Characterization and overexpression of a novel β-agarase from Thalassomonas agarivorans

Authors


Abstract

Aims

The agarase from Thalassomonas agarivorans BCRC 17492 was cloned and overexpressed in Escherichia coli. The characterization of the novel agarase was performed.

Methods and Results

The genomic library of T. agarivorans BCRC 17492 was constructed for screening agarase gene. The novel β-agarase, namely AgaB1, was successfully identified and shared only 57% identity to reported agarase from Alteromonas sp. To characterize the AgaB1 protein, the recombinant AgaB1 can be obtained by heterologous expression in E. coli. The agarase activity of AgaB1 was achieved at 30·25 U per mg at 35°C. According to the analysis of optimal conditions, the highest activity of AgaB1 was attained at 40°C, pH 7·4 and 200 mmol l−1 NaCl, and half-life of AgaB1 can be maintained for almost 1 h at 40°C. Further determination of substrate hydrolysis indicated that AgaB1 had possession of both endo- and exolytic activity, and neoagarobiose was the major hydrolysis product by TLC and high-performance liquid chromatograph/mass spectrometer (HPLC/MS) analysis.

Conclusions

We have successfully cloned and overexpressed the novel β-agarase from T. agarivorans BCRC 17492 in E. coli. The high yield and detailed characterization of recombinant AgaB1 was provided.

Significant and Impact of the study

AgaB1 was the first β-agarase that was cloned and described from Thalassomonas species. In the light of properties of AgaB1, it has the potential as the biocatalyst for industrial applications.

Introduction

Agar obtained from the cell wall of some red algae is a polysaccharide biomass and is composed of agarose and agaropectin (Chi et al. 2012). Agarose can be hydrolysed by α-agarase and β-agarase into agaro-oligosaccharides and neoagaro-oligosaccharides, respectively. Neoagaro-oligosaccharides have been applied in various areas, including antioxidative activity, inhibition of lipid peroxidation, scavenging free radicals, moisturizing skin and whitening effect on melanoma cells (Wu et al. 2005; Lee et al. 2008; Ajisaka et al. 2009). 3,6-Anhydro-L-galactose (L-AHG), the monomer unit of agarose via hydrolysis of neoagaro-oligosaccharides, also has been proved with the activities of lowering melanin production and anti-inflammatory (Yun et al. 2013). In the aspect of food application, they can be employed as low-calorie additives, inhibitor of bacterial growth and slowing down the degradation of starch (Giordano et al. 2006). Recently, biofuel is a popular topic due to exhausted petroleum and environmental benefits. Because the galactose and L-AHG of fermentable sugar can be acquired from agarose by β-agarase and neoagarobiose hydrolase hydrolysis, ethanol has been successfully produced by fermentation of treated agarose (Kim et al. 2012, 2013). Therefore, several researches have been explored on characterization of agarase for industrial application (Fu et al. 2008; Xie et al. 2013).

β-Agarases can be classified into four families, namely glycoside hydrolase-16 (GH16), glycoside hydrolase-50 (GH50), glycoside hydrolase-86 (GH86) and glycoside hydrolase-118 (GH118) according to the Carbohydrate-Active enzymes Database (Cantarel et al. 2009). A number of studies have been devoted on GH16 family that can hydrolyse agar and neoagarohexaose, and the main product is neoagarotetraose (Ohta et al. 2004a,b; Dong et al. 2007a). Moreover, the three-dimensional structures of β-agarases from Saccharophagus degradans and Zobellia galactanivorans have been carried out (Allouch et al. 2003; Henshaw et al. 2006). Catalytic glycoside hydrolase module at N-terminal polypeptides and noncatalytic carbohydrate-binding module at C-terminal polypeptides are recognized, respectively. Ten GH50 agarases presenting either exolytic or both endo- and exolytic activity have been characterized (Fu and Kim 2010; Chi et al. 2012). In contrast to GH16 family, catalytic glycoside hydrolase module of GH50 is assumed to be located at the C-terminus by in silico analysis (Ekborg et al. 2006). The neoagarobiose is the major hydrolysis product of GH50 agarase. However, AgaACN41 from Vibrio sp. and rHZ2 from Agarivorans sp. are exceptions, which generate neoagarotetraose as the end product and designate as endohydrolytic agarases (Liao et al. 2011; Lin et al. 2012). The minority of β-agarases is distributed in GH86 and GH118 families that are belonging to endohydrolytic agarases.

Thalassomonas genus generally isolated from seawater and marine sediments is Gram-negative bacteria (Yi et al. 2004; Thompson et al. 2006; Park et al. 2011). Two species, Thalassomonas ganghwensis and Thalassomonas loyana, even can be tolerated and grown as high as 8% sodium chloride. Only one agarase, AgaA33, has been cloned and characterized from Thalassomonas sp. JAMB-A33 (Hatada et al. 2006). AgaA33 encoding endohydrolytic α-agarase yields the major product of agarotetraose. In addition, it has been successfully overexpressed in heterologous hosts, Bacillus subtilis and Saccharomyces cerevisiae. The specific activities of recombinant AgaA33 are 40·7 and 0·18 unit per mg in B. subtilis and S. cerevisiae, respectively.

Thalassomonas agarivorans BCRC 17492 is isolated from seawater in An-Ping Harbour, Taiwan (Jean et al. 2006). In terms of phylogeny and phenotype, it is proposed for the novel species as the type strain and is capable of agar hydrolysis as well as Thalassomonas agariperforans M-M1(T) (Park et al. 2011). In this study, we constructed a genomic library from T. agarivorans BCRC 17492 and a novel β-agarase, named as agaB1, was successfully cloned. The properties of recombinant AgaB1 produced from E. coli were further described.

Materials and methods

Genomic library construction of Thalassomonas agarivorans

Thalassomonas agarivorans BCRC 17492 was cultivated in BCRC medium 615 (Polypepton-Yeast (PY) broth medium: 2 g polypepton, 0·5 g bacto-yeast extract, 30 g NaCl, 5 g MgCl2·6H2O, 0·005 g CaCl2, 0·005 g Na2MoO4·7H2O, 0·004 g CuCl2·2H2O, 0·006 g FeCl3·6H2O and 6 g Tris per litre; adjusted to pH 8·0) at 26°C for 3 days. The bacterial cells were centrifuged and harvested at 3000 g for 20 min. The cells were resuspended in B1 buffer (50 mmol l−1 Tris-HCl, pH 8·0; 50 mmol l−1 EDTA, pH 8·0; 0·5% Tween-20; 0·5% Triton X-100) for chromosome DNA extraction. Lysozyme (3 mg ml−1) and RNase (20 mg ml−1) were used in T. agarivorans at 37°C for 1 h. Then, proteinase K (1 mg ml−1) and sodium lauroyl sarcosine (10 mg ml−1) were added and incubated at 50°C for 2 h. After that, DNA was purified by phenol/chloroform extraction and precipitated by isopropanol.

To construct a genomic shotgun library of Tagarivorans, the chromosomal DNA was sheared by Hydroshear® DNA Shearer (BST Scientific, Singapore Science Park II, Singapore) to DNA fragment between 1 to 5 kb. The DNA fragments were fractionated by agarose gel electrophoresis and cut-off between 2 and 4 kb. Then, they were purified by a gel purification kit (Geneaid Biotech Ltd., New Taipei City, Taiwan). Each purified DNA fragment was end-repaired and cloned into the vector CopyControl™ pCC1™ (Epicentre, Madison, WS, USA). The recombinant vector was transformed into E. coli competent cell TransforMax™ EPI300™ (Epicentre).

The E. coli clones were inoculated onto the culture plates with LB medium (containing 0·01% arabinose, 0·4 mmol l−1 IPTG and 12·5 μg ml−1 chloramphenical). After incubation at 37°C overnight, the plates were kept at room temperature for 10 days, and the hydrolysis of agar resulted from the production of agarase was observed by staining with Gran's iodine reagent (0·05 mol l−1 I2 dissolved in 0·12 mol l−1 KI; Belas et al. 1988). The light yellow clear zones shown in the plate indicated the ability of agarase from the clone.

Analysis of agarase gene clone by shotgun sequencing

The clone harbouring the agarase gene was obtained for shotgun sequencing. The plasmid was digested with BamH1, and the DNA insertion fragment was sheared by Hydroshear® DNA Shearer. The resulting fragments were separated by agarose gel electrophoresis and cut-off between 0·75 and 1·0 kb. Each purified DNA fragment was ligated into the cloning vector pEZSeq Amp utilizing pEZSeq™ Amp Blunt Cloning Kit (Lucigen Corporation, Middleton, WI, USA), and the recombinant vector was transformed into E. coli competent cell TransforMax™ EPI300™ for incubation of LB medium containing 100 μg ml−1 ampicillin, 0·4 mmol l−1 IPTG and 90 μg ml−1 X-Gal. Forty-six clones were selected, and the plasmids were sequencing with the primers Z-For: 5′-CGCCAGGGTTTTCCCAGTCACGAC-3′ and Z-Rev: 5′-AGCGGATAACAATTTCACACAGGA-3′. Ninety-two sequences were obtained and assembled by the ContigExpress of the Vector NTI (Invitrogen, Carlsbad, CA, USA).

BLAST and phylogenetic analysis of Agarase gene

The assembled sequence was analysed by the ORF finder software available on NCBI's website (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The putative ORF was further examined by the protein blast program available on NCBI's website (http://www.ncbi.nlm.nih.gov/BLAST/). The phylogenetic tree of putative agarase gene was constructed by the neighbour-joining method (Saitou and Nei 1987) using MEGA 3.1 software (Kumar et al. 2004) with 1000 bootstrap replicates. Accession numbers for the agarase genes of the family glycoside hydrolase 50 were compared as follows: Agarivorans sp. (ADY17919; ABK97391; BAD99519), Alteromonas sp. (BAE97587), Pseudoalteromonas haloplanktis (WP_016709240), Saccharophagus degradans (ABD81904), Streptomyces coelicolor (CAB61811) and Vibrio sp. (ADM25828; BAA03541; BAG71427).

Construction of agaB1 gene expression vector

To amplify the full-length ORF of putative agarase gene, the forward primer 5′-CACCATGCATAATAAAATGAGT-3′ (the underlined sequence is designed for the ligation with pET151/D-TOPO plasmid) and the reverse primer 5′-TTACTGCGCTTTAAAGCG-3′ were utilized by polymerase chain reaction (PCR). The PCR solution contained 250 ng of clone plasmid, 8 μl of 2·5 mmol l−1 dNTP, 10 μl of 10X PCR buffer, 2 μl of 10 μmol l−1 forward primer and reverse primer, and 5 U Taq enzyme (Takara Bio Inc., Shiga, Japan). The PCR condition was following: (i) 95°C for 5 min; (ii) 95°C for 30 s, 50°C for 30 s and 72°C for 2 min per cycle of a total of 30 cycles; (3) 72°C for 10 min; and (4) maintained at 4°C. The PCR product was ligated with pET151/D-TOPO (Invitrogen) and then transformed into E. coli Top10 to obtain the plasmid pAGAB1.

Purification and characterization AgaB1 protein

To overexpress the agarase protein, the plasmid pAGAB1 was transformed into E. coli BL21 (DE3) that was incubated in LB medium (100 μg ml−1 ampicillin) at 37°C for overnight. The transformant E. coli was transferred into LB and grown to OD600 0·8. The culture was added with 0·1 mmol l−1 IPTG and incubated at 25°C for 6 h to induce the expression of the recombinant agarase protein. The cells were harvested by centrifugation at 2000 g for 20 min. They were suspended in buffer B (25 mmol l−1 Tris-HCl, pH 7·4; 1 mmol l−1 EDTA, pH 7·4; 250 mmol l−1 sucrose) and disrupted by lysozyme on ice for 30 min. The mixture was added with 1 unit of Benzonase (Merck, Darmstadt, Germany) and placed on ice for 10 min. The solution containing Triton X-100 (final concentration of 0·1%) and 150 mmol l−1 NaCl was added to the mixture. The mixture was centrifuged at 19 000 g under 4°C for 15 min, and the supernatant was collected. The agarase protein was purified by Ni-NTA purification system (Invitrogen). The purified agarase was determined by Bradford Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA) and monitored by SDS-PAGE (10% gels) following Coomassie Blue.

The agarase activity was assayed by measuring the reducing sugars obtained from the digestion of low-melting agarose with agarase. The purified agarase was mixed with the low-melting agarose solution (dissolved in a 150 mmol l−1 NaCl and 25 mmol l−1 Tris-HCl buffer, pH 7·4) and incubated at 35°C for 15 min. The reaction was interrupted by 60°C incubation. DNS solution (1% 3,5-dinitrosalicylic acid, 30% potassium sodium tartrate, 1·5% NaOH) was added and then heated in boiling water for 5 min. Then, the mixture was measured by a spectrophotometer at OD540. D-galactose (Sigma-Aldrich, St. Louis, MO, USA) was used as the standard of reducing sugar. One unit (U) of agarase activity represents the production of 1 μmol reducing sugars per min under the above-mentioned conditions. The optimal temperature of purified agarase was determined between 30 and 50°C for 15 min. The thermostability of purified agarase was assayed by incubation at 30, 35 and 40°C from 0 to 90 min. The pH profile of purified agarase was detected between 6 and 10 at 40°C for 15 min. The salt optimum of purified agarase was performed from 0 to 200 mmol l−1 at 40°C for 15 min.

The analyses of thin-layer chromatography and liquid chromatographic mass spectrometry of the reaction products

The purified agarase was mixed with low-melting agarase, which incubated between 0·25 and 4 h at 35°C. The mixture was centrifuged at 19 000 g under 4°C for 10 min to remove the incompletely digested high molecular polysaccharides. The supernatant was determined by utilizing a Silica Gel 60 TLC plate (Merck). The neoagarobiose and neoagarohexaose (Sigma-Aldrich) were used as the standard and the n-butanol/acetic acid/H2O (2 : 1 : 1) was employed in mobile phase. The TLC plate was immersed in a cerium sulphate solution (1% Cerium (IV) sulphate and 1 mol l−1 H2SO4) and heated for colour development. The portion of the TLC corresponding to the separated main hydrolysis product was cut off and extracted from the TLC by methanol. The extractive product was concentrated by a pressure-reducing and heating method and identified by a high-performance liquid chromatograph/mass spectrometer (HPLC/MS). The HPLC/MS system consists of a Waters model 600 pump with a controller (Miford, MA, USA) and a Waters-Micromass Quattro LC mass spectrometer (Manchester, UK) with an electrospray ionization (ESI) probe set at positive mode. The capillary voltage was 4·5 kV. The desolvation temperature was 350°C, and the extraction cone voltage was 20 V. The HPLC/MS method was performed on a Luna 3 μ C18(2) column (150 × 2 mm id; Phenomenex, Torrance, CA, USA) with a sample size of 10 μl and flow rate of 0·2 ml min−1. The HPLC parameters were as follows: solvent A, 0·1% (v/v) formic acid in H2O and solvent B, 0·1% (v/v) formic acid in acetonitrile. Gradient was programmed as 5% B for 1 min, linear increase from 5 to 95% B in 29 min, and then linear decrease to 5% B in 10 min.

Evaluation of DNA gel extraction by agarase protein

The Lambda DNA/HindIII marker and pUC19 plasmid were embedded in 1% low-melting agarose. The agarose with DNA was incubated at 70°C for 10 min and treated with agarase at 35°C for 1 h. The mixtures were centrifuged at 19 000 g at 4°C for 10 min to remove the undigested residue. The supernatant was precipitated by ethanol. The precipitated DNA was vacuumed to dry and dissolved in sterile water. The equal amounts of recovered DNA and standard DNA (pUC19 plasmid and Lambda DNA/HindIII marker) were carried out by electrophoresis.

Nucleotide sequence accession number

The nucleotide sequence of the novel β-agarase from T. agarivorans has been submitted to GenBank under the Accession Number KF479457.

Results

Genomic library of Thalassomonas agarivorans and identification of agarase gene

Little attention has been published regarding the agarase of T. agarivorans which was a novel species in the genus Thalassmonas (Jean et al. 2006). A genomic library of T. agarivorans was constructed with the insertion of 2–4 kb DNA fragments. The library clones were incubated on the LB plate and stained by the Gran's iodine reagent (Belas et al. 1988). The E. coli with light yellow clear zone shown in the plate indicated that was capable of producing agarase. The clone contained the putative agarase was designated as the pcc1clone A. The pcc1clone A was further sequenced by shotgun sequencing and assembled by Vector NTI (Invitrogen). The DNA fragment of insertion was 4434 bp. Database search and ORF prediction contributed information on the putative gene. It was found that the putative ORF was a novel β-agarase gene with 2325 bp and was named as agaB1 (GenBank Accession Number KF479457). The agaB1 gene encoded a protein with 774 deduced amino acids and 87 613 Da. The predicted isoelectric point was pH 5·05.

The deduced amino acids of agaB1 gene were performed by BLAST program of NCBI and had the 63% identity to Pseudoalteromonas haloplankis putative agarase (GenBank accession no. WP_016709240). Besides it shared only 57% with the reported agarase from Alteromonas sp. E-1 (Kirimura et al. 1999). The conserved domain corresponding to beta-galactosidase of GH42 family was recognized from 486 to 653 amino acids (Fig. 1a). To assess the relationship of glycoside hydrolase family between agaB1 and known agarases, a phylogeny was constructed by the deduced amino acids. The result showed that agaB1 gene shared the same clade with the GH50 family indicating the β-agarase (Fig. 1b).

Figure 1.

The alignment and phylogeny of deduced amino acids of β-agarase from various organisms. (a) Deduced amino acid sequences alignment of hypothetical catalytic domain of β-agarase from GH50 family. The catalytic domains were compared on the basis of the conserved sequences from 486 to 653 amino acids of AgaB1. The filled triangles indicate the assumed active sites of Glutamate and Aspartate. (b) Phylogenetic tree of β-agarase from Thalassomonas agarivorans BCRC 17492 and various bacteria. The phylogeny of β-agarase based on the full open reading frame was constructed. Accession numbers for the β-agarase genes were described in the bracket. The tree was constructed by the neighbour-joining method using MEGA 3.1 software. Bootstrap values were shown in the nodes according to the 1000 replications.

Construction and characterization of recombinant agarase protein

To characterize the AgaB1 protein, agaB1 gene was cloned and overexpressed in E. coli. The pET151/D-TOPO plasmid with a T7 promoter and a His-tag fusion of N-terminus was employed (Fig. 2a). After protein purification using nickel column, AgaB1 was demonstrated as a single band on the SDS-PAGE with an approximate molecular mass of 90 kDa, which was in agreement with the prediction (Fig. 2b). 2·1 Milligram per 100 ml of purified AgaB1 was obtained by batch flask culture. Using low-melting agarose (molecular biology grade) as a substrate, the specific activity of AgaB1 was 30·25 U per mg at 35°C. To determine the substrate specificity, various substrates including low-melting agarose (molecular biology grade), agarose for electorphoresis, bacteriological agar for medium preparation, edible agar with food grade, λ-carrageenan, κ-carrageenan and ι-carrageenan were utilized. The results displayed that the digestive activity of AgaB1 to bacteriological agar was the highest one defining as 100%. The relative activities to agarose for electorphoresis, edible agar and low-melting agar were 75·9, 70·9 and 49·3%, respectively. However, no enzymatic activity was found in λ-carrageenan, κ-carrageenan and ι-carrageenan. These results indicate that AgaB1 can hydrolyse different kind of agar substrates, but not λ-carrageenan, κ-carrageenan and ι-carrageenan.

Figure 2.

Overexpression of recombinant AgaB1 protein. (a) Plasmid map of pAGAB1 based on the pET151/D-TOPO for AgaB1 production in Escherichia coli. The pAGAB1 plasmid included the six histidines at N-terminus of AgaB1. (b) The SDS-PAGE analysis of AgaB1 was stained by Coomassie Blue. M indicates the protein standard marker. Only a single band on the SDS-PAGE was observed, presenting the purified AgaB1 via nickel column.

Optimal activity and thermostability of agarase

The temperature, pH, salt profiles and thermostability of AgaB1 activity were analysed. As shown in Fig. 3, the optimal temperature was detected to be 40°C. Nevertheless, the activity of AgaB1 was dramatically dropped to 28% at 45°C and completely lost at 50°C. The thermal resistant of enzyme is important for industrial application (Yeoman et al. 2010). Hence, the temperature stability of enzymes was further detected at 30, 35 and 40°C from 0 to 90 min. The residual activity can be maintained above 79% at 30 and 35°C. The half-life, the time over which the 50% activity is lost, of AgaB1 was retained for 56 min at 40°C according to the linear regression analysis. The pH profile indicated that the optimal pH of AgaB1 activity was around 7·4 and AgaB1 was slightly alkaline. The relative activities at pH 7 and 8 were 91·2 and 89·7%, respectively. At pH 9, the relative activity was fallen to 38·7%. No detectable activity was observed at pH 6 and 10. As Tagarivorans BCRC 17492 was isolated from seawater (Jean et al. 2006), the effect of various sodium chlorides on AgaB1 was examined. The maximal activity of AgaB1 was explored for almost 100% within 150 and 200 mmol l−1. When the NaCl was absent, the residual activity was only 33%. However, after adding 25 mmol l−1 NaCl, the relative activity was rapidly increased to 83·4%.

Figure 3.

The optimal activity of AgaB1 to 0·3% low-melting agarose including temperature, thermostability, pH and salt. (a) The temperature profile of AgaB1 was assayed at 30–50°C and pH 7·4 for 15 min. (b) Temperature stability of the AgaB1 was determined at 30, 35 and 40°C, respectively, and pH 7·4. The enzymes were preheated from 0 to 90 min. The filled square, diamond and triangle indicates 30 (▪), 35 (◆) and 40°C (▲). (c) The pH profile of AgaB1 was assayed at 40°C for 15 min in potassium phosphate buffer with pH ranging from 6 to 7, Tris-Cl buffer with pH ranging from 7·4 to 8 and glycine-NaOH buffer with pH ranging from 9 to 10. (d) The sodium chloride profile of AgaB1 was assayed at 40°C for 15 min from 0 to 200 mmol l−1 NaCl. All values are the averages of three determinations.

Chromatographic analysis of hydrolysis product

The hydrolysis product from agarase was inspected with 20 μg AgaB1 and 1% low-melting agarose at 35°C. The mixture was incubated within the time course of 0·25–4 h. The result was shown in Fig. 4a. At the initial stage of 0·25 h, the significant hydrolysis product was found corresponding to neoagarobiose (NA2). Small amounts of neoagarotetraose (NA4) and neoagarohexaose (NA6) could also be detected with the time prolongation. According to the spot intensity of colour development, the main product was thought to be neoagarobiose with the standard compound. The neoagarohexaose was further utilized as the substrate and mixed with AgaB1 at 35°C for 24 h. Only a single spot was clearly perceived, suggesting the neoagarobiose product (Fig. 4b). To confirm the major product of AgaB1 activity, the hypothetical neoagarobiose was extracted from TLC plate and conducted by HPLC-MS. Only one peak at the retention time of 1·91 min was observed and its mass spectrum exhibited a high intensity of m/z (mass-to-charge ratio) 347 ion. The m/z 347 ion was proposed to be [neoagarobiose + Na]+ due to two major fragments, m/z 203 for [galactose + Na]+ and m/z 185 for [3,6-anhydro-α-l-galactose + Na]+, were observed after MS/MS analysis at 25 eV (Fig. 5). The results of mass analyses demonstrated that the main hydrolysis product of AgaB1 was neoagarobiose.

Figure 4.

The thin-layer chromatographic determination of hydrolysis product of AgaB1. (a) The purified AgaB1 and low-melting agarose were mixed and incubated for 4 h at 35°C. Lane 1–7 indicates the hydrolysis product after 0·25-, 0·5-, 1·0-, 1·5-, 2·0-, 3·0- and 4·0-h reaction. Lane 8 and 9 exhibit the standard of neoagarobiose (NA2) and neoagarohexaose (NA6). The product between NA2 and NA6 was reasonably assumed to be neoagarotetraose (NA4). (b) The purified AgaB1 and neoagarohexaose were mixed and incubated for 24 h at 35°C. Lane 1 indicates the hydrolysis product of AgaB1 activity. Lane 2 and 3 reveal the standard of NA2 and NA6. The TLC was reacted with a cerium sulphate solution (1% Cerium (IV) sulphate and 1 mol l−1 H2SO4) and heated for staining neoagaro-oligosaccharides.

Figure 5.

MS/MS spectrum of the neoagarobiose produced by AgaB1. The spectrum shows the fragment ions of m/z 347, which is the major constituent of the only peak observed at 1·91 min by HPLC. Two major product fragments, m/z 203 for [galactose + Na]+ and m/z 185 for [3,6-anhydro-α-l-galactose + Na]+, were observed together with the parent m/z 347 in MS/MS spectrum.

DNA extraction from low-melting agarose by agarase

Agarase has been used to application of DNA recovery from agarose gel (Fu and Kim 2010). To prove the applicability of agarase, AgaB1 was also employed to extract DNA in this study. pUC19 plasmid and Lambda DNA/HindIII marker were first embedded in the 1% low-melting agarose, which were treated with 1·5 μg and 3 μg of AgaB1 agarase. As shown in Fig. 6, all the pUC19 plasmid and Lambda DNA/HindIII marker DNA samples could be recovered from agarose gel. The result indicated that over 90% recoverable rate of DNA was declared by spectrophotometer measurement. This was consistent with the DNA band intensity by electrophoresis analysis. The result clarified that AgaB1 agarase was suitable for recovering nucleic acid samples from agarose gel.

Figure 6.

DNA recovery from low-melting agarose by AgaB1. The pUC19 plasmid (a) and Lambda DNA/HindIII marker (b) were used to test the DNA recovery by AgaB1. The DNA was embedded in 1% low-melting agarose that was incubated with AgaB1 at 35°C for 1 h. Lane 1 indicates the 250 ng pUC19 plasmid and Lambda DNA/HindIII marker. Lane 2 and 3 reveal the DNA recovery treated with 1·5 and 3 μg AgaB1, respectively. These DNAs were performed by electrophoresis in 1% agarose.

Discussion

A novel β-agarase, AgaB1, was successfully cloned from T. agarivorans BCRC 17492 and shared only 63% identity to Pseudoalteromonas haloplankis putative agarase (GenBank Accession Number. WP_016709240). According to the in silico analysis (Signal P 4·1; Petersen et al. 2011), a putative signal peptide was observed at the N-terminal AgaB1 with 26 amino acids which was similar with other extracellular β-agarase (Xie et al. 2013). This was in agreement with the analysis of T. agarivorans BCRC 17492, revealing diffusion of agarase out from the colonies by staining with iodine reagent (Jean et al. 2006). The conserved domain between 486 and 653 amino acids was thought as catalytic glycoside hydrolase module (Fig. 1a). Because no three-dimensional structure of β-agarase from GH50 family was examined, several characterized β-agarase of GH50 were aligned and compared in this study. Glutamate and aspartate residues usually play an important role in catalytic activity to be accepting and donating protons during hydrolysis process (Allouch et al. 2003; Hehemann et al. 2012). Only three consensus Asp at 515th, 594th and 646th site and one consensus Glu at 517th site were identified. The three-dimensional structure of AgaB1 deduced amino acids were further predicted by (PS)2 software (Chen et al. 2009). The 515th Asp and 517th Glu were adjacent and assumed to be catalytic residues. Further study on the active sites of AgaB1 such as point mutation experiments will clarify the catalytic region of GH50 family. On the other hand, the noncatalytic carbohydrate-binding module of GH50 family, showing agarose- or neoagaro-oligosaccharide-binding function, is believed in locating at N-terminus of β-agarase (Ekborg et al. 2006). Different from the catalytic residues, Glycine, Alanine and Arginine are the foremost amino acids relative to substrate binding (Allouch et al. 2003; Ma et al. 2007). Comparing N-terminus of β-agarase from GH50 family, twelve Gly, five Arg and one Ala were concordant among these reported β-agarases. If the three-dimensional structure will be resolved, it would be helpful to understand the structural characteristic of GH50 family.

To characterize the novel β-agarase AgaB1, we have successfully overexpressed the recombinant AgaB1 in E. coli BL21 (DE3) (Fig. 2). Using batch culture in 500-ml flask, 4·2 mg recombinant AgaB1 protein from 200 ml cultural broth was obtained and 635 U l−1 broth can be assayed at 35°C. On the basis of the previous studies, much attention has been conducted on the heterologous overexpression of agarases in E. coli and B. subtilis (Ohta et al. 2004b, 2005; Lee et al. 2006; Dong et al. 2007b; Xie et al. 2013). Although B. subtilis shows extracellular and extraordinary production of agarase, E. coli is generally as a host for overexpression and characterization due to its useful and convenient tool in most researches. Comparing the β-agarase in GH50 family, the recombinant AgaB1 had high production than that of β-agarase from Agarivorans sp. JA-1 in E. coli heterologous expression but was lower than that of AgaA11 from Agarivorans sp. JAMB-A11 in B. subtilis overexpression (Ohta et al. 2005; Lee et al. 2006). If the AgaB1 is cloned and produced in B. subtilis, high yield of β-agarase would be anticipated.

Several substrates were employed to verify the activity of AgaB1. Bacteriological agar for medium preparation displayed higher digestibility by AgaB1 than that of agarose for electorphoresis, edible agar and low-melting agar. However, no relative carrageenan can be hydrolysed. To further explore whether AgaB1 degrades agarose by endo- or exolytic activity, TLC and HPLC/MS were utilized to realize the hydrolysis production (Figs 4 and 5). These results revealed that neoagarobiose, neoagarotetraose and neoagarohexaose were detectable, and neoagarobiose was the major product of AgaB1 activity. This protein property was resembling in other β-agarase of GH50 family with both endo- and exolytic activity (Fu and Kim 2010; Chi et al. 2012). The optimal temperature and pH of AgaB1 were also similar to these from Agarivorans sp. and Alteromonas sp. in GH50 family (Fig. 3; Kirimura et al. 1999; Ohta et al. 2005; Lee et al. 2006). Even though AgaB1 was heated at 40°C for almost 1 h, the residual activity was remained for 50%. However, the thermostability of AgaB1 was lower than that of AgaA from the strain JAMB-A94 which is thermostable up to 60°C for 15-min incubation (Ohta et al. 2004b). Consequently, the temperature of AgaB1 agarase activity was fit for hydrolysis of agar which its gelling temperature is around 38°C. This can be demonstrated that AgaB1 was also applied in DNA extraction from the agarose gel with high efficiency recovery (Fig. 6).

In conclusion, a novel β-agarase, AgaB1, belonging to GH50 family was successfully cloned from T. agarivorans BCRC 17492 and overexpressed in E. coli. The detailed characterization of AgaB1 was provided including substrate affinity, optimal activity, thermostability and hydrolysis product. Because sufficient quantity of recombinant AgaB1 protein can be obtained from E. coli, the protein crystallization and X-ray structure will be inspected in our future study. The examination of protein structure will verify the active sites and carbohydrate-binding sites of our suggestion in GH50 family. Moreover, it can be contributed to enhance thermostability by rational design using point mutation for extensively industrial applications (Reetz et al. 2006; Blum et al. 2012).

Acknowledgements

Support of the Ministry of Economic Affairs (Taiwan, ROC) (Grant 102-EC-17-A-01-04-0525) to the Food Industry Research and Development Institute (FIRDI) is appreciated.

Conflict of interest

No conflict of interest declared.

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