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Preliminary characterization of a novel β-agarase from Thalassospira profundimonas

SpringerPlus (2016) 5(1)
DOI: 10.1186/s40064-016-2748-6
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Abstract

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.

Figures

  • Fig. 1 Lugol’s dyeing plate of agar-degrading strains
  • Fig. 2 Agarase activity of four agar-degrading strains
  • Fig. 3 Phylogenetic analysis of strain fst-13007. The 16S rDNA sequence of strain fst-13007 was compared with the 16S rDNA sequences deposited in the GenBank database using the BLAST program. The phylogenetic tree of fst-13007 was made by the MEGA 5.0 and Clustalx software, using the neighbour-joining (NJ) tree method by analysing their 16S rDNA sequences. The numbers at the nodes show the bootstrap values obtained from 1000 resampling analyses
  • Table 1 Purification of agarase-fst from T. profundimaris fst-13007
  • Fig. 4 SDS-PAGE of the purified agarase-fst. Lane M Standard molecular mass markers; Lane 1 agarase-fst after DEAE-Sepharose fast flow; Lane 2, agarase-fst after Sephacry1 S-100
  • Fig. 5 Mascot Score Histogram and Protein sequence coverage of agarase-fst. a Mascot Score Histogram of agarase-fst. Ions score is −10 × Log (p), 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
  • Table 2 Effect of  chemicals on  the relative activities of agarase-fst
  • Fig. 6 Effects of temperature and pH on the activity and stability of agarase-fst. a The effect of temperature. The agarase activity assaying was performed at pH 8 (20 mM Tris/HCl) with DNS method at different temperatures (20–60 °C). The maximum value (1.04 U ml−1) observed at 45 °C was considered 100 %. Thermostability of the enzyme was determined at 45 °C after preincubated at different temperatures (20–60 °C) for 1 h. The highest activity obtained was taken to be 100 %. Filled circles, optimum temperature; open squares, thermostability. b The effect of pH. The agarase activity assaying was performed at 45 °C at different pH conditions. The highest activity (0.96 U ml−1) obtained at pH 8 was considered 100 %. 20 mM sodium acetate buffer (pH 3–6), 20 mM Tris/HClbuffer (pH 7–9) and 20 mM glycine–NaOH buffer (pH 10). Filled circles, optimum pH; open squares pH stability

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APA

Zeng, C., Zhang, L., Miao, S., Zhang, Y., Zeng, S., & Zheng, B. (2016). Preliminary characterization of a novel β-agarase from Thalassospira profundimonas. SpringerPlus, 5(1). https://doi.org/10.1186/s40064-016-2748-6

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