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- Title
Cloning, expression, and biochemical characterization of a novel GH16 β-agarase AgaG1 from Alteromonas sp. GNUM-1.
- Authors
Chi, Won-Jae; Park, Da; Seo, Young; Chang, Yong; Lee, Soon-Youl; Hong, Soon-Kwang
- Abstract
Alteromonas sp. GNUM-1 is known to degrade agar, the main cell wall component of red macroalgae, for their growth. A putative agarase gene ( agaG1) was identified from the mini-library of GNUM-1, when extracellular agarase activity was detected in a bacterial transformant. The nucleotide sequence revealed that AgaG1 had significant homology to GH16 agarases. agaG1 encodes a primary translation product (34.7 kDa) of 301 amino acids, including a 19-amino-acid signal peptide. For intracellular expression, a gene fragment encoding only the mature form (282 amino acids) was cloned into pGEX-5X-1 in Escherichia coli, where AgaG1 was expressed as a fusion protein with GST attached to its N-terminal (GST-AgaG1). GST-AgaG1 purified on a glutathione sepharose column had an apparent molecular weight of 59 kDa on SDS-PAGE, and this weight matched with the estimated molecular weight (58.7 kDa). The agarase activity of the purified protein was confirmed by the zymogram assay. GST-AgaG1 could hydrolyze the artificial chromogenic substrate, p-nitrophenyl-β- d-galactopyranoside but not p-nitrophenyl-α- d-galactopyranoside. The optimum pH and temperature for GST-AgaG1 activity were identified as 7.0 and 40 °C, respectively. GST-AgaG1 was stable up to 40 °C (100 %), and it retained more than 70 % of its initial activity at 45 °C after heat treatment for 30 min. The K and V for agarose were 3.74 mg/ml and 23.8 U/mg, respectively. GST-AgaG1 did not require metal ions for its activity. Thin layer chromatography analysis, mass spectrometry, and C-nuclear magnetic resonance spectrometry of the GST-AgaG1 hydrolysis products revealed that GST-AgaG1 is an endo-type β-agarase that hydrolyzes agarose and neoagarotetraose into neoagarobiose.
- Subjects
CLONING; ALTEROMONAS; BACTERIAL transformation; CHIMERIC proteins; NUCLEAR magnetic resonance; NUCLEOTIDE sequence; HYDROLYSIS; AGARASE
- Publication
Applied Microbiology & Biotechnology, 2014, Vol 98, Issue 10, p4545
- ISSN
0175-7598
- Publication type
Article
- DOI
10.1007/s00253-014-5510-4