Characterization of a κ-Carrageenase-producing Marine Bacterium, Isolate ALAB-001
Crimson C. Tayco1, Francis A. Tablizo1, Raymond S. Regalia2 and Arturo O. Lluisma1*
1The Marine Science Institute, University of the Philippines Diliman, Quezon City, Philippines 1101
2Center for Marine Bio-Innovation, School of Biotechnology and Biomolecular Sciences, Faculty of Science, The University of New South Wales, Sydney, Australia 2052
corresponding author: This email address is being protected from spambots. You need JavaScript enabled to view it.
ABSTRACT
Carrageenases are glycoside hydrolases that specifically degrade carrageenan, a highly anionic polysaccharide found in the cell wall of many red algal species. To date, only a few of these enzymes have been characterized, and identifying additional sources is important considering the role of carrageenases in production of carrageenan derivatives. In this paper, we report the characterization of a marine bacterial strain that produces κ-carrageenase. The strain, which we designate as ALAB-001, was isolated from diseased thallus fragments of the red alga Kappaphycus alvarezii, a commercially important source of carrageenan. Genotypic and phenotypic data suggest that the isolate belongs to a relatively poorly-characterized group of bacteria in Alteromonadaceae (Alteromonadales) and is closely related to Marinimicrobium and Microbulbifer. Significant κ-carrageenase activity (175 U/mL) was evident when the isolate was grown in the presence of κ-carrageenan. Activity against starch was also high (180 U/mL), but activity against agar, alginate, cellulose, ι-carrageenan, and λ-carrageenan was significantly lower (25-50 U/mL). Laboratory-scale production of the enzyme using batch cultures of the isolate was achieved by optimizing culture medium, length of culture time and degree temperature. Optimal growth was observed at 25°C, though the isolate survived at 30°C. An in-house developed seawater-based medium containing equal concentrations of yeast extract and tryptone (YETS) yielded the highest cell growth based on total protein concentration (~ 3000 μg/mL) and enzyme activity (~ 45 U/mL).
INTRODUCTION
κ-carrageenases are enzymes that catalyze the hydrolysis of κ-carrageenan, a highly sulfated polysaccharide and a major component of the cell wall matrix in many red algal species. κ-carrageenases are members of the Family 16 glycoside hydrolases based on their overall and catalytic domain structure (Michel et al. 1999). Studies have already demonstrated the structural similarity of κ-carrageenases with other Family 16 glycoside hydrolases such as β-agarase, laminarase, lichenase and xyloglucan transglycosylases . . . . . . . . . . . . .
REFERENCES
ARAKI T, HIGASHIMOTO Y, MORISHITA T. 1999. Purification and characterization of kappa-carrageenase from a marine bacterium, Vibrio CA1004. Fish Sci 65(6): 937-942.
BARBEYRON T, GERARD A, POTIN P, HENRISSAT B, KLOAREG B. 1998. The kappa-carrageenase of the marine bacterium Cytophaga drobachiensis. Structural and phylogenetic relationships within family-16 glycoside hydrolases. Mol Biol Evol 15(5): 528-537.
BARBEYRON T, L’HARIDON S, CORRE E, KLOAREG B, POTIN P. 2001. Zobellia galactanovorans gen. nov., sp. nov., a marine species of Flavobacteriaceae isolated from a red alga, and classification of Cytophaga uliginosa (ZoBell and Upham 1944) Reichenbach 1989 as Zobellia uliginosa gen. nov., comb. nov. Int J Syst Evol Microbiol 51(3): 985-997.
BARBEYRON T, MICHEL G, POTIN P, HENRISSAT B, KLOAREG B. 2000. iota-Carrageenases Constitute a Novel Family of Glycoside Hydrolases, Unrelated to That of kappa-Carrageenases. J Biol Chem 275(45): 35499-35505.
CÁCERES PJ, CARLUCCI MJ, DAMONTE EB, MATSUHIRO B, ZUNIGA EA. 2000. Carrageenans from Chilean samples of Stenogramme interrupta (Phyllophoraceae): structural analysis and biological activity. Phytochemistry 53(1): 81-86.
GAUTHIER G, GAUTHIER M, CHRISTEN R. 1995. Phylogenetic Analysis of the Genera Alteromonas, Shewanella, and Moritella Using Genes Coding for Small-Subunit rRNA Sequences and Division of the Genus Alteromonas into Two Genera, Alteromonas (Emended) and Pseudoalteromonas gen. nov., and Proposal of Twelve New Species Combinations. Int J Syst Bacteriol 45(4): 755-761.
GONZALEZ JM, MAYER F, MORAN MA, HODSON RE, WHITMAN WB. 1997. Microbulbifer hydrolyticus gen. nov., sp. nov., and Marinobacterium georgiense gen. nov., sp. nov., Two Marine Bacteria from a Lignin Rich Pulp Mill Waste Enrichment Community. Int J Syst Bacteriol 47(2): 369-376.
GUIBET M, COLIN S, GENICOT S, KLOAREG B, MICHEL G, HELBERT W. 2007. Degradation of λ-carrageenan by Pseudoalteromonas carrageenovora λ-carrageenase: a new family of glycoside hydrolases unrelated to κ- and ι-carrageenases. Biochem J 404(1): 105-114.
GUINDON S, GASCUEL O. 2003. A Simple, Fast, and Accurate Algorithm to Estimate Large Phylogenies by Maximum Likelihood. Syst Biol 52(5): 696-704.
HIROISHI S, SUGIE K, YOSHIDA T, MORIMOTO J, TANIGUCHI Y, IMAI S, KUREBAYASHI J. 2001. Antitumor effects of Marginisporum crassissimum (Rhodophyceae), a marine red alga. Cancer Lett 167(2): 145-150.
KNUTSEN SH. 1991. Carrageenase production in a culture of Pseudomonas carrageenovora growing on kappa-carrageenan. In: Garcia Reina G, Pedersen M. eds. Seaweed Cellular Biotechnology, Physiology and Intense Cultivation. Proceedings of a COST-48 (Subgroup 1) Workshop; 8-17 February 1991; Marine Plant Biotechnology Laboratory, University of Las Palmas, Canary Islands, Spain: European Cooperation in the Field of Scientific and Technical Research. p. 277-281.
KNUTSEN SH, GRASDALEN H. 1992. Analysis of carrageenans by enzymic degradation, gel filtration and 1H NMR spectroscopy. Carbohydr Polym 19(3): 199-210.
KOBAYASHI T, UCHIMURA K, KOIDE O, DEGUCHI S, HORIKOSHI K. 2012. Genetic and biochemical characterization of the Pseudoalteromonas tetraodonis alkaline kappa-carrageenase. BBB 76(3): 506-511.
KUMAR S, TAMURA K, NEI M. 2004. MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief Bioinform 5(2): 150-163.
LEMOS ML, TORANZO AE, BARJA JL. 1985. Modified Medium for the Oxidation-Fermentation Test in the Identification of Marine Bacteria. Appl Environ Microbiol 49(6): 1541-43.
LIM J, JEON CO, LEE J, SONG S, KIM K, KIM C. 2006. Marinimicrobium koreense gen. nov. and Marinimicrobium agarilyticum sp. nov., novel moderately halotolerant bacteria isolated from tidal flat sediment in Korea. Int J Syst Evol Microbiol 56(3): 653-657.
LIU GL, LI Y, CHI Z. 2011. Purification and characterization of kappa-carrageenase from the marine bacterium Pseudoalteromonas porphyrae for hydrolysis of kappa-carrageenan. Process Biochem 46(1): 265-271.
MARCHESI JR, SATO T, WEIGHTMAN AJ, MARTIN TA, FRY JC, HIOM SJ, WADE W. 1998. Design and Evaluation of Useful Bacterium-Specific PCR Primers That Amplify Genes Coding for Bacterial 16S rRNA. Appl Environ Microbiol 64(2): 795-799.
MICHEL G, BARBEYRON T, FLAMENT D, VERNET T, KLOAREG B, DIDEBERG O. 1999. Expression, purification, crystallization and preliminary X-ray analysis of the ?-carrageenase from Pseudoalteromonas carrageenovora. Acta Crystallogr D Biol Crystallogr 55(4): 918-920.
MOU H, XIAOLU J, HUASHI G. 2003. A κ-carrageenan derived oligosaccharide prepared by enzymatic degradation containing anti-tumor activity. J Appl Phycol 15: 297-303.
POTIN P, SANSEAU A, GALL Y, ROCHAS C, KLOAREG B. 1991. Purification and characterization of a new kappa-carrageenase from a marine Cytophaga-like bacterium. Eur J Biochem 201(1): 241-247.
POTIN P, RICHARD C, BARBEYRON T, HENRISSAT B, GEY C, PETILLOT Y, FOREST E, DIDEBERG O, ROCHAS C, KLOAREG B. 1995. Processing and hydrolytic mechanism of the cgkA-encoded kappa-carrageenase of Alteromonas carrageenovora. Eur J Biochem 228(3): 971-975.
RENN D. 1997. Biotechnology and the red seaweed polysaccharide industry: status, needs and prospects. Trends Biotechnol 15(1): 9-14.
SARWAR G, ODA H, SAKATA T, KAKIMOTO D. 1985. Potentiality of artificial sea water salts for the production of carrageenase by a marine Cytophaga sp. Microbiol Immunol 29(5): 405-411.
SARWAR G, SAKATA T, KAKIMOTO D. 1983. Isolation and characterization of carrageenan-decomposing bacteria from marine environment. J Gen Appl Microbiol 29(2): 145-155.
SMITH PK, KROHN RI, HERMANSON GT, MALLIA AK, GARTNER FH, PROVENZANO MD, FUJIMOTO EK, GOEKE NM, OLSON BJ, KLENK DC. 1985. Measurement of protein using bicinchoninic acid. Anal Biochem 150(1): 76-85.
WEIGL J, YAPHE W. 1966. The enzymic hydrolysis of carrageenan by Pseudomonas carrageenovora: purification of a kappa-carrageenase. Can J Microbiol 12: 939-947.
YUAN H, SONG J. 2005. Preparation, structural characterization and in vitro antitumor activity of kappa-carrageenan oligosaccharide fraction from Kappaphycus striatum. J Appl Phycol 17(1): 7-13.
ZHOU M, MA J, YE H, HUANG K, ZHAO X. 2008. A kappa-carrageenase from a newly isolated Pseudoalteromonas-like bacterium, WZUC10. Biotechnol. Bioprocess Eng 13(5): 545-551.
