Enzymatic Properties and Nucleotide and Amino Acid Sequences of a Thermostable β-Agarase from the Novel Marine Isolate, JAMB-A94

Ohta, Yutaka; Nogi, Yuichi; Miyazaki, Masaru; Li, Zhijun; Hatada, Yuji; Ito, Susumu; Horikoshi, Koki

doi:10.1271/bbb.68.1073

Cited by 79 publications

(57 citation statements)

References 23 publications

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“…Specifically, this involves hydrolysis of ␤-1,4-glycosidic bonds by ␤-agarases and the production of neoagaro-oligosaccharides that have various degrees of polymerization. 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%

“…In contrast, ␤-agaraseproducing bacteria have been reported in several taxonomically diverse genera, including Cytophaga (32), Pseudomonas (10), Vibrio (17), Alteromonas (33), and Agarivorans (21). On the basis of amino acid sequence similarity, ␤-agarases are found in the CAZY database in 3 distinct families of GHs: GH16, GH50, and GH86 (22). Of these, GH16 is the largest family, composed of more than 1,700 functionally heterogeneous members, including endogalactosidases, endoglucanases, -carrageenases, lichenases, and xyloglucanases.…”

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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%

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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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“…Thin-layer chromatography (TLC) analysis of the agarose hydrolysis products of AgaV was performed as described by Ohta et al (19). The hydrolysis reactions were carried out at 40°C in 50 mM sodium phosphate buffer (pH 7.0) containing the purified agarase and 0.25% substrate.…”

Section: Methodsmentioning

confidence: 99%

Cloning, Characterization, and Molecular Application of a Beta-Agarase Gene fromVibriosp. Strain V134

Zhang

Sun

2007

Appl Environ Microbiol

116

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V134, a marine isolate of the Vibrio genus, was found to produce a new beta-agarase of the GH16 family. The relevant agarase gene agaV was cloned from V134 and conditionally expressed in Escherichia coli. Enzyme activity analysis revealed that the optimum temperature and pH for the purified recombinant agarase were around 40°C and 7.0. AgaV was demonstrated to be useful in two aspects: first, as an agarolytic enzyme, the purified recombinant AgaV could be employed in the recovery of DNA from agarose gels; second, as a secretion protein, AgaV was explored at the genetic level and used as a reporter in the construction of a secretion signal trap which proved to be a simple and efficient molecular tool for the selection of genes encoding secretion proteins from both gram-positive and gram-negative bacteria.Agarose is a type of polysaccharide produced by some species of red-purple marine algae and functions as a component of the cell wall. Agar has been extensively used as a common gelling substance in microbiological culturing media and as an ingredient stabilizer in the food industry. In addition to these classical applications, recent research has discovered various new biological and therapeutic properties in many agar derivatives and hence the potential for their application in the medicinal and pharmaceutical industries (32,33). Chemically, agarose is made up of subunits of galactose that form a polymer through alternating 1,3-linked ␤-D-galactopyranose and 1,4-linked 3,6-anhydro-␣-L-galactopyranose; these linkages can be specifically cleaved by certain agarolytic enzymes known as agarases, which are a group of glycoside hydrolases (GH) that catalyze the degradation of agarose polysaccharide into neoagarooligosaccharides. Based on the mechanisms of their actions, agarases are classified as alpha-agarases, which cleave the ␣-1,3-galactosidic linkages in agarose, and betaagarases, which cleave the ␤-1,4 linkages. On the basis of primary sequences, a number of families have been set up under the category of GH, and except perhaps in one case (3), families GH16, GH50, and GH86 encompass all the beta-agarases that have been characterized at the sequence level. Of these three families of beta-agarases, GH16 is the largest and most heterogeneous group, with members differing from one another in substrate specificity, which has served as the basis for the redivision of the GH16 beta-agarases into several subfamilies (1,7,8).Agarases are the natural products of certain agar-degrading organisms found mostly in marine habitats, which is consistent with the fact that agar, being a product of marine algae, is available to and utilized by some marine organisms as a convenient carbon and energy source. Except in a few cases, most of these agarolytic organisms are bacteria. To date, agaraseproducing bacteria of many different genera have been found, including species of Alteromonas (14, 16, 21, 31), Asticcacaulis (9), Bacillus (13, 28), Pseudomonas (6, 11), Pseudoalteromonas (24), Streptomyces (12), and Vibrio (27). However, ...

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“…This enzyme is capable of digesting sunken wooden debris and might be useful for the production of bio-ethanol from raw materials [13]. Thermostable b-agarase has been isolated from a heat-resistant microorganism from a deep-sea mud sample [14]. This commercially available agarase exhibits thermal resistance and high agarolytic activity.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of the bioactivities of water-soluble extracts from twelve deep-sea jellyfish species

et al. 2013

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Many polypeptides isolated from shallow water cnidarian species have been utilized as valuable biochemical tools in both basic and applied biological sciences. Deepwater cnidarian species might be another potential resource for novel biochemical tools. However, because of limited access to cnidarian samples from deep-sea environments, bioactive polypeptides have never before been reported from this group. In this study, we collected twelve deep-sea jellyfish species (nine hydrozoans and three scyphozoans) using a plankton net that was specially designed for collecting deep-sea organisms, and prepared water-soluble extracts, presumably containing polypeptides, of these jellyfishes. The extracts were subjected to cytotoxicity, hemolytic activity, and crustacean lethal toxicity tests. In the cytotoxicity test, six out of the nine tested hydrozoan species showed activity. In the hemolytic activity test, only three hydrozoans showed activity and none of the scyphozoan jellyfishes showed activity. In the crustacean lethality test, two hydrozoan jellyfishes and all three of the tested scyphozoan jellyfishes showed lethal activity. These results revealed a high incidence of watersoluble bioactive substances occurring in these deepsea jellyfishes. Furthermore, all the heat-treated and the methanol-treated crude jellyfish extracts lost their bioactivities. Thus, it is likely that the bioactive compounds in the water-soluble extracts were unstable polypeptides (proteins). This is the first published report on bioactivities in extracts from deep-sea jellyfishes.

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Enzymatic Properties and Nucleotide and Amino Acid Sequences of a Thermostable β-Agarase from the Novel Marine Isolate, JAMB-A94

Cited by 79 publications

References 23 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

Cloning, Characterization, and Molecular Application of a Beta-Agarase Gene fromVibriosp. Strain V134

Evaluation of the bioactivities of water-soluble extracts from twelve deep-sea jellyfish species

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