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 (control). was amplified using the universal eubacterial primer pair 63F (5’-CTGAACGTACACAATCCGGAC-3’) Cell growth was measured as total protein using the and 1387R (5’-CGGAACATGTGTGGCGGG-3’) bicinchoninic acid total protein assay (Smith et al. 1985). (Marchesi et al. 1998) and internal primers 905RK.rc To determine optimum physical parameters for culture, (5'- TRAAACTCA AAKGAATTG AC-3') and 400+R (5'- growth of the isolate was observed at 25°C and 30°C TGCTGCCTCCCGTAGGAG TCT-3'). The polymerase with NaCl concentrations ranging from 1 to 10% using chain reaction (PCR) amplification of the marker made marine broth (Pronadisa) as a base medium. Growth and use of the following profile: denaturation at 94°C for enzyme activity were measured at 24-hour intervals during 3 minutes; 30 amplification cycles of 30 seconds at a two-week period to determine the duration of culture 94°C, 30 seconds at 60°C, and 45 seconds at 72°C; and for carrageenase production. a final extension for 4 minutes at 72°C. Samples of the PCR amplicons were sent to 1st Base (Singapore) for Enzyme Purification and Characterization sequencing. The sequences (raw reads) obtained were To prepare the carrageenase from the culture medium assembled to a final single sequence using the software for downstream laboratory application, concentration DNA Baser (Heracle Software, Germany). Using the final and purification of the enzyme were carried out via sequence as the query sequence, similar 16S rRNA gene ultrafiltration. The supernatant was harvested by sequences in Genbank were searched and downloaded separating suspended bacterial cells from the culture using the NCBI online tool Basic Local Alignment and medium through centrifugation at 4,750 rpm for 60 Search Tool (BLAST; http://blast.ncbi.nlm.nih.gov/Blast. minutes at 4°C. Ultrafiltration via tangential flow cgi). Alignment of the sequences was carried out using filtration was performed using a 10 kDa Pellicon device ClustalW (as a component of the software MEGA 4.0, (Millipore) with the following flow rates: 32 hp in and Kumar et al. 2004). A phylogenetic tree was inferred from 30 hp out. κ-Carrageenase activity of the concentrated the aligned sequences using the maximum likelihood enzyme against various substrates was quantified after method as implemented in the software PhyML (Guindon concentration. A simple carbohydrate digestion assay was and Gascuel 2003), which was run with the following performed using several modified DNS enzyme activity parameters: Tree Topology Search: Subtree Pruning assays using the polysaccharides agar, alginate, cellulose, and Regrafting (SPR); Starting Tree: generated using ι-carrageenan, λ-carrageenan, κ-carrageenan, and starch BioNJ; Model of Nucleotide Substitution: General-Time as substrates. Reversible (GTR); Parametric Bootstrap Analysis; other parameters were set to be optimized by the program. κ-Carrageenase activity was measured using the 3, 5-dinitrosalicylic acid (DNS) enzyme activity assay or the Growth of ALAB-001 and Production of reducing sugar assay. The procedure was as follows: 500 κ-carrageenase in Batch Cultures μL of standard κ-carrageenan solution (3 g/L κ-carrageenan Optimum cultivation conditions were determined by in 0.10 M NaCl and 5 mM NaHCO3) was digested using testing the effects of several culture parameters. To 50 μL of the harvested enzyme solution for 15 minutes, determine the most suitable medium for growth and then 500 μL of DNS was added and the resulting mixture κ-carrageenase production, cell density and κ-carrageenase was heated at 95 to 100°C for 5 minutes. Absorbance of activity of the isolate were observed as the isolate was the mixture was read at 540 nm. The mass of reducing grown in five different culture media. Three of these were sugar liberated from the digestion was calculated from the as previously reported in the literature: Knutsen’s medium absorbance using a standard curve which was prepared (KNS) (Knutsen 1991), used for carrageenase production using standard solutions of D-galactose. Enzyme activity from Pseudoalteromonas carrageenovora; ZD medium was calculated using the formula:

mass of D  galactose liberated (ug)  total assay volume (μL)  dilution factor enzyme activity ﴾ U ﴿  mL volume of enzyme (μL)  lenght of digestion time (min)

47 Philippine Journal of Science Tayco et al.: Characterization of a κ-Carrageenase-producing Vol. 142 No. 1, June 2013 Marine Bacterium, Isolate ALAB-001

In this particular formula, 1 unit of enzyme is equivalent to activity against a variety of polysaccharides, it is primarily the protein that produces 1 µg of D-galactose per minute a κ-carrageenan degrader. Figure 2 shows how growth of under the described assay conditions (Knutsen 1991). the isolate (expressed in total protein concentration) and κ-carrageenase activity varied with the kind of media used in culturing the isolate. Highest cell growth based on total protein concentration (~ 3000 μg/mL) and enzyme RESULTS AND DISCUSSION activity (~ 45 U/mL) was observed when the isolate was grown in YETS. However, enzyme activity relative to Growth Requirements, Enzymatic Activity, and total protein concentration is higher in the isolate cultured κ-carrageenase Production using KNS and SSM media, though the values obtained The strain ALAB-001 exhibited the ability to form from the said set-ups were substantially lower than those depressions or pits on κ-carrageenan-solidified growth obtained from YETS. MBC and ZDM appeared to have medium and was thus isolated and purified. When grown inhibitory effects on enzyme activity as depicted by the at various temperatures, the isolate exhibited optimal low enzyme activity values observed despite having high growth at 25°C, although it also survived at temperatures total protein concentrations. up to 30°C. It was observed to grow in seawater-based growth media or in media supplemented with 1% to 10% NaCl showing its halophilic nature. Activities of the concentrated enzyme from the isolate grown on YETS using various substrates (seaweed- and non-seaweed-derived polysaccharides) are shown in Figure 1. The enzyme showed highest activity (175 U/ mL) against κ-carrageenan but the isolate was also able to hydrolyze starch (as control carbon source) at similar rates (180 U/mL). It was able to hydrolyze ι- and λ-carrageenan, agar, alginate and cellulose at relatively lower rates (25 to 50 U/mL). These data suggest that although the isolate has Figure 2. Enzyme activity and growth (measured using total protein concentration) of ALAB-001 in 200 various bacterial media supplemented with 0.20% κ-carrageenan: Knutsen’s medium (KNM), Sarwar salts medium (SSM), commercially available marine 150 broth (Pronadisa) (MBC), ZD medium (ZDM), yeast extract, and tryptone in seawater (YETS). Highest growth and enzyme activity was observed when 100 ALAB-001 is grown in YETS.

50 The data showed that growth on YETS was significantly

Enzyme Activity (U/mL) higher than growth on the other media, suggesting that 0 the YETS medium is superior compared to the medium described in the literature (based on cell growth and Agar

Starch enzyme activity measurements). Though the enzyme Alginate

Cellulose activity relative to total protein concentration is higher in KNS and SSM, these media were not able to adequately

iota-carrageena support the growth of ALAB-001; thus, the overall total protein concentration and enzyme activity is still much kappa-Carrageenan lambda-Carrageenan lower in these set-ups than in YETS. Moreover, YETS is a Substrate richer source of nutrients compared to the other media, as Figure 1. Activities of ALAB-001 supernatant against various it contains organic compounds (yeast extract and tryptone) polysaccharides: ι-carrageenan, agar (Ag), alginate (Alg), whereas the latter media are mostly composed of inorganic cellulose (Cl), λ-carrageenan, and κ-carrageenan with salts. Enzyme activity and cell counts observed over a starch as positive control. The spike in κ-carrageenan period of 14 days reached their maximum after 8 days in activity shows that the enzyme produced by ALAB-001 culture (Figure 3). is primarily κ-carrageenase.

48 Philippine Journal of Science Tayco et al.: Characterization of a κ-Carrageenase-producing Vol. 142 No. 1, June 2013 Marine Bacterium, Isolate ALAB-001

Figure 3. Growth of ALAB-001 in YETS and changes in enzyme activity during culture. Maximum growth of ALAB-001 was observed after 8 days in culture. On the other hand, maximum enzyme activity was also observed after 8 days.

Table 1. Closest BLAST hits of ALAB-001 based on 16S rRNA gene sequence. Hits show three (3) genera of marine bacteria including a Microbulbifer sp., several Marinimicrobium sp. and a number of Pseudomonas sp. Closest hit (Microbulbifer sp. MY05) showed 99% sequence identity. Max Total Query Max Accession Number Description E-value Score Score Coverage Identity AY862188.1 Microbulbifer sp. MY05 2442 2442 100% 0.0 99% DQ822530.1 Bacterium QM42 2425 2425 99% 0.0 99% GQ872424.1 Marinimicrobium sp. M5c 2204 2204 100% 0.0 96% AB052968.1 Pseudomonas sp. PE1 2132 2132 100% 0.0 95% AB052965.1 Pseudomonas sp. ND137 2102 2102 100% 0.0 94% AB052969.1 Pseudomonas sp. PE2 2093 2093 100% 0.0 94% GU291859.1 Marinimicrobium sp. HMD3005 2036 2036 99% 0.0 94% AY839869.2 Marinimicrobium koreense strain M9 2030 2030 100% 0.0 94% GQ920839.1 Marinimicrobium sp. SX15 2021 2021 100% 0.0 93%

Genotypic Characteristics and Phylogeny of ALAB-001 ALAB-001 was positioned outside of these major clusters PCR amplification of a 16S rRNA gene region from and grouped with two agarolytic strains, bacterium strain ALAB-001 yielded a 1,288 bp nucleotide sequence, QM42 and a strain identified as Microbulbifer sp. MY05. which was deposited at GenBank under the accession This group is related to another clade of strains identified as number HQ318776. Phylogenetic analysis using the species of Pseudomonas and Marinimicrobium. ALAB-001 aforementioned gene sequence, together with similar appears to be the first member of this group reported as (at sequences downloaded from Genbank nucleotide database least, primarily) a κ-carrageenase producer. It must be noted as well as sequences of representative carrageenase- and that the taxonomic identification of a number of strains agarase-producing strains (also obtained from the same (identified as Pseudomonas or Microbulbifer strains) in database, Table 1), yielded a phylogenetic tree shown these two groups seems dubious since many Pseudomonas, in Figure 4; Prochlorococcus was used as the outgroup. Marinimicrobium, and Microbulbifer strains form separate well-supported clades). In addition, characterization of Three major clades can be recognized in the tree, namely: these strains has not yet been reported in the scientific Marinimicrobium, Microbulbifer, and Pseudomonas. literature; hence, their taxonomic status cannot be verified.

49 Philippine Journal of Science Tayco et al.: Characterization of a κ-Carrageenase-producing Vol. 142 No. 1, June 2013 Marine Bacterium, Isolate ALAB-001

Figure 4. Cladogram based­ on 16S rRNA gene sequences inferred using the maximum likelihood method showing ALAB-001 to be clustered with a relatively uncharacterized group of agarolytic bacteria including an unidentified isolate, Bacteria QM42, and Microbulbifer sp. MY05. Prochlorococcus was used as the outgroup. Sequence, 1288 bp in length, is submitted to GenBank with accession number HQ318776.

The finding of κ-carrageenase activity in ALAB-001 relatives on the tree (QM42 and MY05) are agar-degraders supports the notion that the capability to degrade red suggests that carrageenase and/or agarase activity may not algal cell wall polysaccharides (agar and carrageenan, necessarily be correlated with clade membership. in particular) is likely common in Alteromonadaceae. However, so far only a handful of strains were reported to be Phenotypic and Biochemical Characteristics of carrageenase-producing; these strains thus appear scattered ALAB-001 on the tree (Fig. 4). Whether the presence of a carrageenase- In culture, colonies appeared as colorless and transparent, producer in a clade indicates that closely related strains punctiform or pin-point at the early stages of incubation. also possess carrageenan-degradative capabilities remains As the colonies grow, they gradually become irregularly to be seen. The observation that ALAB-001 has low shaped with undulate or lobate edge. The colonies further agarase activity notwithstanding the fact that its two closest

50 Philippine Journal of Science Tayco et al.: Characterization of a κ-Carrageenase-producing Vol. 142 No. 1, June 2013 Marine Bacterium, Isolate ALAB-001

Figure 5. Electromicrograph of ALAB-001 showing its characteristic rod-shaped cell that seems slightly elongated.

Figure 6. Morphological characteristics of ALAB-001 colonies on κ-carrageenan-solidifed marine broth plates after 7 (a) and 30 (b) days of incubation.

51 Philippine Journal of Science Tayco et al.: Characterization of a κ-Carrageenase-producing Vol. 142 No. 1, June 2013 Marine Bacterium, Isolate ALAB-001 progressed to form a deep and wide depression around 8 to flagella (Table 2). However, ALAB-001 differs from both 10 mm in diameter after 3 to 4 days of incubation to about Microbulbifer and Marinimicrobium in other characteristics, 12 to 15 mm in diameter after 7 days of incubation. In particularly catalase and nitrate reductase activities, as the about 30 days, the colonies appeared opaque, possibly due type strains of both Microbulbifer and Marinimicrobium to mixing with the liquefied κ-carrageenan in the medium do not exhibit these activities. The latter observation (Figure 6A and 6B). ALAB-001 has an observably slower suggests that despite close phylogenetic affinity of ALAB- growth rate and cultures appear to be viable up to 30 days. 001 to the two genera as revealed by 16S rDNA sequence and phenotypic/biochemical characteristics, the ALAB- Oxidative-fermentative (OF) test revealed that ALAB-001 001 isolate may significantly differ from Microbulbifer was capable of oxidative acid production from glucose. and Marinimicrobium strains in certain aspects of their It tested positive for catalase, oxidase, β-galactosidase metabolism. However, the observations noted above indicate and hydrolysis of common sugars such as glucose and that ALAB-001 is a member of a group that includes arabinose. Furthermore, isolate ALAB-001 was also Microbulbifer, Marinimicrobium and Pseudomonas, and capable of utilizing a number of substrates including that the ability to degrade seaweed polysaccharides may be dextrin, glycogen, Tween 40, Tween 80, and D-galactose. a common characteristic of members of the group. Figure 5 shows the image of ALAB-001 cells taken using a scanning electron microscope. The isolate is rod- shaped, approximately 3 μm long, and without flagellum, and these characteristics are consistent with the results ACKNOWLEDGEMENTS obtained using light microscopy. This study was supported by research grants to AOL from Morphologically, ALAB-001 is similar to Microbulbifer, the Philippine Department of Science and Technology Marinimicrobium and all carrageenase-producing strains (DOST) through the Philippine Council for Aquatic and in having the characteristic rod shape, and absence of Marine Research and Development (PCAMRD). We are

Table 2. Phenotypic and biochemical characteristics of ALAB-001. Phenotypic and biochemical characteristics of ALAB- 001 were compared with Marinimicrobium agarilyticum and Microbulbifer hydrolyticus. Upon comparison, ALAB-001 proved to be different from the two type strains in several respects including morphology, extracellular structures and physiology. Isolate

Trait Marinimicrobium Microbulbifer Carrageenase-producers ALAB-001 agarilyticum hydrolyticus (Barbeyron et al. 2001, (Lim et al. 2006) (Michel et al. 1999) Sarwar et al. 1983) Phenotypic: Cellular Morphology Rod Short Rod Rod Long Rod Flagella None Single None None Biochemical: Catalase + - - + Oxidase + + + + Nitrate Reduction + - - + Hydrolysis of: Agar + + - + Gelatin + - + + Tween 80 - + + ND Aesculin + + + ND Acid production from: Glucose + + - ND Fructose - + ND ND Lactose + + ND ND Mannitol - - ND ND Mannose + + ND ND Sucrose + + - ND 52 Philippine Journal of Science Tayco et al.: Characterization of a κ-Carrageenase-producing Vol. 142 No. 1, June 2013 Marine Bacterium, Isolate ALAB-001 particularly grateful to Dr. Estrella Alabastro, the former GUIBET M, COLIN S, GENICOT S, KLOAREG B, DOST Secretary, for the long-term support she had given MICHEL G, HELBERT W. 2007. Degradation of to this research. 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