Purification and Characterization of an Extracellular .ALPHA.-Neoagarooligosaccharide Hydrolase from Bacillus sp. MK03.

Suzuki, Hisashi; Sawai, Yoshinori; Suzuki, Tohru; Kawai, Keiichi

doi:10.1263/jbb.93.456

Cited by 10 publications

(16 citation statements)

References 29 publications

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“…The major products of ␤-agarases are neoagarohexaose (22) and neoagarotetraose, which are further cleaved to neoagarobiose (8). Finally, the ␣-neoagarobiose hydrolase cleaves neoagarobiose into the monomeric sugars D-galactose and 3,6-anhydro-␣-L-galactose, which may be subsequently metabolized in the cell by an unknown metabolic pathway (30).…”

Section: Discussionmentioning

confidence: 99%

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

Temuujin

Chi

Chang

et al. 2011

Journal of Bacteriology

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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...

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Section: Discussionmentioning

confidence: 99%

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

Temuujin

Chi

Chang

et al. 2011

Journal of Bacteriology

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“…2, left). While many scientists have tried to investigate the α-neoagarobiose hydrolase (NABH) responsible for the reaction, only a few biochemical experiments have been able to demonstrate the activity of NABH in agarolytic microorganisms (van der Meulen and Harder 1976;Suzuki et al 2002;Day and Yaphe 1975;Sugano et al 1994). Although genetic information for NABHs is not available, NABH can be categorized into two biological classes depending on its cellular location: cytosolic NABH (Sugano et al 1994;van der Meulen and Harder 1976) and extracellular or periplasmic NABH (Suzuki et al 2002;Day and Yaphe 1975).…”

Section: Enzymology Of Agar Degradationmentioning

confidence: 99%

Agar degradation by microorganisms and agar-degrading enzymes

Chi

Chang

Hong

2012

Appl Microbiol Biotechnol

224

143

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Agar is a mixture of heterogeneous galactans, mainly composed of 3,6-anhydro-L-galactoses (or L-galactose-6-sulfates) D-galactoses and L-galactoses (routinely in the forms of 3,6-anhydro-L-galactoses or L-galactose-6-sulfates) alternately linked by β-(1,4) and α-(1,3) linkages. It is a major component of the cell walls of red algae and has been used in a variety of laboratory and industrial applications, owing to its jellifying properties. Many microorganisms that can hydrolyze and metabolize agar as a carbon and energy source have been identified in seawater and marine sediments. Agarolytic microorganisms commonly produce agarases, which catalyze the hydrolysis of agar. Numerous agarases have been identified in microorganisms of various genera. They are classified according to their cleavage pattern into three types-α-agarase, β-agarase, and β-porphyranase. Although, in a broad sense, many other agarases are involved in complete hydrolysis of agar, most of those identified are β-agarases. In this article we review agarolytic microorganisms and their agar-hydrolyzing systems, covering β-agarases as well as α-agarases, α-neoagarobiose hydrolases, and β-porphyranases, with emphasis on the recent discoveries. We also present an overview of the biochemical and structural characteristics of the various types of agarases. Further, we summarize and compare the agar-hydrolyzing systems of two specific microorganisms: Gram-negative Saccharophagus degradans 2-40 and Gram-positive Streptomyces coelicolor A3(2). We conclude with a brief discussion of the importance of agarases and their possible future application in producing oligosaccharides with various nutraceutical activities and in sustainably generating stock chemicals for biorefinement and bioenergy.

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“…Neoagarobiose hydrolase (NABH) is known to hydrolyze the -(1,3)-linkage in neoagarobiose, yielding the monomeric sugars -d-galactose and 3,6-anhydro--l-galactose, and is regarded as a essential enzyme in bacteria that use agar as a sole carbon source (Ekborg et al, 2006). There have been several studies of the biochemical activity of NABH, but no information at the molecular level such as the sequence, structure or molecular mechanism of the protein is available (Day & Yaphe, 1975;Sugano et al, 1994;Suzuki et al, 2002). Here, we report preliminary crystallization studies of NABH, which will provide information on the saccharification of AP for bioenergy production and help in understanding the metabolic fate of AP in agarolytic bacteria.…”

Section: Introductionmentioning

confidence: 99%

Crystallization and preliminary X-ray analysis of neoagarobiose hydrolase fromSaccharophagus degradans2-40

Lee

et al. 2009

Acta Cryst Sect F

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Many agarolytic bacteria degrade agar polysaccharide into the disaccharide unit neoagarobiose [O-3,6-anhydro-alpha-L-galactopyranosyl-(1-->3)-D-galactose] using various beta-agarases. Neoagarobiose hydrolase is an enzyme that acts on the alpha-1,3 linkage in neoagarobiose to yield D-galactose and 3,6-anhydro-L-galactose. This activity is essential in both the metabolism of agar by agarolytic bacteria and the production of fermentable sugars from agar biomass for bioenergy production. Neoagarobiose hydrolase from the marine bacterium Saccharophagus degradans 2-40 was overexpressed in Escherichia coli and crystallized in the monoclinic space group C2, with unit-cell parameters a = 129.83, b = 76.81, c = 90.11 A, beta = 101.86 degrees . The crystals diffracted to 1.98 A resolution and possibly contains two molecules in the asymmetric unit.

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Purification and Characterization of an Extracellular .ALPHA.-Neoagarooligosaccharide Hydrolase from Bacillus sp. MK03.

Cited by 10 publications

References 29 publications

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

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

Agar degradation by microorganisms and agar-degrading enzymes

Crystallization and preliminary X-ray analysis of neoagarobiose hydrolase fromSaccharophagus degradans2-40

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