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Applied Microbiology and Biotechnology

, Volume 92, Issue 4, pp 749–759 | Cite as

Overexpression and biochemical characterization of DagA from Streptomyces coelicolor A3(2): an endo-type β-agarase producing neoagarotetraose and neoagarohexaose

  • Uyangaa Temuujin
  • Won-Jae Chi
  • Soon-Youl Lee
  • Yong-Keun Chang
  • Soon-Kwang Hong
Biotechnologically Relevant Enzymes and Proteins

Abstract

The DagA product of Streptomyces coelicolor is an agarase with a primary translation product (35 kDa) of 309 amino acids, including a 30-amino acid signal peptide. Although dagA expression in Streptomyces lividans under the control of its own set of promoters was previously reported, its enzymatic properties have never been elucidated. To develop an improved expression system for dagA, three types of strong promoters for the Streptomyces host were linked to dagA, and their efficiencies in DagA production were compared in S. lividans TK24. All of the transformants with dagA grew at improved rates and produced larger amounts of DagA in the modified R2YE medium containing 0.5% agar as the sole carbon source. Of the three transformants, the S. lividans TK24/pUWL201-DagA (ermE promoter) produced the highest agarase activity (A 540 = 4.24), and even the S. lividans TK24/pHSEV1-DagA (tipA promoter) and S. lividans TK24/pWHM3-DagA (sprT promoter) produced higher agarase activity (A 540 = 0.24 and 0.12, respectively) than the control (A 540 = 0.01) in the modified R2YE medium. The mature form of DagA protein (32 kDa) was successfully purified by one-step affinity column chromatography by using agarose beads with excellent yield. The purified DagA was found to exhibit maximal agarase activity at 40°C and pH 7.0. The K m, V max, and K cat values for agarose were 2.18 mg/ml (approximately 1.82 × 10−5 M), 39.06 U/mg of protein, and 9.5 × 103/s, respectively. Thin layer chromatography (TLC) analysis, matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry, and Fourier transform nuclear magnetic resonance (FT-NMR) spectrometry of the hydrolyzed products of agarose by DagA revealed that DagA is an endo-type β-agarase that degrades agarose into neoagarotetraose and neoagarohexaose.

Keywords

Streptomyces coelicolor DagA β-Agarase Neoagarotetraose Neoagarohexaose 

Notes

Acknowledgment

This work was supported by a grant no. SA00004146 from the Next-Generation BioGreen 21 Program, Rural Development Administration, Republic of Korea.

Supplementary material

253_2011_3347_MOESM1_ESM.docx (168 kb)
S1 13C-NMR spectrum of the hydrolyzed product of agarose produced by DagA (DOCX 168 kb)
253_2011_3347_MOESM2_ESM.docx (639 kb)
S2 TLC chromatogram depending on the reaction time. The hydrolysis reactions were carried out at 40°C for 60 min in 50 mM sodium phosphate buffer (pH 7.0) containing 0.3% agarose to measure changes in the viscosity. Simultaneously, the reaction samples were separated on a Silica Gel 60 TLC plate. G, D-(+)-galactose; G2, 4-β-galactobiose; G4, 3α4β3α-galactotetraose (DOCX 639 kb)

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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Uyangaa Temuujin
    • 1
  • Won-Jae Chi
    • 1
  • Soon-Youl Lee
    • 2
  • Yong-Keun Chang
    • 3
  • Soon-Kwang Hong
    • 1
  1. 1.Division of Bioscience and BioinformaticsMyongji UniversityYonginSouth Korea
  2. 2.Department of BiotechnologyHankyong National UniversityAnseongSouth Korea
  3. 3.Department of Chemical and Biomolecular EngineeringKorea Advanced Institute of Science and TechnologyTaejonSouth Korea

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