Cloning, Expression, and Characterization of a Glycoside Hydrolase Family 118 Β-Agarase from Agarivorans Sp. JA-1

J. Microbiol. Biotechnol. (2012), 22(12), 1692–1697 http://dx.doi.org/10.4014/jmb.1209.09033 First published online October 10, 2012 pISSN 1017-7825 eISSN 1738-8872

Cloning, Expression, and Characterization of a Glycoside Hydrolase Family 118 β-Agarase from Agarivorans sp. JA-1

Lee, Dong-Geun, Myong Je Jeon, and Sang-Hyeon Lee*

Major in Bioscience and Biotechnology, Graduate School, Silla University, Busan 617-736, Korea Received: September 11, 2012 / Revised: September 19, 2012 / Accepted: September 20, 2012

We report a glycoside hydrolase (GH)-118 β-agarase from agarase is also an excellent tool for preparing red algae a strain of Agarivorans, in which we previously reported protoplasts [2], recovery of DNA and cells from agarose recombinant expression and characterization of the GH- gels [26], production of bioenergy substrates [14], and 50 β-agarase. The GH comprised an open reading frame production of galactose and anhydro-galactose [27]. Agar of 1,437 base pairs, which encoded a protein of 52,580 can be hydrolyzed by acids, α-agarase, or β-agarase; however, daltons consisting of 478 amino acid residues. Assessment only β-agarase generates functional neoagarooligosaccharides of the entire sequence showed that the enzyme had 97% [28]. Hence, β-agarase-producing microorganisms have nucleotide and 99% amino acid sequence similarities to been identified and reported [1, 5, 17, 22]. Mutations those of GH-118 β-agarase from Pseudoalteromonas sp. improve β-agarase characteristics for industrial applications CY24, which belongs to a different order within the same [13, 19]. class. The gene corresponding to a mature protein of 440 β-Agarases are members of the glycosyl hydrolase (GH) amino acids was inserted, recombinantly expressed in families, such as GH-16, -50, -86, and -118 (http:// Escherichia coli, and purified to homogeneity with affinity www.cazy.org). Previously, authors have reported that chromatography. It had maximal activity at 35oC and pH GH-50 β-agarase of Agarivorans sp. JA-1 is 109,450 7.0 and had 208.1 units/mg in the presence of 300 mM daltons (Da) and consists of 995 amino acid residues. The NaCl and 1 mM CaCl2. More than 80% activity was gene corresponding to a mature protein of 976 amino acids maintained after 2 h exposure to 35oC; however, < 40% has been expressed in E. coli and Bacillus subtilis, and activity remained at 45oC. The enzyme hydrolyzed tyrosinase inhibition activity and the whitening effect of its agarose to yield neoagarooctaose as the main product. neoagarooligosaccharide products have been evaluated, This enzyme could be useful for industrial production of without cellular toxicity [17, 18]. functional neoagarooligosaccharides. Agarivorans is a recently described genus [16], which Kewwords: β-Agarase, Agarivorans, cloning, expression, was named for its agar-degrading ability. It is a Gram- GH-118 negative, strictly aerobic, and agar-hydrolyzing species. This genus is a member of the class Gammaproteobacteria and currently contains only a single type species, Agarivorans albus. Some reports are available on agarases Agar is a building block of the cell walls in some red from Agarivorans strains [8, 11, 17, 21]. Agarolytic algae and is composed of agarose and agaropectin. Agarivorans strains have been isolated from the surface of Neoagarooligosaccharides are generated from agar and seaweeds [6], seawater [17], the gut of turban shells [8], delay starch degradation, reduce the caloric value of food and internal organs of marine creatures such as sea slugs, [9], inhibit bacterial growth [15], produce a whitening effect limpets, and sea squirts [16]. The reported agarases from [17, 18], have high antioxidative properties [29], and are Agarivorans belong to GH-16 [7] and GH-50 [11, 17] and efficient skin moisturizers [22]. Hence, neoagarooligosaccharides have molecular masses >100 kDa. However, no β-agarase have prospective applications in the food, pharmaceutical, has been reported from the GH-86 and GH-118 families of and cosmetic industries based on their physiological and Agarivorans. biological activities. Besides oligosaccharide production, In this study, we describe the cloning, recombinant expression, and characterization of GH-118 β-agarase *Corresponding author Phone: +82-51-999-5624; Fax: +82-51-999-5636; from Agarivorans sp. JA-1, which has been reported to E-mail: [email protected] have a GH-50 β-agarase [17]. 1693 Lee et al.

MATERIALS AND METHODS centrifugation at 5,000 ×g for 5 min after IPTG induction for 3 h and suspended in 30 ml of ice-cold column buffer [20 mM Tris/HCl Bacterial Strains and Culture Conditions (pH 7.4), 0.5 M NaCl, 0.2% Triton X-100, and 2 mM EDTA]. After Agarivorans sp. JA-1 was originally isolated from seawater from the cell disruption by sonication, the sample was centrifuged at 20,000 northeast coast of Cheju Island, Korea [17]. Escherichia coli DH5α ×g for 20 min, and the supernatant was loaded onto a chitin bead (F’ supE44 hsdS20 recA13 ara-14 proA2 lacY1 galK2 rpsL20 xyl-5 column (20 ml set volume) (New England Biolabs) equilibrated mtl-1 leuB6 thi-1) was used as the host for cloning, and Escherichia with column buffer. The column was washed with the same buffer, coli BL21 (DE3) (leuA8 metB5 hsrM1) was used as the host for β- and then equilibrated with a cleavage buffer (column buffer with o agarase AgaJA2 expression. E. coli cells were routinely grown at 30 mM DTT) at 4 C overnight. Proteins were eluted with column 37oC in Luria-Bertani (LB) broth (Difco, Detroit, MI, USA) and buffer to a total volume of 50 ml. The amount of protein was supplemented with ampicillin (100 µg/ml) when required. measured using the BCA Protein Assay Reagent (Pierce Biotechnology, Rockford, IL, USA), utilizing bovine serum albumin as the standard. Molecular Cloning and DNA Sequencing of the β-Agarase Gene The methods used for molecular cloning were based on those of Enzyme Assay Sambrook et al. [23]. Genomic DNA of Agarivorans sp. JA-1 was Agarase activity was determined by enzymatic production of reducing harvested using a Wizard Genomic DNA Purification kit (Promega, sugars from agarose [24]. Recombinant AgaJA2 was incubated in Madison, WI, USA). Plasmid DNA was isolated by the alkaline 50 mM Tris-HCl (Sigma, St. Louis, MO, USA) (pH 7.0) buffer lysis method of Sambrook et al. [23]. The β-agarase agaJA2 gene containing 300 mM NaCl, 1 mM CaCl2, and 0.2% (w/v) molten o of Agarivorans sp. JA-1 was amplified using polymerase chain agarose at 40 C for 30 min. The enzyme reaction was stopped by 2+ reaction (PCR) primers, which were devised based on the β-agarase- adding Cu reagent and used for determining the reducing sugar C gene of Vibrio sp. PO-303 [4], in addition to the genomic DNA content. The mixture was boiled for 10 min and cooled at room of Agarivorans sp. JA-1 as a template using Pyrobest DNA temperature, and arsenomolybdate reagent was added. The amount polymerase (Takara Bio Inc., Otsu, Japan). The forward primer was of reducing sugar liberated was measured using D-galactose as the A_sp-b-agaJA2-F (5'-ATGTTAAAGCGCCACCAAGCTTCAAGG-3'), standard. One unit of enzyme activity was defined as the amount of and the reverse primer was A_sp-b-agaJA2-R (5'-CTATTGGCA protein that produces 1 µmol of reducing sugar per minute under the AGTATAACCTGATACAAC G-3'). The amplified DNA was ligated assay conditions. into the pGEM-T Easy Vector (Promega), resulting in pGEMTe- A_sp-b-agaJA2, and the recombinant plasmid was introduced into Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis E. coli DH5α cells. DNA sequencing was carried out by BioNex (SDS-PAGE) Inc. (Seoul, Korea). The sequence analysis was carried out using the SDS-PAGE was performed by the Laemmli method with an 11% DS_Gene ver. 1.5 program (Accelrys Inc., San Diego, CA, USA). polyacrylamide gel. The enzyme solution was mixed with sample buffer and boiled for 5 min before being placed on the gel. The gels Expression and Purification of Recombinant β-Agarase were stained for protein with GelCode Blue Stain Reagent (Pierce). The mature β-agarase agaJA2 gene was amplified using PCR primers: A_sp-b-agaJA2E-F (5'-CATATGGCTAACTATACGGCC Effects of Temperature and pH on Enzyme Activity AGCAATGCC-3' (incorporated NdeI restriction site is underlined)) The optimal temperature for β-agarase activity was examined in the and A_sp-b-agaJA2E-R (5'-CTCGAGTTGGCAAGTATAACCTGA buffer used under standard assay conditions at various temperatures. TACAAC-3' (inserted XhoI restriction site is underlined)), and The thermostability of AgaJA2 was evaluated by measuring the pGEMTe-A_sp-b-agaJA2 as a template using Pyrobest DNA residual activity of the enzyme after an incubation at different polymerase (Takara Bio). Amplified DNA was ligated into the temperatures for 0.5, 1.0, 1.5, and 2.0 h. The optimal pH of β- pGEM-T Easy Vector, resulting in pGEMTe-A_sp-b-agaJA2E. The agarase was determined in various buffers. The buffers used were recombinant plasmid was introduced into E. coli DH5α cells. The 50 mM sodium acetate buffer, pH 4.0-6.0; 50 mM Tris-HCl, pH integrity of the construct was verified by restriction analysis and 6.0-8.0; and 50 mM TAPS buffer, pH 8.0-10.0. sequencing. pGEMTe-A_sp-b-agaJA2E, carrying the mature β-agarase agaJA2 Chromatographic Analysis of the Agarose Hydrolysis Products gene, was digested with NdeI and XhoI, and a 1.3 kb DNA fragment Hydrolyzed products of agarose by β-agarase were identified using was ligated to corresponding sites of the pTXB1 E. coli expression thin-layer chromatography (TLC). Enzymatic hydrolysis of agarose o vector (New England Bio-labs Inc., Beverly, MA, USA). The (USB Inc., Cleveland, OH, USA) was carried out at 40 C in 50 mM recombinant plasmid was introduced into E. coli DH5α cells, and Tris-HCl (pH 7.0) buffer containing 300 mM NaCl, 1 mM CaCl2, cells harboring the recombinant plasmid were grown overnight, and 0.2% (w/v) agarose. The agarose was molten owing to heating o o collected by centrifugation at 5,000 ×g for 5 min, and subjected to at 95 C and was used as molten substrate at 45 C. The reaction plasmid preparation. The integrity of the recombinant plasmid was mixtures were applied to silica gel 60 TLC plates (Merck, Darmstadt, confirmed by restriction digestion using NdeI and XhoI and Germany), and the plates were developed using a solvent system designated pTXB1-A_sp-b-agaJA2. E. coli BL21(DE3) cells were composed of n-butanol:acetic acid:H2O [2:1:1 (v/v)]. Spots were transformed using the pTXB1-A_sp-b-agaJA2 β-agarase expression visualized by spraying 10% (v/v) H2SO4 followed by heating o plasmid and grown in 1 L of LB broth supplemented with ampicillin (80 C). D-Galactose (Sigma), neoagarotetraose (V-Labs Inc., St. at 37oC for 12 h. IPTG (final concentration, 0.3 mM) was added to Covington, LA, USA), and neoagarohexaose (Sigma) were used as the medium to induce the T7 promoter. The cells were harvested by standards. RECOMBINANT EXPRESSION OF GH-118 β-AGARASE 1694

RESULTS

Cloning of the β-Agarase agaJA2 Gene from Agarivorans sp. JA-1 The novel agarase gene from Agarivorans sp. JA-1 was cloned and sequenced. The gene comprised 1,437 bp with a G + C content of 45.4%. The gene began with ATG and ended with TAC. It encoded a protein of 478 amino acids with a molecular mass of 52,580 Da. The nucleotide sequence was 97% identical to the Vibrio sp. PO-303 β- agarase agaC gene (Accession No. BAF03590) [4] and the β-agarase agaB gene (AAQ56237) of Pseudoalteromonas sp. CY24 [20]. The amino acid sequence of β-agarase AgaJA2 was 99% similar with the β-agarase AgaC (BAF03590) from Vibrio sp. PO-303 [4] and β-agarase AgaB (AAQ56237) from Pseudoalteromonas sp. CY24 [20]. This result Fig. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the novel agarase from Agarivorans sp. JA-1 (SDS-PAGE) of recombinant β-agarase AgaJA2 from E. coli should be classified as part of the GH family 118 [4]. The cells harboring pTXB1-A_sp-b-agaJA2E. putative signal peptide sequence is first 38 amino acids Lane M, size markers; lane C, cell-free extract; lane P, purified enzyme by affinity chromatography. The arrow indicates the position of the recombinant from methionine to alanine. β-agarase AgaJA2. The nucleotide and amino acid sequences reported in this paper have been submitted to the GenBank under the accession number JX840781. retained >80% activity after 2 h exposure at the indicated temperature. However, it maintained <40% activity after o o Expression and Purification of Recombinant β-Agarase 1 h at 45 C and 55 C (Fig. 3). AgaJA2 Production of recombinant β-agarase was examined using TLC Analysis of the Hydrolysis Products E. coli BL21 (DE3) as a host and pTXB1 as the vector. E. The enzyme reaction products were analyzed over time by coli BL21 (DE3) cells harboring pTXB1-A_sp-b-agaJA2 TLC (Fig. 4) and quantified by NIH image software. The produced a high amount of β-agarase (Fig. 1). The enzyme hydrolyzed agarose to generate neoagarooctaose recombinant β-agarase was purified 231-fold after affinity (NA8) within 0.1 h. We assumed it was NA8 from the chromatography, with a specific activity of 208.1 U/mg relative ratio to the front, from Ma et al. [20] and Dong et and a final yield of 50.1% (Table 1). SDS-PAGE of the al. [4], although we did not use NA8 as a standard, because purified enzyme exhibited a single band with an apparent NA8 is not commercially available. The amount of the molecular mass of 48 kDa (Fig. 1). This value agreed with NA8 increased with the passage of time (2-12 h); however, that estimated from the DNA sequence. no neoagarooligosaccharides shorter than NA8 were Effects of Temperature and pH on Enzyme Activity produced. The ratio of the shortest product NA8 was and Stability 26.7% of total products after 24 h incubation. The optimal temperature for β-agarase AgaJA2 activity was 35oC (Fig. 2A). Enzyme activity was >80% at 25oC and 45oC and <10% over 55oC, compared with enzyme DISCUSSION activity (100%) at 35oC. The optimal pH for β-agarase AgaJA2 activity was approximately 7.0 (Fig. 2B). Enzyme We have previously cloned and sequenced the novel GH activity was about 80% at pH 6.0 and <60% over pH 8.0, family 50 β-agarase gene from the marine bacterium compared with enzyme activity (100%) at pH 7. β-Agarase Agarivorans sp. JA-1, which was isolated from the sea AgaJA2 activity was stable at 25oC and 35oC. The enzyme near Cheju Island, Korea [17, 18]. Here, we cloned,

Table 1. Purification of β-agarase AgaJA2 from E. coli cells harboring pTXB1-A_sp-b-agaJA2E. Purification step Total protein (mg/l) Total activity (U) Specific activity(U/mg) Yield (%) Cell-free extract 554.1 498.7 0.9 100 Affinity chromatography 1.2 249.7 208.1 50 1695 Lee et al.

Fig. 3. Effect of temperature on stability of recombinant β- agarase AgaJA2. The enzyme (0.2 unit/ml) was preincubated at the indicated temperatures for 0.5, 1, 1.5, and 2 h in 50 mM Tris-HCl (pH 7.0) buffer. Samples were applied to measure residual activity under the standard conditions of the enzyme assay. The values are percentages of enzyme activity (100%) observed at pH 7.0 without heating.

Fig. 2. Effects of temperature and pH on recombinant β-agarase AgaJA2 activity. (A) Temperature dependence of enzyme activity. Values on the ordinate are shown as percentages of enzyme activity (100%) observed at 35oC (B) pH dependence of enzyme activity. The buffers used were 50 mM sodium acetate buffer (black rectangles, pH 4.0-6.0), 50 mM Tris-HCl buffer (black triangles, pH 6.0-8.0), and 50 mM TAPS buffer (black circles, pH 8.0-10.0). The values on the ordinate are shown as percentages of enzyme activity (100%) observed at pH 7.0. sequenced, recombinantly expressed, and characterized another β-agarase gene from the same Agarivorans sp. JA-1 strain. The recombinant β-agarase was produced using pTXB1 as the vector and E. coli BL21 (DE3) as the host. The enzyme amino acid sequence showed high homology with that of the extracellular β-agarase AgaB precursor Fig. 4. Thin-layer chromatography (TLC) of the agarose hydrolysis products by recombinant β-agarase AgaJA2. (AAQ56237) from Pseudoalteromonas sp. CY24 [20] and o The reactions were carried out at 40 C in 50 mM Tris-HCl (pH 7.0) buffer β-agarase AgaC from Vibrio sp. PO-303 [4]. Hehemann et containing 300 mM NaCl, 1 mM CaCl2, and 1% agarose with 0.34 U/ml al. [10] reported a comparative metagenome analyses of enzyme for the indicated times. The reaction mixtures were developed symbiotic microbes from the human gut and showed that by TLC. S, standards; G, D-galatose; NA4, neoagarotetraose; NA6, Bacteroides plebeius in the Japanese gut had porphyranases neoagarohexaose; NA8, neoagarooctaose. and agarases, whereas B. plebeius in the guts of North American individuals lacked these enzymes. Furthermore, them is 97%. Furthermore, 99% similarities were observed those genes matched those of Zobellia galactanivorans, in our amino acid sequences (data not shown), which strongly and Hehemann et al. [10] mentioned that they originated suggest that these genes were horizontally transferred, as from horizontal gene transfer. All AgaJA2, AgaB the Agarivorans, Pseudoalteromonas, and Vibrio genera (AY293310), and AgaC (AB218419) genes have the same are in different orders but belong to the same subclass of number of nucleotides, and the minimal similarity among Gammaproteobacteria. All matched agarases belonged to RECOMBINANT EXPRESSION OF GH-118 β-AGARASE 1696 family GH-118 at the CAZy site (http://www.cazy.org). β- been used at various pHs [4]. Compared with the enzyme Agarases were sorted into four families of GH-16, -50, - activity (100%) of AgaJA2 at 35oC, about 80% activity 86, and -118 (http://www.cazy.org/) based on amino acid remained at 25oC and 35oC even after a 2 h exposure (Fig. 3). similarity. Until now GH-16 [8] and GH-50 [17, 18] have AgaB of Pseudoalteromonas sp. CY24 [20] retains >90% been reported from Agarivorans. However, no reports are activity at 35oC. available on the GH-118 β-agarase from an Agarivorans The time-course analysis revealed that strain. Thus, AgaJA2 is the first family GH-118 agarase neoagarooligosaccharides longer than NA8 were mainly derived from Agarivorans, and it is reasonable to presume produced at the initial phase (Fig. 4). NA8 or longer that AgaJA2 might possess novel properties compared neoagarooligosaccharides were produced until 24 h, and with those of other GH family β-agarases in Agarivorans. the amount of NA8 was increased with the passage of β-Agarase in the family GH-118 was first reported from time. AgaJA2 was shorter (N-terminal, 38 amino acids) Vibrio sp. PO-303 in 2006 [4]. Since then, only six than that of AgaC [4], but the sequence of the remaining sequences have been reported at CAZy and only two amino acids was the same. However, Dong et al. [4] found agarases, Pseudoalteromonas sp. CY24 and Vibrio sp. PO- that agarose is hydrolyzed into neoagaro-octaose (NA8), 303, have been characterized. CAZymes that cleave the -decaose (NA10), -dodecaose (NA12), and -tetradecaose agarose β-1,4 glycosidic bond, namely β-agarase, belong (NA14) at 0.05 U/ml, whereas NA8 is produced predominantly to GH families 16, 50, 86, and 118. CAZymes that cleave in addition to NA6 and NA4 at 30 U/ml AgaC. Some the α-1,3 glycosidic bond, namely α-agarase, are members family GH 16 agarases degrade agarose and agarose of families 96 and 117. oligosaccharides composed of at least six sugars to yield At least three agarases were found in Agarivorans NA4 as the main product [10, 12]. GH-16 β-agarase from sp. JA-1. GH-50 β-agarase was previously cloned and Agarivorans albus YKW-34 mainly yielded NA4 [7]. GH- recombinantly expressed and is 109 kDa, consisting of 995 50 β-agarase from Agarivorans sp. JA-1 mainly produces amino acid residues [17, 18]. The agaJA2 gene of NA4 and NA6 [17], whereas that from Vibrio sp. strain Agarivorans sp. JA-1 comprised 1,437 bp and encoded a JT0107 produces NA2 [25]. GH-86 β-agarase from protein of 478 amino acids with a molecular mass of 53 Pseudoalteromonas atlantica T6c degrades agarose to kDa (JX840781). The molecular mass of another agarase largely yield NA6 [3]. Ma et al. [20] reported that AgaB was about 47 kDa and is under investigation. Two agarases from GH-118 cannot degrade NA8, and that NA10 is the have been reported from Agarivorans albus YKW-34, shortest substrate oligomer that can be recognized and namely GH-16 β-agarase with 453 amino acids (49 kDa) hydrolyzed by AgaB. They suggested that the peculiar and GH-50 β-agarase of 50 kDa [7, 8]. Ohta et al. [21] structure of β-agarase AgaB is more suitable to hydrolyze reported that Agarivorans sp. JAMB-A11 expresses four long oligosaccharides such as NA12. Here, we have agarases of 45, 60, 105, and 150 kDa. Hu et al. [11] cloned, recombinantly expressed, and characterized GH- reported that Agarivorans sp. HZ105 possesses three 118 β-agarase from a strain of the Agarivorans genus, extracellular GH-50 β-agarases of 105, 58, and 54 kDa. As from which we earlier reported recombinant expression mentioned above, Agarivorans species tend to possess and characterization of a GH-50 β-agarase. A combination multiple agarases. It would be beneficial to degrade and of these agarases would be useful for the production of utilize agar, which has rigid and complex structures. functional neoagarooligosaccharides. The optimal temperature and pH for AgaB are 40oC and 6.0 [20], respectively, whereas those are 35oC and 7.0 for AgaJA2, respectively. AgaB showed stable activity at low Acknowledgment temperatures and maintained >90% activity to 35oC. AgaJA2 showed stable (85% and 96%) activity at 25oC This work was supported by the Marine and Extreme and 45oC, respectively (Fig. 2). 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