characterization of a κ-carrageenase-producing marine...

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Philippine Journal of Science142 (1): 45-54, June 2013ISSN 0031 - 7683Date Received: ?? Feb 20??

Key Words: κ-carrageenan, κ-carrageenase, carrageenan-degrading bacteria, Kappaphycus alvarezii

*Corresponding author: [email protected]

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-Inn ovation, 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).

Characterization of a κ-Carrageenase-producing Marine Bacterium, Isolate ALAB-001

INTRODUCTIONκ-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 (Lemos et al. 1985).

Only a handful of published reports describe the isolation of κ-carrageenase-producing marine bacteria and demonstrate their ability to produce carrageenase in culture. Bacterial species reported in the scientific literature include Pseudomonas carrageenovora (Weigl and Yaphe 1966), Cytophaga strain 1k-C783 (Sarwar

Tayco et al.: Characterization of a κ-Carrageenase-producing Marine Bacterium, Isolate ALAB-001

Philippine Journal of ScienceVol. 142 No. 1, June 2013

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et al. 1983), Alteromonas fortis (Potin et al. 1995), Pseudoalteromonas carrageenovora (Gauthier et al. 1995), Vibrio sp. CA-1004 (Araki et al. 1999), ‘Cytophaga drobachiensis’ / Zobellia galactanivorans (Barbeyron et al. 1998, Barbeyron et al. 2001), Pseudoalteromonas-like bacterium (Zhou et al. 2008), Pseudoalteromonas porphyrae (Liu et al. 2010), and Pseudoaltermonas tetraodonis (Kobayashi et al. 2012). These studies suggest that the enzyme is synthesized by marine bacteria that belong to at least two distantly related lineages, Proteobacteria and Bacteroidetes, although most of the isolates described in the reports belong to the former group.

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 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: one in κ-carrageenan, two in ι-carrageenan and three in λ-carrageenan. Although carrageenan is principally used in the industry as gelling, emulsifying, stabilizing and texturing agents, studies have revealed other potential applications, particularly, in health and biomedicine. For example, oligosaccharides derived from κ-carrageenan (using carrageenases) have been shown to exhibit anti-tumor activity, particularly, those with a molecular weight of 1726 Da (Mou et al. 2003). Although the mechanism of anti-tumor activity is still unclear, the researchers concluded that oligo-carrageenan could be a potent anti-tumor substance. Similar studies (Caceres et al. 2000; Yuan and Song 2005) also found significant anti-tumor activity of certain fractions of carrageenans and oligo-carrageenans. These studies indicate that carrageenan-derivatives, i.e., oligo-carrageenans obtained via degradation of carrageenan, possess significant potential for biomedical and physiological applications.

Carrageenan-derivatives can be obtained using two different methods. The first method employs acid hydrolysis of carrageenan in order to generate oligo-carrageenans. The downside of this procedure, however, is that acid hydrolysis produces degradation products with considerably varied molecular weights. The second method, on the other hand, utilizes enzymes that catalyze the hydrolysis of carrageenan (e.g. ĸ-carrageenase) into its oligosaccharide components. Since enzymes have specific activities, this approach is more likely to produce carrageenan-derivatives with uniform molecular weights which can be more advantageous since the observed physiological activities of oligo-carrageenans are associated with their molecular weights.

Although carrageenases are also known to be produced

by marine invertebrates feeding on carrageenophytes, using them as sources of the enzyme carrageenase will necessitate the establishment of a hatchery and culture facility as well as the development of a laborious process of crude extraction from visceral organs. Conversely, bacteria, as sources of carrageenase, are very easy to handle in the laboratory, do not require large storage space, and since the enzyme is secreted in the medium, harvesting of the enzyme with relatively fewer contaminants is easy. Hence, as far as laboratory enzyme production is concerned, bacteria are still preferred sources of κ-carrageenase over marine invertebrates. In this paper, we report the characterization of ALAB -001, a κ-carrageenase-producing bacterial isolate.

MATERIALS AND METHODS

Bacterial isolateA carrageenan-degrading marine bacterial strain, designated as ALAB-001, was previously isolated via standard microbiological strategy. Diseased thallus fragments of the red seaweed Kappaphycus alvarezii (collected from a seaweed farm in Calatagan, Batangas, Philippines) were swabbed onto solid κ-carrageenan-(1.5%)-sterile seawater media and the plates were then screened for bacterial colonies that manifest plate depression-forming activity. Pure cultures of the depression-forming bacterial isolates were obtained by repeated streaking and picking on plates and then maintained by regular spot inoculation on carrageenan-solidifed medium prepared using marine broth (Pronadisa) with 1.5% κ-carrageenan (MBC) (Shemberg Corporation, Philippines).

Phenotypic, Biochemical, and Phylogenetic Characterization To determine the cellular morphology of the isolate, light microscopy of Gram-stained specimens was carried out and a sample of the bacterium (1 mL, OD600 = 0.1) was sent to an electron microscopy facility at the University of the Philippines at Los Banos, Laguna, Philippines for photomicrography. Colonial characteristics were observed by spot inoculating the isolate on MBC plates. To determine the biochemical properties, oxidative or fermentative (OF) behavior was determined via a modified OF medium for marine bacteria (Lemos et al. 1985); substrate utilization was determined using BIOLOG GN2 (Biolog, Inc.). The morphology and physiology of isolate ALAB-001 were compared with two related strains Microbulbifer (Gonzalez et al. 1997) and Marinimicrobium (Lim et al. 2006), both belonging to Alteromonadales (Proteobacteria). ALAB-001 was also



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compared with several carrageenase-producing bacteria, in particular Zobellia galactanovorans (Barbeyron et al. 2001) and isolates described by Sarwar et al. (1983). These carrageenase-producing bacteria are members of the two distantly-related taxa, Alteromonadales (Proteobacteria) and Flavobacteriaceae (Bacteroidetes).

Genomic DNA (gDNA) was extracted using the QIamp DNA mini kit (Qiagen). The 16S rRNA gene was amplified using the universal eubacterial primer pair 63F (5’-CTGAACGTACACAATCCGGAC-3’) and 1387R (5’-CGGAACATGTGTGGCGGG-3’) (Marchesi et al. 1998) and internal primers 905RK.rc (5'- TRAAACTCA AAKGAATTG AC-3') and 400+R (5'- TGCTGCCTCCCGTAGGAG TCT-3'). The polymerase chain reaction (PCR) amplification of the marker made use of the following profile: denaturation at 94°C for 3 minutes; 30 amplification cycles of 30 seconds at 94°C, 30 seconds at 60°C, and 45 seconds at 72°C; and a final extension for 4 minutes at 72°C. Samples of the PCR amplicons were sent to 1st Base (Singapore) for sequencing. The sequences (raw reads) obtained were assembled to a final single sequence using the software DNA Baser (Heracle Software, Germany). Using the final sequence as the query sequence, similar 16S rRNA gene sequences in Genbank were searched and downloaded using the NCBI online tool Basic Local Alignment and Search Tool (BLAST; http://blast.ncbi.nlm.nih.gov/Blast.cgi). Alignment of the sequences was carried out using ClustalW (as a component of the software MEGA 4.0, Kumar et al. 2004). A phylogenetic tree was inferred from the aligned sequences using the maximum likelihood method as implemented in the software PhyML (Guindon and Gascuel 2003), which was run with the following parameters: Tree Topology Search: Subtree Pruning and Regrafting (SPR); Starting Tree: generated using BioNJ; Model of Nucleotide Substitution: General-Time Reversible (GTR); Parametric Bootstrap Analysis; other parameters were set to be optimized by the program.

Growth of ALAB-001 and Production of κ-carrageenase in Batch CulturesOptimum cultivation conditions were determined by testing the effects of several culture parameters. To determine the most suitable medium for growth and κ-carrageenase production, cell density and κ-carrageenase activity of the isolate were observed as the isolate was grown in five different culture media. Three of these were as previously reported in the literature: Knutsen’s medium (KNS) (Knutsen 1991), used for carrageenase production from Pseudoalteromonas carrageenovora; ZD medium

(ZDM) (Barbeyron et al. 2000), used for carrageenase production from Zobellia galactanovorans; and, Sarwar salts medium (SSM) (Sarwar et al. 1985), used for carrageenase production from Cytophaga sp. In addition, we also used an in-house formulated culture medium referred to as YETS, composed of equal concentrations of yeast extract and tryptone (5 g/L each) in seawater supplemented with 1.5 % carrageenan. MBC was used as a basal salt medium (control).

Cell growth was measured as total protein using the bicinchoninic acid total protein assay (Smith et al. 1985). To determine optimum physical parameters for culture, growth of the isolate was observed at 25°C and 30°C with NaCl concentrations ranging from 1 to 10% using marine broth (Pronadisa) as a base medium. Growth and enzyme activity were measured at 24-hour intervals during a two-week period to determine the duration of culture for carrageenase production.

Enzyme Purification and CharacterizationTo prepare the carrageenase from the culture medium for downstream laboratory application, concentration and purification of the enzyme were carried out via ultrafiltration. The supernatant was harvested by separating suspended bacterial cells from the culture medium through centrifugation at 4,750 rpm for 60 minutes at 4°C. Ultrafiltration via tangential flow filtration was performed using a 10 kDa Pellicon device (Millipore) with the following flow rates: 32 hp in and 30 hp out. κ-Carrageenase activity of the concentrated enzyme against various substrates was quantified after concentration. A simple carbohydrate digestion assay was performed using several modified DNS enzyme activity assays using the polysaccharides agar, alginate, cellulose, ι-carrageenan, λ-carrageenan, κ-carrageenan, and starch as substrates.

κ-Carrageenase activity was measured using the 3, 5-dinitrosalicylic acid (DNS) enzyme activity assay or the reducing sugar assay. The procedure was as follows: 500 μL of standard κ-carrageenan solution (3 g/L κ-carrageenan in 0.10 M NaCl and 5 mM NaHCO3) was digested using 50 μL of the harvested enzyme solution for 15 minutes, then 500 μL of DNS was added and the resulting mixture was heated at 95 to 100°C for 5 minutes. Absorbance of the mixture was read at 540 nm. The mass of reducing sugar liberated from the digestion was calculated from the absorbance using a standard curve which was prepared using standard solutions of D-galactose. Enzyme activity was calculated using the formula:

enzyme activity ﴾ UmL ﴿

mass of D galactose liberated (ug) total assay volume (μL) dilution factorvolume of enzyme (μL) lenght of digestion time (min)



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In this particular formula, 1 unit of enzyme is equivalent to the protein that produces 1 µg of D-galactose per minute under the described assay conditions (Knutsen 1991).

RESULTS AND DISCUSSION

Growth Requirements, Enzymatic Activity, and κ-carrageenase Production The strain ALAB-001 exhibited the ability to form depressions or pits on κ-carrageenan-solidified growth medium and was thus isolated and purified. When grown at various temperatures, the isolate exhibited optimal growth at 25°C, although it also survived at temperatures 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

activity against a variety of polysaccharides, it is primarily a κ-carrageenan degrader. Figure 2 shows how growth of 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 activity (~ 45 U/mL) was observed when the isolate was grown in YETS. However, enzyme activity relative to total protein concentration is higher in the isolate cultured using KNS and SSM media, though the values obtained from the said set-ups were substantially lower than those obtained from YETS. MBC and ZDM appeared to have inhibitory effects on enzyme activity as depicted by the low enzyme activity values observed despite having high total protein concentrations.

Figure 1. Activities of ALAB-001 supernatant against various polysaccharides: ι-carrageenan, agar (Ag), alginate (Alg), cellulose (Cl), λ-carrageenan, and κ-carrageenan with starch as positive control. The spike in κ-carrageenan activity shows that the enzyme produced by ALAB-001 is primarily κ-carrageenase.

Figure 2. Enzyme activity and growth (measured using total protein concentration) of ALAB-001 in various bacterial media supplemented with 0.20% κ-carrageenan: Knutsen’s medium (KNM), Sarwar salts medium (SSM), commercially available marine broth (Pronadisa) (MBC), ZD medium (ZDM), yeast extract, and tryptone in seawater (YETS). Highest growth and enzyme activity was observed when ALAB-001 is grown in YETS.

The data showed that growth on YETS was significantly higher than growth on the other media, suggesting that the YETS medium is superior compared to the medium described in the literature (based on cell growth and enzyme activity measurements). Though the enzyme activity relative to total protein concentration is higher in KNS and SSM, these media were not able to adequately support the growth of ALAB-001; thus, the overall total protein concentration and enzyme activity is still much lower in these set-ups than in YETS. Moreover, YETS is a richer source of nutrients compared to the other media, as it contains organic compounds (yeast extract and tryptone) whereas the latter media are mostly composed of inorganic salts. Enzyme activity and cell counts observed over a period of 14 days reached their maximum after 8 days in culture (Figure 3).

Substrate

iota

-car

rage

ena

Aga

r

Alg

inat

e

Cel

lulo

se

lam

bda-

Car

rage

enan

kapp

a-C

arra

geen

an

Star

ch

Enzy

me

Act

ivity

(U/m

L)

0

50

100

150

200



49

Genotypic Characteristics and Phylogeny of ALAB-001PCR amplification of a 16S rRNA gene region from ALAB-001 yielded a 1,288 bp nucleotide sequence, which was deposited at GenBank under the accession number HQ318776. Phylogenetic analysis using the aforementioned gene sequence, together with similar sequences downloaded from Genbank nucleotide database as well as sequences of representative carrageenase- and agarase-producing strains (also obtained from the same database, Table 1), yielded a phylogenetic tree shown in Figure 4; Prochlorococcus was used as the outgroup.

Three major clades can be recognized in the tree, namely: Marinimicrobium, Microbulbifer, and Pseudomonas.

ALAB-001 was positioned outside of these major clusters and grouped with two agarolytic strains, bacterium strain QM42 and a strain identified as Microbulbifer sp. MY05. This group is related to another clade of strains identified as species of Pseudomonas and Marinimicrobium. ALAB-001 appears to be the first member of this group reported as (at least, primarily) a κ-carrageenase producer. It must be noted that the taxonomic identification of a number of strains (identified as Pseudomonas or Microbulbifer strains) in these two groups seems dubious since many Pseudomonas, Marinimicrobium, and Microbulbifer strains form separate well-supported clades). In addition, characterization of these strains has not yet been reported in the scientific literature; hence, their taxonomic status cannot be verified.

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.

Accession Number Description Max Score

Total Score

Query Coverage E-value Max

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%



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The finding of κ-carrageenase activity in ALAB-001 supports the notion that the capability to degrade red algal cell wall polysaccharides (agar and carrageenan, in particular) is likely common in Alteromonadaceae. However, so far only a handful of strains were reported to be carrageenase-producing; these strains thus appear scattered on the tree (Fig. 4). Whether the presence of a carrageenase-producer in a clade indicates that closely related strains also possess carrageenan-degradative capabilities remains to be seen. The observation that ALAB-001 has low agarase activity notwithstanding the fact that its two closest

relatives on the tree (QM42 and MY05) are agar-degraders suggests that carrageenase and/or agarase activity may not necessarily be correlated with clade membership.

Phenotypic and Biochemical Characteristics of ALAB-001In culture, colonies appeared as colorless and transparent, punctiform or pin-point at the early stages of incubation. As the colonies grow, they gradually become irregularly shaped with undulate or lobate edge. The colonies further

Figure 4. Cladogram b ased 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.



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

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



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progressed to form a deep and wide depression around 8 to 10 mm in diameter after 3 to 4 days of incubation to about 12 to 15 mm in diameter after 7 days of incubation. In about 30 days, the colonies appeared opaque, possibly due to mixing with the liquefied κ-carrageenan in the medium (Figure 6A and 6B). ALAB-001 has an observably slower growth rate and cultures appear to be viable up to 30 days.

Oxidative-fermentative (OF) test revealed that ALAB-001 was capable of oxidative acid production from glucose. It tested positive for catalase, oxidase, β-galactosidase and hydrolysis of common sugars such as glucose and arabinose. Furthermore, isolate ALAB-001 was also capable of utilizing a number of substrates including dextrin, glycogen, Tween 40, Tween 80, and D-galactose.

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 obtained using light microscopy.

Morphologically, ALAB-001 is similar to Microbulbifer, Marinimicrobium and all carrageenase-producing strains in having the characteristic rod shape, and absence of

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.

Trait

Isolate

ALAB-001Marinimicrobium

agarilyticum(Lim et al. 2006)

Microbulbifer hydrolyticus

(Michel et al. 1999)

Carrageenase-producers(Barbeyron et al. 2001,

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

flagella (Table 2). However, ALAB-001 differs from both Microbulbifer and Marinimicrobium in other characteristics, particularly catalase and nitrate reductase activities, as the type strains of both Microbulbifer and Marinimicrobium do not exhibit these activities. The latter observation suggests that despite close phylogenetic affinity of ALAB-001 to the two genera as revealed by 16S rDNA sequence and phenotypic/biochemical characteristics, the ALAB-001 isolate may significantly differ from Microbulbifer and Marinimicrobium strains in certain aspects of their metabolism. However, the observations noted above indicate that ALAB-001 is a member of a group that includes Microbulbifer, Marinimicrobium and Pseudomonas, and that the ability to degrade seaweed polysaccharides may be a common characteristic of members of the group.

ACKNOWLEDGEMENTSThis study was supported by research grants to AOL from the Philippine Department of Science and Technology (DOST) through the Philippine Council for Aquatic and Marine Research and Development (PCAMRD). We are



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particularly grateful to Dr. Estrella Alabastro, the former DOST Secretary, for the long-term support she had given to this research. The ALAB-series of carrageenase- and agarase-producing marine bacteria (such as the ALAB-001 isolate) isolated in our laboratory are so named in recognition of her support.

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characterization of a κ-carrageenase-producing marine...

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