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 × 10(3)/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.

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 ␣-(1,3) and ␤-(1,4) linkages between the 3,6-anhydro-L-galactoses and D-galactoses of agar must be hydrolyzed by ␣/␤-agarases. In S. coelicolor, DagA was confirmed to be an endo-type ␤-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 ␤-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. ␤-Agarase activity was detected in transformant culture broth using the artificial chromogenic substrate p-nitrophenyl-␤-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 ␤-agarase that degrades agarose, neoagarotetraose, and neoagarohexaose into neoagarobiose.A gar, an important polysaccharide found in the cell walls of the Gracilariales and the Gelidiales red algae, is composed of 2 different components: agarose and agaropectin. Agarose consists of a linear chain of alternating residues of 3-O-linked ␤-Dgalactopyranose and 4-O-linked 3,6-anhydro-␣-L-galactose (7). Because agarose has excellent gelling ability, stabilizing properties, and high viscosity, it has been widely used for food, cosmetic, and pharmaceutical applications (2). Although agaropectin is presumably substituted by sulfate esters, pyruvate acetal, and methyl ethers on similar backbone units, its exact structure is still obscure (7). Recently, efforts have been focused on using terrestrial or marine plant biomasses, and the red alga is a good biomass resource candidate because of its easy cultivation in a broad range of oceans and rapid growth. A biomass must be efficiently degraded by chemical or enzymatic treatment for utilization, and the enzymatic process is preferred because it is more environmentally friendly and produces a smaller amount of toxic by-products than the chemical process.Agarases are classified into 2 groups on the basis of their mode of action: ␣-agarases (EC 3.2.1.158) and ␤-agarases (EC 3.2.1.81). ␤-Agarases hydrolyze ␤-1,4 linkages in agarose and produce neoagaro-oligosaccharides with D-galactose residues at t...

Victivallis vadensis ATCC BAA-548 is a Gram-negative, anaerobic bacterium that was isolated from a human fecal sample. From the genomic sequence of V. vadensis, one gene was found to encode agarase; however, its enzymatic properties have never been characterized. The gene encoding the putative agarase (NCBI reference number ZP_01923925) was cloned by PCR and expressed in E. coli Rosetta-gami by using the inducible T(7) promoter of pET28a(+). The expressed protein with a 6×His tag at the N-terminus was named His6-VadG925 and purified as a soluble protein by Ni(2+)-NTA agarose affinity column chromatography. The purification of the enzyme was 26.8-fold, with a yield of 73.2% and a specific activity of 1.02 U/mg of protein. The purified His6-VadG925 produced a single band with an approximate MW of 155 kDa, which is consistent with the calculated value (154,660 Da) including the 6×His tag. Although VadG925 and many of its homologs were annotated as agarases, it did not hydrolyze agarose. Instead, purified His(6)-VadG925 hydrolyzed an artificial chromogenic substrate, p-nitrophenyl-β-D-galactopyranoside, but not p-nitrophenyl-α-D-galactopyranoside. The optimum pH and temperature for this β-galactosidase activity were pH 7.0 and 40°C, respectively. The K(m) and V(max) of His6-VadG925 towards p-nitrophenyl-β-D-galactopyranoside were 1.69 mg/ml (0.0056 M) and 30.3 U/mg, respectively. His6-VadG925 efficiently hydrolyzed lactose into glucose and galactose, which was demonstrated by TLC and mass spectroscopy. These results clearly demonstrated that VadG925 is a novel β-galactosidase that can hydrolyze lactose, which is unusual because of its low homology to validated β-galactosidases.

The sco6545 gene of Streptomyces coelicolor A3(2) was nominated as a putative cellulase with 863 mature-form amino acids (90.58 kDa). We overexpressed and purified Sco6545 and demonstrated that the protein is not a cellulase but a xyloglucanspecific glycosyl hydrolase which cleaves xyloglucan at unbranched glucose residues.

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