Philippine Journal of Science 142 (1): 45-54, June 2013 ISSN 0031 - 7683 Date Received: ?? Feb 20?? 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 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). Key Words: κ-carrageenan, κ-carrageenase, carrageenan-degrading bacteria, Kappaphycus alvarezii INTRODUCTION with other Family 16 glycoside hydrolases such as β-agarase, laminarase, lichenase and xyloglucan κ-carrageenases are enzymes that catalyze the hydrolysis transglycosylases (Lemos et al. 1985). of κ-carrageenan, a highly sulfated polysaccharide and a major component of the cell wall matrix in many red algal Only a handful of published reports describe the species. κ-carrageenases are members of the Family 16 isolation of κ-carrageenase-producing marine bacteria glycoside hydrolases based on their overall and catalytic and demonstrate their ability to produce carrageenase domain structure (Michel et al. 1999). Studies have already in culture. Bacterial species reported in the scientific demonstrated the structural similarity of κ-carrageenases literature include Pseudomonas carrageenovora (Weigl and Yaphe 1966), Cytophaga strain 1k-C783 (Sarwar *Corresponding author: [email protected] 45 Philippine Journal of Science Tayco et al.: Characterization of a κ-Carrageenase-producing Vol. 142 No. 1, June 2013 Marine Bacterium, Isolate ALAB-001 et al. 1983), Alteromonas fortis (Potin et al. 1995), by marine invertebrates feeding on carrageenophytes, Pseudoalteromonas carrageenovora (Gauthier et al. using them as sources of the enzyme carrageenase will 1995), Vibrio sp. CA-1004 (Araki et al. 1999), ‘Cytophaga necessitate the establishment of a hatchery and culture drobachiensis’ / Zobellia galactanivorans (Barbeyron et facility as well as the development of a laborious al. 1998, Barbeyron et al. 2001), Pseudoalteromonas- process of crude extraction from visceral organs. like bacterium (Zhou et al. 2008), Pseudoalteromonas Conversely, bacteria, as sources of carrageenase, are porphyrae (Liu et al. 2010), and Pseudoaltermonas very easy to handle in the laboratory, do not require large tetraodonis (Kobayashi et al. 2012). These studies storage space, and since the enzyme is secreted in the suggest that the enzyme is synthesized by marine bacteria medium, harvesting of the enzyme with relatively fewer that belong to at least two distantly related lineages, contaminants is easy. Hence, as far as laboratory enzyme Proteobacteria and Bacteroidetes, although most of the production is concerned, bacteria are still preferred isolates described in the reports belong to the former sources of κ-carrageenase over marine invertebrates. In group. this paper, we report the characterization of ALAB -001, a κ-carrageenase-producing bacterial isolate. Polysaccharides from marine rhodophytes, particularly carrageenan and agar, are major raw materials for a number of industries worldwide (Renn 1997). Carrageenan is a highly sulphated polysaccharide made MATERIALS AND METHODS up of D-galactose units linked by α (1→3) and β (1→4) glycosidic bonds. It exists in different forms depending on the number of sulphate substituents per disaccharide unit: Bacterial isolate one in κ-carrageenan, two in ι-carrageenan and three in A carrageenan-degrading marine bacterial strain, λ-carrageenan. Although carrageenan is principally used designated as ALAB-001, was previously isolated via in the industry as gelling, emulsifying, stabilizing and standard microbiological strategy. Diseased thallus texturing agents, studies have revealed other potential fragments of the red seaweed Kappaphycus alvarezii applications, particularly, in health and biomedicine. For (collected from a seaweed farm in Calatagan, Batangas, example, oligosaccharides derived from κ-carrageenan Philippines) were swabbed onto solid κ-carrageenan- (using carrageenases) have been shown to exhibit anti- (1.5%)-sterile seawater media and the plates were tumor activity, particularly, those with a molecular weight then screened for bacterial colonies that manifest of 1726 Da (Mou et al. 2003). Although the mechanism plate depression-forming activity. Pure cultures of the of anti-tumor activity is still unclear, the researchers depression-forming bacterial isolates were obtained concluded that oligo-carrageenan could be a potent anti- by repeated streaking and picking on plates and then tumor substance. Similar studies (Caceres et al. 2000; maintained by regular spot inoculation on carrageenan- Yuan and Song 2005) also found significant anti-tumor solidifed medium prepared using marine broth (Pronadisa) activity of certain fractions of carrageenans and oligo- with 1.5% κ-carrageenan (MBC) (Shemberg Corporation, carrageenans. These studies indicate that carrageenan- Philippines). derivatives, i.e., oligo-carrageenans obtained via degradation of carrageenan, possess significant potential Phenotypic, Biochemical, and Phylogenetic for biomedical and physiological applications. Characterization To determine the cellular morphology of the isolate, light Carrageenan-derivatives can be obtained using two microscopy of Gram-stained specimens was carried out different methods. The first method employs acid and a sample of the bacterium (1 mL, OD = 0.1) was hydrolysis of carrageenan in order to generate oligo- 600 sent to an electron microscopy facility at the University carrageenans. The downside of this procedure, however, of the Philippines at Los Banos, Laguna, Philippines is that acid hydrolysis produces degradation products for photomicrography. Colonial characteristics were with considerably varied molecular weights. The second observed by spot inoculating the isolate on MBC plates. method, on the other hand, utilizes enzymes that catalyze To determine the biochemical properties, oxidative the hydrolysis of carrageenan (e.g. ĸ-carrageenase) or fermentative (OF) behavior was determined via a into its oligosaccharide components. Since enzymes modified OF medium for marine bacteria (Lemos et have specific activities, this approach is more likely to al. 1985); substrate utilization was determined using produce carrageenan-derivatives with uniform molecular BIOLOG GN2 (Biolog, Inc.). The morphology and weights which can be more advantageous since the physiology of isolate ALAB-001 were compared with observed physiological activities of oligo-carrageenans two related strains Microbulbifer (Gonzalez et al. 1997) are associated with their molecular weights. and Marinimicrobium (Lim et al. 2006), both belonging Although carrageenases are also known to be produced to Alteromonadales (Proteobacteria). ALAB-001 was also 46 Philippine Journal of Science Tayco et al.: Characterization of a κ-Carrageenase-producing Vol. 142 No. 1, June 2013 Marine Bacterium, Isolate ALAB-001 compared with several carrageenase-producing bacteria, (ZDM) (Barbeyron et al. 2000), used for carrageenase in particular Zobellia galactanovorans (Barbeyron et al. production from Zobellia galactanovorans; and, Sarwar 2001) and isolates described by Sarwar et al. (1983). These salts medium (SSM) (Sarwar et al. 1985), used for carrageenase-producing bacteria are members of the two carrageenase production from Cytophaga sp. In addition, distantly-related taxa, Alteromonadales (Proteobacteria) we also used an in-house formulated culture medium and Flavobacteriaceae (Bacteroidetes). referred to as YETS, composed of equal concentrations of yeast extract and tryptone (5 g/L each) in seawater Genomic DNA (gDNA) was extracted using the supplemented with 1.5 % carrageenan. MBC was used QIamp DNA mini kit (Qiagen). The 16S rRNA gene as a basal salt medium