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Kousaku Murata - One of the best experts on this subject based on the ideXlab platform.
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Comparative characterization of three bacterial exo-type alginate lyases.
International Journal of Biological Macromolecules, 2016Co-Authors: Makoto Hirayama, Wataru Hashimoto, Kousaku Murata, Shigeyuki KawaiAbstract:Alginate, a major acidic polysaccharide in brown macroalgae, has attracted attention as a carbon source for production of ethanol and other chemical compounds. Alginate is monomerized by exo-type alginate lyase into an unsaturated uronate; thus, this enzyme is critical for the saccharification and utilization of alginate. Although several exo-type alginate lyases have been characterized independently, their activities were not assayed under the same conditions or using the same unit definition, making it difficult to compare enzymatic properties or to select the most suitable enzyme for saccharification of alginate. In this study, we characterized the three bacterial exo-type alginate lyases under the same conditions: A1-IV of Sphingomonas sp. strain A1, Atu3025 of Agrobacterium tumefaciens, and Alg17c of Saccharophagus degradans. A1-IV had the highest specific activity as well as the highest productivity of uronate, whereas Alg17c had the lowest activity and productivity. Only dialyzed Atu3025 and Alg17c were tolerant to freezing. Alg17c exhibited a remarkable halotolerance, which may be advantageous for monomerization of alginate from marine brown algae. Thus, each enzyme exhibited particular desirable and undesirable properties. Our results should facilitate further utilization of the promising polysaccharide alginate.
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a biosystem for alginate metabolism in agrobacterium tumefaciens strain c58 molecular identification of atu3025 as an exotype family pl 15 alginate lyase
Research in Microbiology, 2006Co-Authors: Akihito Ochiai, Wataru Hashimoto, Kousaku MurataAbstract:The Gram-negative bacterium Sphingomonas sp. strain A1 (strain A1) has a peculiar biosystem to directly import and depolymerize a macromolecule, alginate, which is encoded by a cluster of genes on the genome. We identified five clustered ORFs homologous to some genes of the strain A1 cluster in the genome of Agrobacterium tumefaciens strain C58 (strain C58). These ORFs are Atu3021, Atu3022, Atu3023, and Atu3024, encoding a putative sugar ABC transporter system and Atu3025, which encodes a putative alginate lyase. We analyzed the involvement of this gene cluster in alginate metabolism. Strain C58 cells grew significantly on low-molecular-weight (LMW) alginate (average molecular weight, 1000), and we detected specific alginate-induced expression of Atu3024 and Atu3025. This strain does not grow on alginate (average molecular weight, 25 600), suggesting that the strain C58 gene cluster is involved in importing and degrading LMW alginate. One protein, Atu3025, purified from strain C58, was identified as an alginate lyase, and the enzyme overexpressed in Escherichia coli was further characterized. Atu3025 released monosaccharides specifically from alginate most efficiently at pH 7.3 and 30 °C through a β-elimination reaction, indicating that Atu3025 is an exotype alginate lyase potentially involved in the assimilation of LMW alginate in strain C58.
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direct evidence for sphingomonas sp a1 periplasmic proteins as macromolecule binding proteins associated with the abc transporter molecular insights into alginate transport in the periplasm
Biochemistry, 2005Co-Authors: Keiko Momma, Yumiko Mishima, Wataru Hashimoto, Bunzo Mikami, Kousaku MurataAbstract:A Gram-negative bacterium, Sphingomonas sp. A1, has a macromolecule (alginate) import system consisting of a pit on the cell surface and an alginate-specific ATP-binding cassette importer in the inner membrane. Transport of alginate from the pit to the ABC importer is probably mediated by two periplasmic binding protein homologues (AlgQ1 and AlgQ2). Here we describe characteristics of binding of AlgQ1 and AlgQ2 to alginate and its oligosaccharides through surface plasmon resonance biosensor analysis, UV absorption difference spectroscopy, and X-ray crystallography. Both AlgQ1 and AlgQ2 were inducibly expressed in the periplasm of alginate-grown cells of strain A1. Biosensor analysis indicated that both proteins specifically bind alginate with a high degree of polymerization (>100) and that dissociation constants for alginate with an average molecular mass of 26 kDa are 2.3 × 10-7 M for AlgQ1 and 1.5 × 10-7 M for AlgQ2. An in vitro ATPase assay using the membrane complex, including the alginate ABC importe...
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molecular identification of oligoalginate lyase of sphingomonas sp strain a1 as one of the enzymes required for complete depolymerization of alginate
Journal of Bacteriology, 2000Co-Authors: Wataru Hashimoto, Keiko Momma, Osamu Miyake, Shigeyuki Kawai, Kousaku MurataAbstract:A bacterium, Sphingomonas sp. strain A1, can incorporate alginate into cells through a novel ABC (ATP-binding cassette) transporter system specific to the macromolecule. The transported alginate is depolymerized to di- and trisaccharides by three kinds of cytoplasmic alginate lyases (A1-I [66 kDa], A1-II [25 kDa], and A1-III [40 kDa]) generated from a single precursor through posttranslational autoprocessing. The resultant alginate oligosaccharides were degraded to monosaccharides by cytoplasmic oligoalginate lyase. The enzyme and its gene were isolated from the bacterial cells grown in the presence of alginate. The purified enzyme was a monomer with a molecular mass of 85 kDa and cleaved glycosidic bonds not only in oligosaccharides produced from alginate by alginate lyases but also in polysaccharides (alginate, polymannuronate, and polyguluronate) most efficiently at pH 8.0 and 37°C. The reaction catalyzed by the oligoalginate lyase was exolytic and thought to play an important role in the complete depolymerization of alginate in Sphingomonas sp. strain A1. The gene for this novel enzyme consisted of an open reading frame of 2,286 bp encoding a polypeptide with a molecular weight of 86,543 and was located downstream of the genes coding for the precursor of alginate lyases (aly) and the ABC transporter (algS, algM1, and algM2). This result indicates that the genes for proteins required for the transport and complete depolymerization of alginate are assembled to form a cluster.
Mansoor M Amiji - One of the best experts on this subject based on the ideXlab platform.
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enzyme immobilization in novel alginate chitosan core shell microcapsules
Biomaterials, 2004Co-Authors: Ehab S Taqieddin, Mansoor M AmijiAbstract:Abstract Alginate–chitosan core-shell microcapsules were prepared in order to develop a biocompatible matrix for enzyme immobilization, where the protein is retained either in a liquid or solid core and the shell allows permeability control over substrates and products. The permeability coefficients of different molecular weight compounds (vitamin B2, vitamin B12, and myoglobin) were determined through sodium tripolyphosphate (Na-TPP)-crosslinked chitosan membrane. The microcapsule core was formed by crosslinking sodium alginate with either calcium or barium ions. The crosslinked alginate core was uniformly coated with a chitosan layer and crosslinked with Na-TPP. In the case of calcium alginate, the phosphate ions of Na-TPP were able to extract the calcium ions from alginate and liquefy the core. A model enzyme, β -galactosidase, was immobilized in the alginate core and the catalytic activity was measured with o -nitrophenyl- β - d -galactopyranoside (ONPG). Change in the activity of free and immobilized enzyme was determined at three different temperatures. Na-TPP crosslinked chitosan membranes were found to be permeable to solutes of up to 17,000 Da molecular weight. The enzyme loading efficiency was higher in the barium alginate core (100%) as compared to the calcium alginate core (60%). The rate of ONPG conversion to o -nitrophenol was faster in the case of calcium alginate–chitosan microcapsules as compared to barium alginate–chitosan microcapsules. Barium alginate–chitosan microcapsules, however, did improve the stability of the enzyme at 37°C relative to calcium alginate–chitosan microcapsules or free enzyme. This study illustrates a new method of enzyme immobilization for biotechnology applications using liquid or solid core and shell microcapsule technology.
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enzyme immobilization in novel alginate chitosan core shell microcapsules
Biomaterials, 2004Co-Authors: Ehab S Taqieddin, Mansoor M AmijiAbstract:Alginate-chitosan core-shell microcapsules were prepared in order to develop a biocompatible matrix for enzyme immobilization, where the protein is retained either in a liquid or solid core and the shell allows permeability control over substrates and products. The permeability coefficients of different molecular weight compounds (vitamin B2, vitamin B12, and myoglobin) were determined through sodium tripolyphosphate (Na-TPP)-crosslinked chitosan membrane. The microcapsule core was formed by crosslinking sodium alginate with either calcium or barium ions. The crosslinked alginate core was uniformly coated with a chitosan layer and crosslinked with Na-TPP. In the case of calcium alginate, the phosphate ions of Na-TPP were able to extract the calcium ions from alginate and liquefy the core. A model enzyme, beta-galactosidase, was immobilized in the alginate core and the catalytic activity was measured with o-nitrophenyl-beta-D-galactopyranoside (ONPG). Change in the activity of free and immobilized enzyme was determined at three different temperatures. Na-TPP crosslinked chitosan membranes were found to be permeable to solutes of up to 17,000Da molecular weight. The enzyme loading efficiency was higher in the barium alginate core (100%) as compared to the calcium alginate core (60%). The rate of ONPG conversion to o-nitrophenol was faster in the case of calcium alginate-chitosan microcapsules as compared to barium alginate-chitosan microcapsules. Barium alginate-chitosan microcapsules, however, did improve the stability of the enzyme at 37 degrees C relative to calcium alginate-chitosan microcapsules or free enzyme. This study illustrates a new method of enzyme immobilization for biotechnology applications using liquid or solid core and shell microcapsule technology.
L D Stasiuk - One of the best experts on this subject based on the ideXlab platform.
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organic facies in devonian and mississippian strata of western canada sedimentary basin relation to kerogen type paleoenvironment and paleogeography
Bulletin of Canadian Petroleum Geology, 2004Co-Authors: L D Stasiuk, M G FowlerAbstract:Abstract Petrographic analyses of dispersed organic matter (including macerals and palynomorphs), siliceous and calcareous microfossil assemblages and microtextures (e.g. stromatolitic) have been used to define and interpret five organic facies and regionally map their distribution for the following informal groupings of potential hydrocarbon source rocks in the Western Canada Sedimentary Basin: Upper Devonian Woodbend group, Upper Devonian Winterburn group and Upper Devonian to Lower Mississippian black shales of the Exshaw and Bakken formations. Five petrographic organic facies (A-E) are defined for the potential source rocks based on assemblages of Alginites, acritarchs, sporinites, siliceous microfossils and algal mat microtextures. Organic facies A, B (prasinophyte Alginites and acritarchs) and C (coccoidal Alginite), represent accumulation in relatively deep (basin), intermediate (shelf-platform), and shallow water depths (bank-reef margin to lagoonal). Organic facies D is defined by siliceous microfossils (e.g. Radiolaria) and accumulated in deep basinal to outer shelf settings immediately east of an ancient Pacific Ocean, or south of an ancient Arctic Ocean. This facies may reflect regions of upwelling which extended into intracratonic and epicontinental settings. Organic facies E, characterized by stromatolitic microtextures with or without coccoidal Alginite, only occur within Upper Devonian Winterburn Group shallow water, restricted shelf to lagoonal dolostones associated with evaporites. As a whole, the regional distribution of organic facies is related to paleogeography, paleobathymetry or paleostructure in the source rocks. Surprisingly, petrographic organic facies do not show strong positive correlation with kerogen type as defined by Hydrogen-Oxygen indices or TOC-S2 plots.
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confocal laser scanning fluorescence microscopy of botryococcus Alginite from boghead oil shale boltysk ukraine selective preservation of various micro algal components
Organic Geochemistry, 1999Co-Authors: L D StasiukAbstract:Abstract Confocal laser scanning fluorescence microscopy (LSM) has exceptional potential for resolving micrometer scale morphological details within fluorescing macerals (e.g. Alginite) of hydrocarbon source rocks and oil shales. This investigation of well preserved Botryococcus Alginites from a Paleogene boghead oil shale from central Ukraine clearly illustrates that LSM can effectively resolve microalgal cellular organization. A dominance of highly resistant, outermost cell walls in Botryococcus confirms that selective preservation was an effective process during kerogen formation. Three dimensional serial section compilations of images taken through Botryococcus Alginites reveals a number of cellular features including: (i) micro-layering within resistant outer cell walls; (ii) preservation of resistant, very thin, outer walls of apical cells and; (iii) stacks of successive thimble-shaped layers and funnel-shaped cups which comprise the resistant stalk framework of fossilized compound colonies. LSM also discloses preservation of pairs of ‘reproducing’ Botryococcus cells consisting of outer resistant walls enclosing mainly unstructured, granular organic matter in the ‘cell contents region’. Some serial images from these areas do, however, show evidence for cellular organization and possible selective preservation of possible resistant biomacromolecules derived from aplanosphores or zoospores.
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oil prone Alginite macerals from organic rich mesozoic and palaeozoic strata saskatchewan canada
Marine and Petroleum Geology, 1994Co-Authors: L D StasiukAbstract:Abstract Hydrogen-rich, oil-prone remnants of algae, or Alginite macerals, are examined from organic-rich, carbonate and siliciclastic Phanerozoic intervals at Saskatchewan, Canada using incident light fluorescence microscopy. Upper Cambrian Alginites from siliciclastic rocks contribute to Types II–III kerogen (as defined by Rock-Eval pyrolysis) and consist mainly of unicellular, Prasinophyte Leiosphaeridia , unique agglomerations of Synsphaeridium sphaeromorphs and two types of coccoidal Gloeocapsomorpha Alginite. Middle Ordovician platform to basin shales host mainly disseminated Gloeocapsomorpha prisca Alginite macerals (Types I–II kerogen). Upper Ordovician platformal carbonates contain disseminated and stromatolitic varieties of G. prisca Alginite (Type I kerogen), with the latter occasionally forming wholly organic, well preserved microtextures. Concentrations of Nostocaceae -like filamentous Alginite (Type II kerogen) occur locally in Upper Ordovician carbonates and form unique, brecciated and crinkled stromatolites. Middle Devonian Type II kerogen-rich intervals from a platform setting are dominated by stromatolitic Nodularia -like filamentous Alginite, whereas abundant Leiosphaeridia and Tasmanites Alginite characterize inter-reef, basinal palaeoenvironments. Middle and Upper Devonian organic-rich intervals associated with evaporites contain mainly stromatolitic filamentous Alginite with only minor amounts of Prasinophyte Alginite. Black shales of Devonian-Mississippian age contain abundant Leiosphaeridia and Tasmanites Alginites within a Type II amorphous kerogen network. The smallest Leiosphaeridia occur in inner shelf rocks whereas the largest forms characterize outer shelf to basin Devonian-Mississippian shales. Mesozoic marine marls and shales contain Prasinophyte Alginite dominated by small unnamed sphaeromorphs, Leiosphaeridia, Tasmanites and minor Pterosphaeridia (Types II–III kerogen). Fresh to brackish water colonial Botryococcus Alginite (as part of a Type II kerogen) occurs initially in Mesozoic strata deposited in nearshore palaeoenvironments.
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fluorescence properties of palaeozoic oil prone Alginite in relation to hydrocarbon generation williston basin saskatchewan canada
Marine and Petroleum Geology, 1994Co-Authors: L D StasiukAbstract:Abstract Alginite macerals, the oil-prone microscopic components of hydrocarbon source rocks, fluoresce in the visible region when irradiated with ultraviolet light. The fluorescence properties ( λ max and red/green quotient) of coccoidal, unicellular and filamentous Alginite macerals were investigated in Palaeozoic potential hydrocarbon source rocks, northern Williston Basin, Saskatchewan, Canada. This study documents the variation in fluorescence properties of Alginite during thermal maturation and the onset of petroleum generation. Values of λ max (nm) of disseminated, coccoidal Gloeocapsomorpha Alginite from Upper Cambrian siliciclastics and Upper Ordovician carbonates show different magnitudes of red shift with increasing thermal maturity. Gloeocapsomorpha prisca disseminated Alginite and G. prisca stromatolitic Alginite from Upper Ordovician kukersites exhibit unique fluorescence properties with increasing thermal maturity. The former undergoes a greater red shift in fluorescence than the latter over an 1800 m change in depth of burial. Devonian and Mississippian unicellular Prasinophyte Leiosphaeridia and Tasmanites Alginite show a minimal change in fluorescence properties ( λ max 450–500 nm) between vitrinite reflectance values of 0.35 and 0.65%Ro. Beyond 0.65%Ro vitrinite reflectance, Prasinophyte Alginites show a marked red shift to a λ max of 560–600 nm. This phenomenon is coincident with peak hydrocarbon generation from a Type II filamentous Alginite-bituminite kerogen within Middle Devonian carbonate source rocks. A comparison of the fluorescence properties of Palaeozoic Prasinophytes with the fluorescence properties of Mesozoic Prasinophytes reveals that the latter undergo a substantially greater red shift in λ max with increasing thermal maturity relative to the former. This conspicuous difference is interpreted as a reflection of the well documented, distinct difference in species and abundance of Leiosphaeridia and Tasmanites Alginite between the geological eras.
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reflected light microscopy and micro ftir of upper ordovician gloeocapsomorpha prisca Alginite in relation to paleoenvironment and petroleum generation saskatchewan canada
Organic Geochemistry, 1993Co-Authors: L D Stasiuk, B D Kybett, Stephen BendAbstract:Abstract Gloeocapsomorpha prisca Alginites from Upper Ordovician Type I kerogen in hydrocarbon source rocks (kukersites), Yeoman Formation, Saskatchewan, Canada, have been studied using incident light microscopy and transmission micro-Fourier transform infrared spectroscopy. Gloeocapsomorpha prisca coccoidal Alginite are classified as three maceral varieties: (i) small agglomerations of thin-walled disseminated A, (ii) larger agglomerations of thick-walled disseminated B; and (iii) a stromatolitic variety. Each Alginite maceral variety is interpreted as a stage in the life cycle of the algal precursor resulting from progressive normal growth controlled by chemical and physical changes in the paleowater column. Thickened cell walls of disseminated B and stromatolitic G. prisca are characterized by strong i.r. absorbances in the OH region and a higher aromatic C—H absorption compared to the early growth stage of the disseminated A Alginite at the same level of thermal maturity. This characteristic is attributed to the presence of highly aliphatic and resistant outer cell wall biopolymers formed by the algal precursors in response to a variation in either oxygen supply or fluctuating salinity levels within the paleoenvironment and is outlined in a proposed paleoenvironmental model. The rate of reflectance increase in oil and the degree of fluorescence red shift (lambda max) with increasing depth of burial and thermal maturation is greater for G. prisca disseminated varieties compared to the stromatolitic maceral variety. Aromatic C—H absorption (3050 cm −1 ) per unit area for disseminated A and B Alginite increases at approximately the same rate with increasing thermal maturity. In contrast the aliphatic C absorption bands (2920 and 2850 cm −1 ) decrease with increasing maturity in G. prisca disseminated A Alginite whereas aliphatic absorption per unit area increases in disseminated B Alginites with increasing thermal maturation. The aliphatic absorption bands also increase with increasing thermal maturation for stromatolitic G. prisca , but in a pattern unlike disseminated B. A hydrocarbon generation model for G. prisca Alginite proposes that the molecular structure of the disseminated B variety undergoes a transformation from an alkyl long chain structure into a more cyclic bitumen-like maceral prior to peak generation. In contrast, the smaller, thin-walled, disseminated A Alginite,representing the early growth stage, probably generates free bitumen from diagenesis through to catagenesis. This is supported by a reduction in aliphatics with increasing thermal maturity and by the formation of oily-bitumen that evolves from the Alginite during petrographic analysis. The stromatolitic G. prisca Alginite remains morphologically very rigid throughout the diagenetic range of thermal maturities.
Ehab S Taqieddin - One of the best experts on this subject based on the ideXlab platform.
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enzyme immobilization in novel alginate chitosan core shell microcapsules
Biomaterials, 2004Co-Authors: Ehab S Taqieddin, Mansoor M AmijiAbstract:Abstract Alginate–chitosan core-shell microcapsules were prepared in order to develop a biocompatible matrix for enzyme immobilization, where the protein is retained either in a liquid or solid core and the shell allows permeability control over substrates and products. The permeability coefficients of different molecular weight compounds (vitamin B2, vitamin B12, and myoglobin) were determined through sodium tripolyphosphate (Na-TPP)-crosslinked chitosan membrane. The microcapsule core was formed by crosslinking sodium alginate with either calcium or barium ions. The crosslinked alginate core was uniformly coated with a chitosan layer and crosslinked with Na-TPP. In the case of calcium alginate, the phosphate ions of Na-TPP were able to extract the calcium ions from alginate and liquefy the core. A model enzyme, β -galactosidase, was immobilized in the alginate core and the catalytic activity was measured with o -nitrophenyl- β - d -galactopyranoside (ONPG). Change in the activity of free and immobilized enzyme was determined at three different temperatures. Na-TPP crosslinked chitosan membranes were found to be permeable to solutes of up to 17,000 Da molecular weight. The enzyme loading efficiency was higher in the barium alginate core (100%) as compared to the calcium alginate core (60%). The rate of ONPG conversion to o -nitrophenol was faster in the case of calcium alginate–chitosan microcapsules as compared to barium alginate–chitosan microcapsules. Barium alginate–chitosan microcapsules, however, did improve the stability of the enzyme at 37°C relative to calcium alginate–chitosan microcapsules or free enzyme. This study illustrates a new method of enzyme immobilization for biotechnology applications using liquid or solid core and shell microcapsule technology.
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enzyme immobilization in novel alginate chitosan core shell microcapsules
Biomaterials, 2004Co-Authors: Ehab S Taqieddin, Mansoor M AmijiAbstract:Alginate-chitosan core-shell microcapsules were prepared in order to develop a biocompatible matrix for enzyme immobilization, where the protein is retained either in a liquid or solid core and the shell allows permeability control over substrates and products. The permeability coefficients of different molecular weight compounds (vitamin B2, vitamin B12, and myoglobin) were determined through sodium tripolyphosphate (Na-TPP)-crosslinked chitosan membrane. The microcapsule core was formed by crosslinking sodium alginate with either calcium or barium ions. The crosslinked alginate core was uniformly coated with a chitosan layer and crosslinked with Na-TPP. In the case of calcium alginate, the phosphate ions of Na-TPP were able to extract the calcium ions from alginate and liquefy the core. A model enzyme, beta-galactosidase, was immobilized in the alginate core and the catalytic activity was measured with o-nitrophenyl-beta-D-galactopyranoside (ONPG). Change in the activity of free and immobilized enzyme was determined at three different temperatures. Na-TPP crosslinked chitosan membranes were found to be permeable to solutes of up to 17,000Da molecular weight. The enzyme loading efficiency was higher in the barium alginate core (100%) as compared to the calcium alginate core (60%). The rate of ONPG conversion to o-nitrophenol was faster in the case of calcium alginate-chitosan microcapsules as compared to barium alginate-chitosan microcapsules. Barium alginate-chitosan microcapsules, however, did improve the stability of the enzyme at 37 degrees C relative to calcium alginate-chitosan microcapsules or free enzyme. This study illustrates a new method of enzyme immobilization for biotechnology applications using liquid or solid core and shell microcapsule technology.
Wataru Hashimoto - One of the best experts on this subject based on the ideXlab platform.
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Comparative characterization of three bacterial exo-type alginate lyases.
International Journal of Biological Macromolecules, 2016Co-Authors: Makoto Hirayama, Wataru Hashimoto, Kousaku Murata, Shigeyuki KawaiAbstract:Alginate, a major acidic polysaccharide in brown macroalgae, has attracted attention as a carbon source for production of ethanol and other chemical compounds. Alginate is monomerized by exo-type alginate lyase into an unsaturated uronate; thus, this enzyme is critical for the saccharification and utilization of alginate. Although several exo-type alginate lyases have been characterized independently, their activities were not assayed under the same conditions or using the same unit definition, making it difficult to compare enzymatic properties or to select the most suitable enzyme for saccharification of alginate. In this study, we characterized the three bacterial exo-type alginate lyases under the same conditions: A1-IV of Sphingomonas sp. strain A1, Atu3025 of Agrobacterium tumefaciens, and Alg17c of Saccharophagus degradans. A1-IV had the highest specific activity as well as the highest productivity of uronate, whereas Alg17c had the lowest activity and productivity. Only dialyzed Atu3025 and Alg17c were tolerant to freezing. Alg17c exhibited a remarkable halotolerance, which may be advantageous for monomerization of alginate from marine brown algae. Thus, each enzyme exhibited particular desirable and undesirable properties. Our results should facilitate further utilization of the promising polysaccharide alginate.
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a biosystem for alginate metabolism in agrobacterium tumefaciens strain c58 molecular identification of atu3025 as an exotype family pl 15 alginate lyase
Research in Microbiology, 2006Co-Authors: Akihito Ochiai, Wataru Hashimoto, Kousaku MurataAbstract:The Gram-negative bacterium Sphingomonas sp. strain A1 (strain A1) has a peculiar biosystem to directly import and depolymerize a macromolecule, alginate, which is encoded by a cluster of genes on the genome. We identified five clustered ORFs homologous to some genes of the strain A1 cluster in the genome of Agrobacterium tumefaciens strain C58 (strain C58). These ORFs are Atu3021, Atu3022, Atu3023, and Atu3024, encoding a putative sugar ABC transporter system and Atu3025, which encodes a putative alginate lyase. We analyzed the involvement of this gene cluster in alginate metabolism. Strain C58 cells grew significantly on low-molecular-weight (LMW) alginate (average molecular weight, 1000), and we detected specific alginate-induced expression of Atu3024 and Atu3025. This strain does not grow on alginate (average molecular weight, 25 600), suggesting that the strain C58 gene cluster is involved in importing and degrading LMW alginate. One protein, Atu3025, purified from strain C58, was identified as an alginate lyase, and the enzyme overexpressed in Escherichia coli was further characterized. Atu3025 released monosaccharides specifically from alginate most efficiently at pH 7.3 and 30 °C through a β-elimination reaction, indicating that Atu3025 is an exotype alginate lyase potentially involved in the assimilation of LMW alginate in strain C58.
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direct evidence for sphingomonas sp a1 periplasmic proteins as macromolecule binding proteins associated with the abc transporter molecular insights into alginate transport in the periplasm
Biochemistry, 2005Co-Authors: Keiko Momma, Yumiko Mishima, Wataru Hashimoto, Bunzo Mikami, Kousaku MurataAbstract:A Gram-negative bacterium, Sphingomonas sp. A1, has a macromolecule (alginate) import system consisting of a pit on the cell surface and an alginate-specific ATP-binding cassette importer in the inner membrane. Transport of alginate from the pit to the ABC importer is probably mediated by two periplasmic binding protein homologues (AlgQ1 and AlgQ2). Here we describe characteristics of binding of AlgQ1 and AlgQ2 to alginate and its oligosaccharides through surface plasmon resonance biosensor analysis, UV absorption difference spectroscopy, and X-ray crystallography. Both AlgQ1 and AlgQ2 were inducibly expressed in the periplasm of alginate-grown cells of strain A1. Biosensor analysis indicated that both proteins specifically bind alginate with a high degree of polymerization (>100) and that dissociation constants for alginate with an average molecular mass of 26 kDa are 2.3 × 10-7 M for AlgQ1 and 1.5 × 10-7 M for AlgQ2. An in vitro ATPase assay using the membrane complex, including the alginate ABC importe...
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molecular identification of oligoalginate lyase of sphingomonas sp strain a1 as one of the enzymes required for complete depolymerization of alginate
Journal of Bacteriology, 2000Co-Authors: Wataru Hashimoto, Keiko Momma, Osamu Miyake, Shigeyuki Kawai, Kousaku MurataAbstract:A bacterium, Sphingomonas sp. strain A1, can incorporate alginate into cells through a novel ABC (ATP-binding cassette) transporter system specific to the macromolecule. The transported alginate is depolymerized to di- and trisaccharides by three kinds of cytoplasmic alginate lyases (A1-I [66 kDa], A1-II [25 kDa], and A1-III [40 kDa]) generated from a single precursor through posttranslational autoprocessing. The resultant alginate oligosaccharides were degraded to monosaccharides by cytoplasmic oligoalginate lyase. The enzyme and its gene were isolated from the bacterial cells grown in the presence of alginate. The purified enzyme was a monomer with a molecular mass of 85 kDa and cleaved glycosidic bonds not only in oligosaccharides produced from alginate by alginate lyases but also in polysaccharides (alginate, polymannuronate, and polyguluronate) most efficiently at pH 8.0 and 37°C. The reaction catalyzed by the oligoalginate lyase was exolytic and thought to play an important role in the complete depolymerization of alginate in Sphingomonas sp. strain A1. The gene for this novel enzyme consisted of an open reading frame of 2,286 bp encoding a polypeptide with a molecular weight of 86,543 and was located downstream of the genes coding for the precursor of alginate lyases (aly) and the ABC transporter (algS, algM1, and algM2). This result indicates that the genes for proteins required for the transport and complete depolymerization of alginate are assembled to form a cluster.