Identification and Biochemical Characterization of Sco3487 from Streptomyces coelicolor A3(2), an Exo- and Endo-Type beta-Agarase-Producing Neoagarobiose

Abstract

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 alpha-(1,3) and beta-(1,4) linkages between the 3,6-anhydro-L-galactoses and D-galactoses of agar must be hydrolyzed by alpha/beta-agarases. In S. coelicolor, DagA was confirmed to be an endo-type beta-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 beta-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. beta-Agarase activity was detected in transformant culture broth using the artificial chromogenic substrate p-nitrophenyl-beta-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 degrees C, respectively. The K(m) and V(max) for agarose were 4.87 mg/ml (4 x 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 beta-agarase that degrades agarose, neoagarotetraose, and neoagarohexaose into neoagarobiose.