Identification and biochemical characterization of Sco3487 from Streptomyces coelicolor A3(2), an exo- and endo-type β-agarase-producing neoagarobiose.
Streptomyces coelicolor can degrade agar, the main cell wall component of red macroalgae, for growth. To constitute a crucial carbon source for bacterial growth, the alternating α-(1,3) and β-(1,4) linkages between the 3,6-anhydro-L-galactoses and D-galactoses of agar must be hydrolyzed by α/β-agarases. In S. coelicolor, DagA was confirmed to be an endo-type β-agarase that degrades agar into neoagarotetraose and neoagarohexaose. Genomic sequencing data of S. coelicolor revealed that Sco3487, annotated as a putative hydrolase, has high similarity to the glycoside hydrolase (GH) GH50 β-agarases. Sco3487 encodes a primary translation product (88.5 kDa) of 798 amino acids, including a 45-amino-acid signal peptide. The sco3487 gene was cloned and expressed under the control of the ermE promoter in Streptomyces lividans TK24. β-Agarase activity was detected in transformant culture broth using the artificial chromogenic substrate p-nitrophenyl-β-D-galactopyranoside. Mature Sco3487 (83.9 kDa) was purified 52-fold with a yield of 66% from the culture broth. The optimum pH and temperature for Sco3487 activity were 7.0 and 40°C, respectively. The K(m) and V(max) for agarose were 4.87 mg/ml (4 × 10(-5) M) and 10.75 U/mg, respectively. Sco3487 did not require metal ions for its activity, but severe inhibition by Mn(2+) and Cu(2+) was observed. Thin-layer chromatography analysis, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and Fourier transform-nuclear magnetic resonance spectrometry of the Sco3487 hydrolysis products revealed that Sco3487 is both an exo- and endo-type β-agarase that degrades agarose, neoagarotetraose, and neoagarohexaose into neoagarobiose.
Division of Bioscience and Bioinformatics, Myongji University, Yongin, Gyeonggi-do, South Korea.
Uyangaa
U
Temuujin
Won-Jae
WJ
Chi
Yong-Keun
YK
Chang
Soon-Kwang
SK
Hong
D000362:Agar / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism*; D005697:Galactosides / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D006026:Glycoside Hydrolases / Q000145:classification / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism; D012685:Sepharose / Q000378:metabolism; D048372:Streptomyces coelicolor / Q000235:genetics* / Q000378:metabolism*; D048170:Streptomyces lividans / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004187:Disaccharides; D005697:Galactosides; D009844:Oligosaccharides; C081798:neoagarotetraose; C068544:neoagarobiose; D000362:Agar; D012685:Sepharose; D006026:Glycoside Hydrolases; C019624:agarase