Preliminary characterization of a novel β-agarase from Thalassospira profundimonas

Background The objective of this study was to characterize the agarase from a newly isolated agarolytic bacterium Thalassospira profundimaris fst-13007. Results Agarase-fst was purified to homogeneity which apparent molecular weight was 66.2 kDa. Its activity was optimal at 45 °C and pH 8 and was stable at pH 5–9 or 30–50 °C. Agarase-fst required Mn2+ for agarase activity and inhibition by Cu2+, Fe3+ and EDTA. Tests of hydrolysis pattern and substrate specificity, TLC analysis and mass spectrometry of the hydrolysis products revealed that it is an endo-type β-agarase hydrolyzing agarose into neoagarobiose, neoagarotetraose and neoagarohexaose. Results of MALDI-TOF-TOF/MS indicate that it lack of homology to previously identified proteins and present conserved domain of β-agarase. Conclusion Agarase-fst from T. profundimaris fst-13007 was confirmed to be a novel endo-type β-agarase.

In this work, a novel agarase was firstly purified from a newly isolated agarolytic bacterium, Thalassospira profundimaris.

Materials
DEAE-Sepharose Fast Flow and Sephacryl S-100 (AKTA purifier) were purchased from GE Inc., Sweden. MALDI-TOF-TOF/MS (ABI 5800 plus) were purchased from Applied Biosystems, USA. MS Agilent LC1290-Triple-Q 6410 was purchased from Agilent Technologies, USA. Other chemicals were purchased from Sigma Inc., USA.

Isolation and identification of fst-13007
Seawater sample, collected from the offshore sea of Xiamen (northern latitude 24°27′, east longitude 118°04′, 2.7 % of salinity, 20.9 °C), China, were initially cultivated at 28 °C in a marine medium 2216 contained 1.50 % (w/v) agarose as the single carbon source. After incubation at Open Access *Correspondence: zlongtao@hotmail.com 1 College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian, People's Republic of China Full list of author information is available at the end of the article 37 °C for 24-48 h, colonies that formed depressions on agarose plates were picked out and transferred onto new plates until the colony morphology was unchanged. The plates were stained using Lugol's solution (5 g iodine crystals and 10 g KI in 100 ml distilled water) and agarase activity was visualized by zones of clearing. The colony on the plates showed the highest agarase activity was selected to cultivate for the following investigation. Its 16S rDNA was amplified, sequenced, and compared with the 16S rDNA sequences deposited in the GenBank database with the BLAST program.

Enzyme assay
Agarase activity was measured by determining the amount the reducing sugar according to the DNS method by Miller (1959) with minor modification. Briefly, 50 μl enzymes were mixed with 950 μl Tris/HCl buffer (20 mM, pH 8, buffer A) containing 0.2 % (w/v) agarose as a substrate. After incubation at 45 °C for 30 min, the sample was mixed with 500 μl of 3,5-dinitrosalicylic acid reagent solution. The tubes were heated in boiling water for 10 min, and then cooled in an ice water bath. OD values were measured at 540 nm. The amount of enzyme that released 1 μmol of galactose per min under the assay conditions was defined as one unit (U) of agarase. D-galactose was used as a reference reducing sugar for preparing the standard curve.

Maldi-tof-tof/ms
The agarase band was excised from the SDS-PAGE staining gel. After enzymatic hydrolysis, the agarase was subjected to MALDI-TOF-TOF/MS analysis (ABI 5800 plus) to identify the purified protein. Both the MS and MS/ MS data were integrated and processed with the GPS Explorer V3.6 software (Applied Biosystems, USA) with default parameters. Based on combined MS and MS/MS spectra, proteins were successfully identified based on 95 % or higher confidence interval of their scores in the MASCOT V2.3 search engine (Matrix Science Ltd., London, UK).

Determination of enzymatic characteristics
The optimum temperature of agarase activity was determined in buffer A at different temperatures (20-60 °C). After pre-incubation at temperatures ranging from 20 to 60 °C for 1 h, the temperature stability of enzyme was determined under the defined optimal conditions (45 °C and pH 8). The optimum pH condition was determined at 45 °C using various buffers ranging from pH 3 to 11. For pH stability measurements, the agarase-fst was maintained at 45 °C for 1 h in these buffers before assaying. The effect of metal ions (1 mM), inhibitors (2 mM), and denaturing reagents (2 mM) on enzyme activity was assayed in buffer with various chemicals. All experiments were carried out a minimum of three times.

Determination of substrate specificity
To determine the substrate specificity of agarasefst, p-nitrophenyl-α-d-galactopyranoside and pnitrophenyl-β-d-galactopyranoside were used as artificial chromogenic substrates for enzyme assaying (Chi et al. 2015). The reaction was carried out at 45 °C for 12 h and then stopped by the addition of 1 M Na 2 CO 3 . The release of p-nitrophenol from the hydrolysis of the artificial chromogenic substrate was measured using OD 420 .

Hydrolysis pattern
Kinematic viscosity of the agarase was determined using a NDJ-79 Rotary viscometer (Shanghai Precision Instrument Co. China) and result was shown in Fig. 5.

Enzymatic product analysis
The enzymatic product was analyzed by TLC and the molecular mass distribution was determined using LC-MS/MS. Results were shown in Figs. 6 and 7, respectively.

Isolation and identification of fst-13007
In the primary screening experiment, 4 strains showed agarolytic activity (Fig. 1). The plates were stained using Lugol's solution to check agarolytic ability. The strain A, strain B, strain C and strain D were named fst-13007, fst-13006, fst-13005 and fst-13002, respectively. Fst-13007 formed the biggest hole was selected and identified the highest enzyme activity. Its culture showed agarase activity at 0.84 U ml −1 (Fig. 2).
Its 16S rDNA gene sequence was deposited in Gen-Bank with an Accession Number KU578316, showed 99 % similarity with Thalassospira sp. MCCC 1A00370. The phylogenic relationship with others chosen twelve bacterial species was reconstructed using the neighbourjoining (NJ) tree method. According to the topology, fst-13007 was grouped into the clade composed of annotated T. profundimaris species (Fig. 3). To the best of our knowledge, it is the first time of identifying a strain of T. profundimaris degrading agar, which has been deposited under the number M 2013708 in China Center for Type Culture Collection (CCTCC).

Purification of agarase
The purification of agarase was summarized in Table 1. SDS-PAGE (Fig. 4) revealed that it was successfully purified and appeared on the gel as a single protein band after Sephacry1 S-100. It was purified 18.97-fold with a yield of 13.87 %. The specific activity of purified agarase was 1418 U mg −1 . Its molecular weight was approximately 66.2 kDa (Fig. 4).

Effect of temperature and pH on agarase activities
The temperature dependence of the agarase-fst activity reaches maximum at 45 °C. It was stable up to 45 °C (95 % activity remaining) and retained more than 70 % activity at 50 °C, but was inactivated after 1 h heat treatment at 60 °C (over 10 % remaining) (Fig. 6a). The agarase activity was maximized at pH 8 and remained active at pH values ranging from 4 to 10 (over 49 % remaining) (Fig. 6b), showing good pH stability in neural, mild acid and mild alkali conditions.

Effects of chemicals on agarase activities
As shown in Table 2, the agarase was strongly inhibited by Cu 2+ , Fe 3+ and EDTA. In contrast, it was a little activated by Mn 2+ and Dithiothreitol (DTT). DTT could enhance the activity of agarase which indicated the possible existence of thiol at the catalytic site, as the reducing reagent could protect thiol from being oxidized to a disulfide bond (Fu et al. 2008).

Hydrolysis pattern
The viscosity of reaction mixture significantly decreased from the first 30 min and gradually decreased until the end of the reaction, showing that the agarase-fst is an endo-type agarase (Fig. 7).

Analysis of hydrolysis products
TLC analysis results were shown in Fig. 8. In the beginning of the reaction, the predominant products

Table 1 Purification of agarase-fst from T. profundimaris fst-13007
After 20 min centrifugation at 12,000g and filtration through a 0.45 μm membrane, crude agarase-fst from the culture broth was precipitated by 80 % (w/v) ammonium sulfate saturation and left to stand overnight. After 20 min of centrifugation at 12,000 g, the precipitate was resuspended in 20 mM Tris/HCl buffer (pH 7.5, buffer B) and dialyzed against the same buffer overnight. The dialyzed section was ultrafiltrated through an ultrafilter (5000-10,000 kDa, transmembrane pressure of 0.5 bars). The active fractions were collected and applied onto a DEAE Sepharose Fast Flow (1.6 cm × 10 cm; GE Healthcare) equilibrated with buffer B. The bound proteins were eluted with gradient of 0-1 M NaCl in the same buffer. The flow rate was maintained at 1 ml min −1 . Active fractions were loaded onto Sephacryl S-100 gel column (1.6 cm × 60 cm; GE Healthcare) that had previously been equilibrated with buffer B. The enzyme was eluted with same buffer, at a flow rate of 1 ml min  were NA4and NA6. After 24 h, the NA2 began to be observed. It could be inferred that various lengths of NAOS longer than NA6 were continuously hydrolysed and NAOS with short chain was gradually accumulated. After 48 h, two major spots (NA2 and NA4) and one negligible spot (NA6) were observed. Up to now, where p is the probability that the observed match is a random event. Individual ions scores >64 indicate identity or extensive homology (p < 0.05). Protein scores are derived from ions scores as a non-probabilistic basis for ranking protein hits. b Protein sequence coverage of agarase-fst compared with agarase from Pseudoalteromonas atlantica (gi|1220461). Matched peptides shown in red bold this is the third agarase hydrolyzing agar to NA2 and NA4and NA6, except for another two agarases from Zobellia galactanivorans (Jam et al. 2005) and Microbulbifer Thermotolerans JAMB-A94 (Ohta et al. 2004) respectively.   To determine the molecular mass distribution, the hydrolysis products were analysed by LC-MS/MS. As shown in Fig. 9, peak 1 was revealed to have a m/z ratio of 347 (M + Na) + , which was applicable to NA2, and peak 2 was identified as NA4 [m/z = 653 (M + Na) + ]. The peak 3 with molecular weight of 959 Da corresponded to the molecules of (M + Na) + , which was attributed to NA6 (936 Da). According to the relative peak intensities, hydrolysis products contained 40 % NA2, 53 % NA4 and 7 % NA6.

Conclusions
An external agarase-fst was purified from T. profundimaris and characterized. Based on the biochemical characteristics of the enzyme and lack of homology to previously identified proteins, it can be concluded that the agarase-fst is a novel endo-type β-agarase hydrolyzing agarose into NA2, NA4 and NA6. The bacterium and the agarase will be a new resource for the high valueadded product development in the agar industry. Aliquots of the reaction mixture (10 μl) at 6, 12, 24, and 48 h were spotted onto silica gel 60 plates (Merck, Germany) and developed using a solvent system composed of n-butanol, acetic acid and water (2:1:1, v/v/v). The resulting spots were visualized by spraying with 10 % (v/v) H 2 SO 4 and heating at 95 °C for 10 min. Neoagarobiose (NA2), neoagarotetraose (NA4) and neoagarohexaose (NA6) were used as standards Fig. 9 LC-MS/MS spectrum of the enzymatic products of agarose resulting from agarase-fst. Samples were resuspended with 5 µl 0.1 % (w/v) TFA followed by mixing (1:1, v/v) with a matrix consisting of a saturated solution of α-cyano-4-hydroxy-trans-cinnamic acid in 50 % (w/v) ACN, 0.1 % (w/v) TFA. And 1 μl mixture was spotted on a stainless steel sample target plate. The molecular mass distribution was determined using MS Agilent LC1290-Triple-Q 6410 (Agilent Technologies, USA). Ionization was achieved by atmospheric pressure electrospray ionization in the positive mode with an ESI capillary voltage of 4 kV. The mass spectra were recorded in the range of m/z 50 and1500