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.

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