Preliminary characterization of a novel β-agarase from Thalassospira profundimonas
© The Author(s) 2016
Received: 30 March 2016
Accepted: 4 July 2016
Published: 15 July 2016
The objective of this study was to characterize the agarase from a newly isolated agarolytic bacterium Thalassospira profundimaris fst-13007.
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.
Agarase-fst from T. profundimaris fst-13007 was confirmed to be a novel endo-type β-agarase.
KeywordsAgarase Characterization Purification Thalassospira profundimaris
Agar, composed of agaropectin and agarose, is the main component in cell wall of the red algae. Oligosaccharides derived from agar have many useful properties, such as delayed starch degradation, bacterial growth inhibition, anticancer and antioxidation activities, etc. (Seok et al. 2012). Agarases are glycoside hydrolases (GHs) that catalyse the degrading of agarose, which is classified into β-agarase (EC 22.214.171.124), α-agarases (E.C.126.96.36.199), and β-porphyranases (EC 3.2.1.-) (Fu and Kim 2010). β-agarases hydrolyze agarose at the β-1,4 linkages into series of neoagarooligosaccharides (NAOS) with D-galactose at the reducing end. Nearly 50 agarases have been characterized, and most of them are β-agarases. α-agarases hydrolyze agarose at the α-1, 3 linkages into agarooligosaccharides with 3,6-anhydro-l-galactose at the reducing end (Potin et al. 1993). β-Porphyranases hydrolyze porphyrobiose at the β-1,4 linkages into series of porphyrooligosaccharides with D-galactose at the reducing end (Hehemann et al. 2010). A considerable number of agar-degrading enzymes from 34 microorganisms have been characterised (Chi et al. 2012). While, to the best of our knowledge, only β-agarase from Pseudomonas atlanticacould be purchased in the market, such as from Sigma.
In this work, a novel agarase was firstly purified from a newly isolated agarolytic bacterium, Thalassospira profundimaris.
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 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.
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.
The strain fst-13007 was cultured in 1.5 l medium (NaCl 3 %(w/v), KCl 0.1 %(w/v), CaCl2 0.01 %(w/v), MgSO4 0.05 %(w/v), FeSO4 0.002 %(w/v), Fe2(SO4)3 0.001 %(w/v), tryptone 0.50 %(w/v), yeast extract 0.10 %(w/v), pH 8) supplemented with 0.2 % (w/v) agarose as the sole carbon source at 37 °C for 48 h. The purification was applied to 80 % (w/v) saturation (NH4)2SO4 precipitation, ultrafiltration, DEAE Fast Flow, and Sephacryl S-100 gel column sequentially. Fractions with agarase activity were collected and subjected to enzyme activity assay and electrophoresis (SDS-PAGE).
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 agarase-fst, p-nitrophenyl-α-d-galactopyranoside and p–nitrophenyl-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 Na2CO3. The release of p-nitrophenol from the hydrolysis of the artificial chromogenic substrate was measured using OD420.
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
Results and discussion
Isolation and identification of fst-13007
Purification of agarase
Purification of agarase-fst from T. profundimaris fst-13007
Total protein (mg)
Total activity (U)
Specific activity (U mg−1)
Crude enzyme solution
Effect of temperature and pH on agarase activities
Effects of chemicals on agarase activities
Effect of chemicals on the relative activities of agarase-fst
Relative activity (%)
Metal ions (1 mM)
92.94 ± 1.65
93.29 ± 0.57
95.56 ± 1.01
43.77 ± 1.34
31 ± 0.38
162.54 ± 2.85
97.35 ± 0.93
Other reagents (2 mM)
94.20 ± 2.04
86.88 ± 1.67
134.67 ± 1.13
100.42 ± 0.62
The test of Substrate specificity of agarase-fst showed strong hydrolysis activity (OD540 = 0.87) toward p-nitrophenyl-β-d-galactopyranoside, but negligible activity (OD540 = 0.06) toward p-nitrophenyl-α-d-galactopyranoside, indicating that it cleavage the β-linkage but not the α-linkage. Results suggest that agarase-fst is a β-agarase (Chi et al. 2015), which is consistent with the MALDI-TOF-TOF/MS results stated above.
Analysis of hydrolysis products
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 value-added product development in the agar industry.
LZ conceived of the study and the experimental design and contributed to the manuscript. CZ, SM, YZ, SZ and BZ contributed to the experiments, and CZ contributed to the data analysis. All authors read and approved the final manuscript.
We gratefully acknowledge the financial support from “Regional Demonstration of Marine Economy Innovative Development Project (No. 12PYY001SF08); the Science and Technology Plan of Fujian Province (2014I0008, 2015NZ01010013); China Scholarship for Visiting Scholar 3012.
The authors declare that they have no competing interests.
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