Identification and Biochemical Characterization of Sco3487 from Streptomyces coelicolor A3(2), an Exo- and Endo-Type  -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 ␣-(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...

Identification and biochemical characterization of a novel endo-type β-agarase AgaW from Cohnella sp. strain LGH

“…Agarolytic activity has also been rarely observed in terrestrial bacteria, such as Paenibacillus (Hosoda et al 2003), Pseudomonas (Song et al 2015, and Streptomyces (Temuujin et al 2012). Here, we reported the isolation of a Gram-positive soil bacterium, Cohnella sp.…”

“…The chemical shifts of the anomeric carbon signals in the spectrum were identical to those of a typical pattern for neoagarooligosaccharides (Fu et al 2008;Rochas et al 1986;Temuujin et al 2012).The resonances at 92.360 and 96.182 ppm were signals for the α-and β-anomeric forms of the galactose unit at the reducing end of the neoagarooligosaccharides, respectively. No resonance was detected at 90.8 ppm, which was the characteristic signal of the reducing end of the hydrated form of 3,6-anhydro-L-galactose of the agarooligosaccharides (Fu et al 2008;Rochas et al 1994;Temuujin et al 2012). Thus, the dimer, tetramer, and hexamer agarolytic products were identified as neoagarobiose, neoagarotetraose, and neoagarohexaose, respectively.…”

Agar degradation by microorganisms and agar-degrading enzymes

“…In silico analysis predicted the absence of a signal peptide in Sco3481. When expressed in S. lividans TK24, the protein was present only in the cytosol, quite distinct from the behavior of the two extracellular β-agarases DagA and DagB (Temuujin et al 2011(Temuujin et al , 2012. The purified Sco3481 hydrolyzed neoagarobiose to G and LA and released LA from the non-reducing ends of neoagarotetraose and neoagarohexaose was released in the same way as Aga117F from S. degradans 2-40 (unpublished data).…”

“…Biochemical study of mature DagB (83.9 kDa) revealed that it is both an exo-and endo-β-agarase that degrades agarose, neoagarotetraose, and neoagarohexaose into neoagarobiose. In addition, agarose hydrolysis to neoagarobiose was synergistically enhanced by coincubation of the substrate with DagA and DagB, implying that the two enzymes can hydrolyze agarose in a cooperative way (Temuujin et al 2012).…”

Marine enzymes and food industry: insight on existing and potential interactions

“…Typically, β-agarases are divided into types I and II, depending on substrate specificity and product profile. Thus, type I cleaves agarose into neoagarotetraose, while the major product of type II is neoagarobiose (Temuujin et al, 2012). Several reports on the identification and characterization of β-agarases have been presented recently, that highlight the topicality of this matter.…”