotech. 6, 1-18.