Expression and Characterization of a Novel Thermostable and pH

Article pubs.acs.org/JAFC

Expression and Characterization of a Novel Thermostable and pH-Stable β‑Agarase from Deep-Sea Bacterium Flammeovirga Sp. OC4 Xing-Lin Chen,†,‡,§ Yan-Ping Hou,‡,§ Min Jin,‡,∥,§ Run-Ying Zeng,‡,∥ and He-Tong Lin*,† †

College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China State Key Laboratory Breeding Base of Marine Genetic Resource, Third Institute of Oceanography, State Oceanic Administration, Xiamen, Fujian 361005, China ∥ South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Guangzhou, Guangdong 510000, China ‡

ABSTRACT: A novel gene (aga4436), encoding a potential agarase of 456 amino acids, was identified in the genome of deepsea bacterium Flammeovirga sp. OC4. Aga4436 belongs to the glycoside hydrolase 16 β-agarase family. Aga4436 was expressed in Escherichia coli as a fusion protein and purified. Recombinant Aga4436 showed an optimum agarase activity at 50−55 °C and pH 6.5, with a wide active range of temperatures (30−80 °C) and pHs (5.0−10.0). Notably, Aga4436 retained more than 90%, 80%, and 35% of its maximum activity after incubation at 30 °C, 40 °C, and 50 °C for 144 h, respectively, which exhibited an excellent thermostability in medium-high temperatures. Besides, Aga4436 displayed a remarkable tolerance to acid and alkaline environments, as it retained more than 70% of its maximum activity at a wide range of pHs from 3.0 to 10.0 after incubation in tested pHs for 60 min. These desirable properties of Aga4436 could make Aga4436 attractive in the food and nutraceutical industries. KEYWORDS: deep-sea bacterium, Flammeovirga sp. OC4, β-agarase, thermostable, pH stable

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β-agarases have been successfully applied in agar hydrolysis to produce oligosaccharides, which have potential applications in the food and nutraceutical industries since they possess diverse physiological and biological functions beneficial for the health of human beings.10−13 Moreover, agarases also have been applied to recover deoxyribonucleic acid (DNA) from agarose gel,14 to degrade algae cell wall for protoplast preparation,15 and to investigate the composition and structure of the cell wall of algae. Agarases are mainly found in marine organisms, particular marine microorganisms, such as Pseudoalteromonas,16 Pseudomonas,17 Flammeovirga,18 Cytophaga,19 Alteromonas,20 Bacillus,21 Agarivorans,4 and so on. Up to now, despite the increasing number of newly discovered agarases, few of these enzymes have been commercially used in the food and nutraceutical industries due to their low stability. Thus, finding a novel agarase with high stablility will be of great importance for research and commercial purposes. In our previous study, Flammeovirga sp. OC4, a Gracilaria lemaneiformis degrading strain, was isolated from deep-sea water of the South China Sea (118°01′E, 21°21′N) by in situ enrichment using G. lemaneiformis as the substrate.22 The objective of this study was to find a novel gene in the genome of Flammeovirga sp. OC4, which encodes agarase, and to evaluate the stability of the agarase for potential use in the food and nutraceutical industries.

INTRODUCTION Agar, a type of hydrophilic colloid, is the major cell wall component of marine red algae such as Gracilaria and Gelidium.1 Agar consists of two components including agarose and agaropectin. Agarose, the major gelling component of agar, is a neutral linear polysaccharide composed of repeating residues of 3-O-linked β-D-galactopyranose and 4-O-linked 3,6-anhydroα-L-galactopyranose. Due to its favorable property of thermoreversible aqueous gelation, agar can be widely used as additives in the food industry.2 Agarases are the hydrolytic enzymes that act on the hydrolysis of agar and are the first enzymes in the agar catabolic pathway. Depending on their cleavage sites, agarases can be classified into two distant types, i.e., α-agarases (EC 3.2.1.158), which hydrolyze the α-1,3 bonds to produce agarooligosaccharides with 3,6-anhydro-L-galactopyranose at the reducing end,3 and β-agarases (E.C. 3.2.1.81), which hydrolyze the β-1,4 bonds to produce neoagarooligosaccharides with various degrees of polymerization.4 Most of the reported agarases are β-agarases, with only a few of α-agarases reported so far.5−8 Based on homology of amino acid sequences, β-agarases can been further grouped into four glycoside hydrolase (GH) families, namely, GH16, GH50, GH86, and GH118. GH16 is the largest group of the agarase family, with more than 1000 members, and possesses heterogeneous functions (e.g., carrageenase, lichenase, and so on), while the GH50 and GH86 families solely belong to β-agarases, with significantly fewer family members.9 Agarases have a broad range of applications in the biotechnology, food, and nutraceutical industries. As reported, © 2016 American Chemical Society

Received: Revised: Accepted: Published: 7251

July 4, 2016 September 2, 2016 September 5, 2016 September 5, 2016 DOI: 10.1021/acs.jafc.6b02998 J. Agric. Food Chem. 2016, 64, 7251−7258

Article

Journal of Agricultural and Food Chemistry

Figure 1. The DNA and protein sequence of Aga4436. The predicted signal peptide is marked with a box. The conserved GH16 catalytic domain is underlined. The start and stop codons are in bold. The active residues are indicated with asterisks, and the catalytic residues are shaded in gray.

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the Luria−Bertani (LB) cultures reached 0.6−0.7 at 37 °C. The bacteria were collected by centrifugation at 15000g for 10 min and disrupted by sonication in ice for 30 min at a pulse frequency of 3 s/2 s. After centrifugation for 20 min at 20000g, the supernatant was collected. Then the recombinant Aga4436 proteins in the supernatant were purified with a Ni-nitilotriacetic acid (NTA) affinity column according to the guidelines of the manufacturer (Qiagen, Germany). The obtained recombinant Aga4436 protein was analyzed by glycine− sodium dodecyl sulfate (SDS)−polyacrylamide gel electrophoresis (PAGE) and visualized by coomassie brilliant blue staining. Enzyme Activity Assay. The activity of Aga4436 was determined by measuring the release of the reducing sugars according to the 3,5dinitrosalicylic acid (DNS) method described by Miller23 with minor modifications. Briefly, 100 μL of diluted enzyme solution was incubated with 900 μL of 0.2% (w/v) melted agar [dissolved in phosphate buffered saline (PBS), pH 7.4] at 50 °C for 5 min. The enzymatic reaction was stopped by immersion in boiling water for 5 min to inactivate enzymes. Subsequently, 750 μL of DNS reagent was added to the 250 μL reaction solution, and followed by heating of the mixture in a boiling water bath for 10 min. After cooled to room temperature, the amount of released reducing sugar in the reaction mixture was quantified spectrophotometrically at the wavelength of 550 nm. One unit of enzyme activity was defined as the amount of enzymes which released 1 μmol of reducing sugar per minute under standard assay conditions. All enzymatic assays were performed in triplicate. Characterization of Recombinant Aga4436. The temperature effects on Aga4436 activity were investigated by conducting the enzymatic activity assay in PBS buffer (pH 7.4) at different temperatures ranging from 30 to 80 °C. The temperature effects on the thermostability of recombinant Aga4436 were studied by monitoring the remaining enzyme activity after incubating the Aga4436 in PBS buffer (pH 7.4) at different temperatures (30 °C,

MATERIALS AND METHODS

Sequence Analysis. The conserved domain of Aga4436 was predicted using the NCBI (National Center for Biotechnology Information, USA) Conserved Domain Database. The homology analysis of nucleotide and protein sequences was performed with N-BLAST (nucleotide basic local alignment search tool) and P-BLAST (protein basic local alignment search tool) programs, respectively. The phylogenetic tree was constructed using Molecular Evolutionary Genetics Analysis (MEGA) Program version 5.1 (DNAstar, USA) by the neighbor-joining method based on agarase protein sequences. Overexpression and Purification of Recombinant Aga4436. The Flammeovirga sp. OC4 strain isolated from deep-sea water of the South China Sea was identified and characterized in our previous study.22 Notably, Flammeovirga sp. OC4 can produce oligosaccharides from G. lemaneiformis, when growing on a medium only containing 3% (w/v) G. lemaneiformis and seawater. A putative agarase gene (designated as aga4436), which may contribute to G. lemaneiformis degradation, was identified in the draft genome of Flammeovirga sp. OC4.22 The aga4436 gene was amplified with an upstream primer (5′-GGCCATATGATGAAAAACAATTATCTATTAATC-3′) and a reverse primer (5′-GGCAAGCTTTTAATTGATTAGAAGTTTTTG-3′) from the genomic DNA of Flammeovirga sp. OC4, which contained the cleavage sites for NdeI and HindIII (underlined), respectively. To make the pColdII-aga4436 construct, the amplicon was digested with NdeI and HindIII, and then inserted into expression vector pColdII downstream of the 6× histine tag. For the overexpression and purification of 6× histine tagged Aga4436 recombinant protein, the pColdII-aga4436 construct and the empty pColdII vector were transferred into Escherichia coli BL21 (DE3) cells by chemical transformation, separately. The recombinant E. coli cells were induced for protein expression with 1 mM isopropyl-β-Dthiogalactopyranoside (IPTG) at 16 °C for 10 h when the OD600 nm of 7252

DOI: 10.1021/acs.jafc.6b02998 J. Agric. Food Chem. 2016, 64, 7251−7258

Article

Journal of Agricultural and Food Chemistry

Figure 2. Multialignment of the Aga4436 amino acid sequence with the protein sequences of other agarases. The agarases aligned are from Flammeovirga yaeyamensis (gb|AEK80424.1), Flammeovirga sp. MY04 (gb|ANQ52755.1), Flammeovirga pacif ica (ref|WP_052431868.1), and Tamlana sp. s12 (gi|1057230811). The identical residues of all aligned sequences are shaded black. The conserved active residues and conserved catalytic residues are indicated with asterisks and box, respectively.

Figure 3. Phylogenetic tree of agarases based on amino acid sequence homology. The phylogenetic tree was generated by the neighborjoining method using Mega 5.1 software. The species names are indicated along with accession number of corresponding agarase sequence. Bootstrap values of 1000 trials are presented in the branching points. The scale bar indicating ten nucleotide substitutions per 100 nucleotides is indicated at the bottom. Aga4436 is displayed in bold.

Figure 4. SDS−PAGE analysis of overexpressed (A) and purified (B) recombinant Aga4436. (A) SDS−PAGE of overexpressed Aga4436. Lane M, protein marker. Lane 1, E. coli-pColdII-aga443, total protein, induced. Lane 2, E. coli-pColdII-aga443, total protein, not induced. Lane 3, E. coli-pColdII-aga443, supernatant protein, induced. (B) SDS−PAGE of purified recombinant Aga4436. Lane M, protein marker. Lane 1, purified recombinant Aga4436. For the above plots, the arrows indicate the position of the recombinant Aga4436 band.

40 °C, and 50 °C) in the absence of substrate for various periods (0 h, 48 h, 74 h, 99 h, 120 h, and 144 h). The pH profiles were acquired by determination of Aga4436 activity at different pH values (pH 3.0−10.5) at 50 °C. The following buffers were used to incubate Aga4436: 50 mM Na2HPO4/citric acid solution (pH 3.0−8.0), 50 mM Tris-HCl buffer (pH 7.0−9.0), or 50 mM Gly/ NaOH buffer (pH 9.0−10.6).24,25 The effects of pH on the stability of Aga4436 were evaluated by preincubating Aga4436 in pH 3.0−10.6 solutions at 50 °C for 60 min followed by assaying the residual activity.

To assess the effects of metal ions and chelators on the enzymatic activity of Aga4436 activity, various metal ions and ethylenediaminetetraacetic acid (EDTA) with the final concentrations of 10 mM were added to the reaction solution, and the residual activity of Aga4436 was detected under the standard assay conditions. The tested agents were listed as follows: metal ions (K+, Na+, Sr2+, Mg2+, Cu2+, Ni2+, Co2+, Mn2+, Fe2+, Ba2+, Ca2+, Rb+, and Zn2+), and chelator (EDTA). 7253

DOI: 10.1021/acs.jafc.6b02998 J. Agric. Food Chem. 2016, 64, 7251−7258

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Journal of Agricultural and Food Chemistry

Figure 5. Effects of temperature and pH on recombinant Aga4436. (A) Temperature effects on the activity of recombinant Aga4436. The activity of Aga4436 was detected at the temperature ranges of 30 to 80 °C at pH 6.5. (B) Temperature effects on the stability of recombinant Aga4436. The remaining activity of Aga4436 was measured after incubating Aga4436 in the absence of substrate at 30 °C, 40 °C, or 50 °C for various durations. (C) pH effects on the activity of recombinant Aga4436. pH profiles were determined by incubating Aga4436 at 50 °C in the following buffers: pH 3.0 to 8.0, Na2HPO4/citric acid buffer; pH 7.0 to 9.0, Tris-HCl buffer; pH 9.0 to 10.6, Gly/NaOH buffer. (D) pH effects on the stability of Aga4436. Prior to determination of residual activity, the Aga4436 was first incubated in buffers of desired pH (pH 3.0−10.6) at 50 °C for 60 min. For all of the above plots, values are presented as percentages of the maximum activity of Aga4436 (taken as 100%) and are expressed as mean of three parallel trials with standard deviation. All above Aga4436 characterization experiments were carried out in triplicate. Cleavage Site Assay. The cleavage site of agar by Aga4436 was determined by using the artificial chromogenic substrates p-nitrophenyl-α-D-galactopyranoside and p-nitrophenyl-β-D-galactopyranoside. 200 μL of diluted enzyme solution was mixed with 500 μL of artificial chromogenic substrates, and was incubated at 50 °C for 30 min. The enzymatic reaction was stopped by adding 500 μL of 1 M Na2CO3 to the reaction mixture. The cleavage site was determined by measuring the amount of p-nitrophenol spectrophotometrically at the wavelength of 420 nm, which was released from the hydrolysis of the corresponding artificial chromogenic substrates. Identification of Hydrolysis Products. In order to determine the components of the enzymatic hydrolysates, the hydrolysis products were analyzed by the thin layer chromatography (TLC) and ion

exchange chromatography (IC). The pure neoagarobiose (NA2), NA4, and NA6 (Marineoligo, China) were used as standards for TLC and IC analysis. The TLC analysis of hydrolysis products of agar by Aga4436 was performed according to the methods described by Hou et al.26 Briefly, the recombinant Aga4436 protein was added to 0.2% agar solution [dissolved in Tris-HCl buffer (50 mM, pH 7.0)], and the mixture was incubated at 50 °C for enzymatic reaction. At different time intervals, reaction samples were withdrawn and the enzymatic reactions were stopped by heating the mixture in boiling water for 5 min. After centrifugation at 4 °C at 15000g for 15 min, the supernatant was collected, concentrated, and loaded to silica gel 60 TLC plates (Merck, Darmstadt, Germany). The plates were developed using n-butanol/acetic acid/water (2:1:1, v/v/v) as a solvent system, and the spots were visualized by spraying 10% (v/v) H2SO4 and heating at 100 °C for 10 min. 7254

DOI: 10.1021/acs.jafc.6b02998 J. Agric. Food Chem. 2016, 64, 7251−7258

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Journal of Agricultural and Food Chemistry

Table 1. Comparison of General Enzymatic Properties of Aga4436 and Other Agarases Belonging to Different GH Families agarase name

source

family

Aga4436 AgaYT AgrP RAgaA AgaA AgaG1 AgaG4 AgaP4383

Flammeovirga sp. OC4 Flammeovirga yaeyamensis Pseudoalteromonas sp. AG4 Vibro sp. PO-303 Pseudoalteromonas sp. CY24 Alteromonas sp. GNUM1 Flammeovirga sp. MY04 Flammeovirga pacif ica

GH16 GH16 GH16 GH16 GH16 GH16 GH16 GH86

NA4, NA4, NA4, NA4, NA2, NA2, NA4, NA4,

AgaO AgaA AgaXa

Microbulbifer sp. JAMB-A94 Cellvibrio sp. OA-2007 Catenovulum sp. X3

55 °C/50 °C (80%, 42 h) 6.0/3.0−10.0 40 °C/− 8.0/− 55 °C/55 °C (80%, 1 h) 5.5/− 40 °C/37 °C 7.5/5.5−11.0 (1 h) 40 °C/30 °C for 1 h 6.5/5.0−9.0 40 °C/45 °C for 30 min 7.0/6.0−9.0 (97%, 30 min) 50 °C/50 °C 7.5/6.0−9.0 50 °C/50 °C (100%, 12 h) 60 °C (70%, 9.0/5.0−10.0 (90%, 24 h) 30 min) GH86 45 °C/− 7.5/6.0−9.0 (95%, 30 min) GH86 42.5 °C/40 °C (90%, 30 min) 6.5/− GH118 52 °C/42 °C (95%, 1 h) 7.4/5.0−9.0 (85%, 12 h)

35 43 34

AgWH50A RagaA11

Agarivorans gilus WH0801 Agarivorans sp. JAMB-A11

GH50 GH50

30 °C/30 °C (100%, 1 h) 40 °C/45 °C (85%, 15 min)

NA6 NA2, NA4 NA6, NA8, NA10, NA12 NA4 NA2

AgWH50C

Agarivorans gilvus WH0801

GH50

30 °C/40 °C (35%, 1 h)

NA2

46

temp optima/stability

rel act. (%)

Metal ion (10 mM)

rel act. (%)

control K+ Na+ Sr2+ Mg2+ Cu2+ EDTA

100 88.94 ± 2.12 97.3 ± 3.34 89.19 ± 4.51 83.28 ± 3.75 63.85 ± 1.86 57.89 ± 2.93

Ni2+ Co2+ Mn2+ Fe2+ Ba2+ Ca2+ Rb+

73.6 ± 2.46 131.77 ± 2.73 117.06 ± 3.21 78.8 ± 5.24 82.73 ± 4.89 76.84 ± 3.28 100.86 ± 2.98

6.0/− 7.5−8.0/6.0−11.0 (70%, 30 min) 6.0/−

products

ref

NA6 NA6 NA6 NA6 NA4, NA6 NA4 NA6 NA6

this study 38, 39 16 40 41 42 31 26

44 45

as aga4436), encoding a potential agarase of 456 amino acids with a theoretical molecular mass of 51 kDa, was identified in the genome of Flammeovirga sp. OC4.22 The DNA and amino acid sequence of Aga4436 are shown in Figure 1. According to the SignalP 3.0 prediction, the Aga4436 protein possesses an N-terminal signal peptide of 23 amino acids with the most likely cleavage site between Trp23 and Ser24. Conserved domain analysis suggested an existence of a single conserved family 16 glycoside hydrolases (GH16) catalysis domain immediately following the signal peptide, suggesting that Aga4436 is not a modular enzyme as some β-agarases are (e.g., β-agarases from Pseudomonas atlantica and Aeromonas sp.). Eleven conserved active residues (Ala141, Cys143, Trp145, Glu154, Asp156, Glu159, His172, Ser174, Trp194, Asn326, Glu328) and 3 conserved catalytic residues (Glu154, Asp156, Glu159) were identified in the GH16 domain (Figure 1). The three conserved catalytic residues (Glu154, Asp156, Glu159) act as nucleophile and acid residues in catalysis, respectively, and hence are crucial for enzymatic activity.29,30 Recently, β-agarase, AgaG4, which has an extra peptide within its GH16 catalytic module, was identified from Flammeovirga sp. MY04.31 It was shown that the extra peptide and Tyr276 residue of AgaG4 played an important role in binding and degrading the neoagarooctaose (NA8) substrate. Similarly, Aga4436 contains a peptide that is partially conservative with AgaG4, and it also yields NA4 and NA6 as the final agarose degradation products, suggesting that this peptide of Aga4436 may be also important for NA8 degradation. Although the nucleotide sequence of Aga4436 had no significant identity with the known nucleotide sequence of glycoside hydrolase in the NCBI database (less than 80% identity), the encoded Aga4436 protein shared 74%, 74%, 72%, and 66% similarity with β-agarases of Flammeovirga yaeyamensis (gb|AEK80424.1), Flammeovirga sp. MY04 (gb|ANQ52755.1), Flammeovirga pacif ica (ref|WP_052431868.1), and Tamlana sp. s12 (gi|1057230811), respectively (Figure 2). On basis of the amino acid sequence homology, β-agarases can be classified into four glycoside hydrolase (GH) families, namely, GH16, GH50, GH86, and GH118.26 To determine the subfamily of Aga4436, the phylogenetic tree based on the amino acid sequence of agarases belonging to different groups was constructed to compare the sequence homology. As shown in

Table 2. Effects of Metal Ions and Chemical Agents on Aga4436 Activity metal ion (10 mM)

pH optima/stability

For ion exchange chromatography analysis, the above concentrated hydrolysis products as well as the mixed oligosaccharide standards (NA2, NA4, NA6) were analyzed under the same conditions with an anion exchange chromatograph (DIONEX, Sunnyvale, CA, USA) equipped with a 250 × 4 mm IonPac column (ASII-HC). After the sample was loaded, the column was washed with the mobile phase (100 mM NaOH, 150 Mm NaAc) at a flow rate of 0.25 mL/min for 50 min. The liquid chromatography (LC) plot was acquired by plotting the electrical conductivity of the eluent against the retention time. Accession Numbers. The DNA and protein sequences of Aga4436 are deposited at the GenBank database under the accession number of AJW82062.1. The Flammeovirga sp. OC4 strain providing target gene is available in Marine Culture Collection of China (MCCC, accession number 1A07090).

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RESULTS AND DISCUSSION Sequence Analysis of Aga4436. The genus Flammeovirga, first described in 1997 by Nakagawa and co-workers, consists of only five species to date.27 All species within genus Flammeovirga were isolated from marine environments, and were agarolytic bacteria that could produce large gelase fields and deep craters in agar plates.28 Flammeovirga sp. OC4 was isolated from deep-sea water of the South China Sea by in situ enrichment using G. lemaneiformis as substrates. It could grow with G. lemaneiformis as the sole carbon and nitrogen source, and produce oligosaccharides from G. lemaneiformis.22 The agarolytic property of Flammeovirga sp. OC4 strain likely plays a role in the predation of alga by marine creatures, and may participate in the carbon cycle of marine ecosystems. Based on gene prediction, a 1,371-bp open reading frame (ORF) (GenBank accession number AJW82062.1, designated 7255

DOI: 10.1021/acs.jafc.6b02998 J. Agric. Food Chem. 2016, 64, 7251−7258

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Journal of Agricultural and Food Chemistry

Figure 6. Identification of hydrolysis products. (A) TLC analysis of hydrolysis products. Lanes 1, 2: standard markers (NA4, neoagarotetraose; NA6, neoagarohexaose). Lanes 3−8: end products after incubation of Aga4436 with agarose at 50 °C for 20 min, 40 min, 1 h, 2 h, 4 h, and 24 h, respectively. Due to the uneven staining at the edge of the plate, the NA4 of lane 8 appeared like two points in the plate. However, there was actually only one point at the site of NA4 in lane 8. (B) IC analysis of oligosaccharide standards (left) and hydrolysis products after 24 h enzymatic reaction (right). Peaks are labeled with identified oligosaccharides and retention times.

characterized to be most active at 40 °C (Table 1).32,33 However, the GH16 β-agarases Aga4436 described in this study exhibited a temperature optimum at 55 °C, which was significantly higher than most other reported GH16 β-agarases. Due to the cold marine environments where agarases are isolated from, most agarases are not stable at high temperatures for a long time, which greatly hinders their application in industries (Table 1). Notably, Aga4436 exhibited an excellent thermostability at medium-high temperatures, being stable at 30 and 40 °C. Furthermore, Aga4436 retained more than 90%, 80%, and 35% of its maximum activity after incubation at 30 °C, 40 °C, and 50 °C for as long as 144 h, respectively (Figure 5B). The pH profiles showed that Aga4436 had a wide active pH spectrum from pH 5.0 to 10.0, with an optimum pH observed at pH 6.0 (Figure 5C). In contrast to neutral or alkaline pH stability of most β-agarases (Table 1),2 Aga4436 showed a strong pH stability over a broad pH range from 3.0 to 10.0, which is much wider than that for most β-agarases.34,35 Aga4436 displayed an excellent tolerance to acid and alkaline environments, as it retained more than 70% of its maximum activity at a wide range of pH values from 3.0 to 10.0 after incubation at tested pH values for 60 min. Remarkably, no obvious enzymatic activity loss of Aga4436 was observed after its incubation at pH 9.0−10.0 for 60 min (Figure 5D). The effects of various additives, mainly metal ions, on the activity of Aga4436 were investigated, and the results are

Figure 3, Aga4436 clustered with representative GH16 agarases, further confirming that Aga4436 was one of the GH16 family β-agarases. Overexpression and Purification of Recombinant Aga4436 Protein. For better characterization, aga4436 gene was cloned into the expression vector pColdII and overexpressed in E. coli cells as a 6× histine tagged fusion protein. SDS−PAGE results revealed the existence of a new protein with an approximate molecular weight of 54 kDa (corresponding to the size of 6× His tagged Aga4436 recombinant protein) in induced recombinant E. coli cells but not in noninduced E. coli cells, indicating the successful overexpression of recombinant Aga4436 protein in E. coli (Figure 4A). Ni+ affinity column purification results showed a single pure band of recombinant Aga4436 protein in SDS−PAGE gel, which was suitable for downstream enzymatic property characterization (Figure 4B). Characterization of the Recombinant Aga4436. The temperature effects on the activity of recombinant Aga4436 were evaluated at a wide temperature range from 30 to 80 °C (Figure 5A). The optimum temperature for the maximum activity of Aga4436 was determined at 55 °C. Recombinant Aga4436 was active over a broad temperature range from 30 to 80 °C, since it retained more than 60% of its maximum activity at 30−65 °C and retained more than 30% of its maximum activity at 65−80 °C (Figure 5A). To this date, most of the reported β-agarases within the GH16 family have been 7256

DOI: 10.1021/acs.jafc.6b02998 J. Agric. Food Chem. 2016, 64, 7251−7258

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Journal of Agricultural and Food Chemistry summarized in Table 2. Among the metal ions tested, Mn2+ and Co2+ could promote the activity of Aga4436, especially Co2+ could significantly enhance the activity of Aga4436 by 31%. In contrast, several metal ions (K+, Sr2+, Mg2+, Cu2+, Ni2+, Fe2+, Ba2+, Ca2+) exerted strong negative effects on Aga4436 activity. Besides, EDTA significantly inhibited the enzymatic activity of Aga4436, implying the crucial roles of certain divalent metal ions in the Aga4436 activity. Previous studies have revealed the existence of a conserved calcium-binding site in GH16 β-agarases by structure analysis.36 Studies have indicated that calcium ions could stabilize the GH16 β-agarases instead of participating in the catalytic activity.37 However, no calciumbinding site was identified in Aga4436 amino acid sequence by sequence analysis. Besides, the calcium ions exerted strong inhibitory effects instead of stimulating effects on Aga4436 activity, suggesting that calcium ions were not necessary for the stability of Aga4436. Under optimal conditions, the recombinant Aga4436 showed a substrate conversion rate of 31.8% toward agar of enzymatic reaction for 5 min. To determine the cleavage site of agar by Aga4436, the enzymatic activities of Aga4436 toward artificial chromogenic substrates (p-nitrophenyl-β-D-galactopyranoside and p-nitrophenyl-α-D-galactopyranoside) were examined. The cleavage site can be determined spectrophotometrically by the release of p-nitrophenol due to the hydrolysis of the artificial chromogenic substrates. As expected, Aga4436 could catalyze the hydrolysis of p-nitrophenyl-β-D-galactopyranoside (absorbance at 420 nm was 0.612) but not p-nitrophenyl-α-D-galactopyranoside (absorbance at 420 nm was 0.014), indicating that it cleaved the β-1,4 bonds but not the α-1,3 bonds in the agar. To investigate the hydrolysis modes of Aga4436, TLC analysis was first performed to separate and identify the hydrolysates produced from agar by Aga4436 for various reaction times (20 min, 40 min, 1 h, 2 h, 4 h, and 24 h). As shown in Figure 6A, Aga4436 can hydrolyze agar to generate diverse products in early stage (20 min post reaction), including NA4, NA6, and oligosaccharides larger than hexamer. As the reaction time went on, the amount of small oligosaccharides (NA4 and NA6) increased, while the amount of large oligosaccharides (larger than hexamer) decreased. After hydrolysis for 24 h, the end products from agar were NA4 and NA6, which was similar to other β-agarases (Table 1). The hydrolysis products from the sample of enzymatic reaction for 24 h were further analyzed with anion exchange liquid chromatography. As shown in Figure 6B, the hydrolysis products were NA4 and NA6, which were consistent with the TLC results. In conclusion, a novel β-agarase gene aga4436, which was isolated from the deep-sea bacterium Flammeovirga sp. OC4, was cloned, expressed, and characterized. Recombinant Aga4436 protein exhibited outstanding properties, which meet the industrial demands of high thermostability, wide pH stability, and good environmental adaption. Since the gelling temperature of agar is about 38 °C, the high optimum temperature and good thermostability of Aga4436 are beneficial for efficient DNA recovery from agar gel as well as industrial production of oligosaccharides from agar.

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Funding

This work was financially supported by Public Science and Technology Research Funds Projects of Ocean, China (Grant 201505026-2), Xiamen South Ocean Research Center Project of China (Grant 13GZP003NF09), and the Construction Projects of Top University at Fujian Agriculture and Forestry University of China (Grants 612014042, 612014043). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED GH, glycoside hydrolase; DNA, deoxyribonucleic acid; NCBI, National Center for Biotechnology Information; N-BLAST, nucleotide basic local alignment search tool; P-BLAST, protein basic local alignment search tool; MEGA, molecular evolutionary genetics analysis; IPTG, isopropyl-β-D-thiogalactopyranoside; LB, Luria−Bertani; NTA, nitilotriacetic acid; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; DNS, 3,5-dinitrosalicylic acid; PBS, phosphate buffer saline; EDTA, ethylenediaminetetraacetic acid; MCCC, Marine Culture Collection of China; ORFs, open reading frames; TLC, thin layer chromatography; NA4, neoagarotetraose; NA6, neoagarohexaose; NA2, neoagarobiose

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X.-L.C., Y.-P.H., and M.J. contributed equally to this work. 7257

DOI: 10.1021/acs.jafc.6b02998 J. Agric. Food Chem. 2016, 64, 7251−7258

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DOI: 10.1021/acs.jafc.6b02998 J. Agric. Food Chem. 2016, 64, 7251−7258