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Masao Tokunaga - One of the best experts on this subject based on the ideXlab platform.
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Distinct Characteristics of Single Starch-Binding Domain SBD1 Derived from Tandem Domains SBD1-SBD2 of Halophilic Kocuria varians Alpha-Amylase
The Protein Journal, 2012Co-Authors: Rui Yamaguchi, Hiroko Tokunaga, Matsujiro Ishibashi, Tsutomu Arakawa, Masao TokunagaAbstract:Kocuria varians alpha-amylase contains tandem starch-binding domains SBD1-SBD2 (SBD12) that possess typical halophilic characteristics. Recombinant tandem domains SBD12 and single domain SBD1, both with amino-terminal hexa-His tag, were expressed in and purified to homogeneity from Escherichia coli . The circular dichroism (CD) spectrum of His-SBD12 was characterized by a positive peak at 233 nm ascribed to the aromatic stacking. Although the signal occurred in the far UV region, it is an indication of tertiary structure folding. CD spectrum of single domain His-SBD1 exhibited the same peak position, signal intensity and spectral shape as those of His-SBD12, suggesting that the aromatic stacking must occur within the domain, and that two SBD domains in SBD12 and SBD1 has a similar folded structure. This structural observation was consistent with the biological activity that His-SBD1 showed binding activity against raw starch granules and amylose resin with 70–80% efficiency compared with binding of equimolar His-SBD12. Although the thermal unfolding rate of SBD12 and SBD1 were similar, the refolding rates of SBD12 and SBD1 from thermal melting were greatly different: His-SBD12 refolded slowly (T_1/2 = ~84 min), while refolding of single domain His-SBD1 was found to be 20-fold faster (T_1/2 = 4.2 min). The possible mechanism of this large difference in refolding rate was discussed. Maltose at 20 mM showed 5–6 °C increase in thermal melting of both His-SBD12 and His-SBD1, while its effects on the time course of unfolding and refolding were insignificant.
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halophilic characterization of starch binding domain from Kocuria varians α amylase
International Journal of Biological Macromolecules, 2012Co-Authors: Rui Yamaguchi, Yasuhiro Inoue, Hiroko Tokunaga, Matsujiro Ishibashi, Tsutomu Arakawa, Junichi Sumitani, Takashi Kawaguchi, Masao TokunagaAbstract:The tandem starch-binding domains (KvSBD) located at carboxy-terminal region of halophilic α-amylase from moderate halophile, Kocuria varians, were expressed in E. coli with amino-terminal hexa-His-tag and purified to homogeneity. The recombinant KvSBD showed binding activity to raw starch granules at low to high salt concentrations. The binding activity of KvSBD to starch was fully reversible after heat-treatment at 85°C. Circular dichroism and thermal scanning experiments indicated that KvSBD showed fully reversible refolding upon cooling after complete melting at 70°C in the presence of 0.2-2.0M NaCl. The refolding rate was enhanced with higher salt concentration.
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Salt-dependent thermo-reversible α-amylase: cloning and characterization of halophilic α-amylase from moderately halophilic bacterium, Kocuria varians
Applied Microbiology and Biotechnology, 2011Co-Authors: Rui Yamaguchi, Hiroko Tokunaga, Matsujiro Ishibashi, Tsutomu Arakawa, Masao TokunagaAbstract:A moderately halophilic bacterium, Kocuria varians , was found to produce active α-amylase ( K. varians α-amylase (KVA)). We have observed at least six different forms of α-amylase secreted by this bacterium into the culture medium. Characterization of these KVA forms and cloning of the corresponding gene revealed that KVA comprises pre-pro-precursor form of α-amylase catalytic domain followed by the tandem repeats, which show high similarity to each other and to the starch binding domain (SBD) of other α-amylases. The observed six forms were most likely derived by various processing of the protein product. Recombinant KVA protein was successfully expressed in Escherichia coli as a fusion protein and was purified with affinity chromatography after cleavage from fusion partner. The highly acidic amino acid composition of KVA and the highly negative electrostatic potential surface map of the modeled structure strongly suggested its halophilic nature. Indeed, KVA showed distinct salt- and time-dependent thermal reversibility: when α-amylase was heat denatured at 85°C for 3 min in the presence of 2 M NaCl, the activity was recovered upon incubation on ice (50% recovery after 15 min incubation). Conversely, KVA denatured in 0.1 M NaCl was not refolded at all, even after prolonged incubation. KVA activity was inhibited by proteinaceous α-amylase inhibitor from Streptomyces nitrosporeus , which had been implicated to inhibit only animal α-amylases. KVA with putative SBD regions was found to digest raw starch.
Isabel Pardo - One of the best experts on this subject based on the ideXlab platform.
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ability of Kocuria varians lth 1540 to degrade putrescine identification and characterization of a novel amine oxidase
Journal of Agricultural and Food Chemistry, 2015Co-Authors: Sara Callejon, Ramon Sendra, Sergi Ferrer, Isabel PardoAbstract:This work describes the identification and characterization of an amine oxidase from Kocuria varians LTH 1540 (syn. Micrococcus varians) primarily acting on putrescine. Data from MALDI-TOF MS/MS and the identification of Δ1-pyrroline as degradation product from putrescine indicate that the enzyme is a flavin-dependent putrescine oxidase (PuO). Properties of partially purified enzyme have been determined. The enzyme oxidizes diamines, putrescine and cadaverine, and, to a lesser extent, polyamines, such as spermidine, but not monoamines. The kinetic constants (Km and Vmax) for the two major substrates were 94 ± 10 μM and 2.3 ± 0.1 μmol/min·mg for putrescine and 75 ± 5 μM and 0.15 ± 0.02 μmol/min·mg for cadaverine. Optimal temperature and pH were 45 °C and 8.5, respectively. Enzyme was stable until 50 °C. K. varians PuO is sensitive to human flavin-dependent amine oxidase inhibitors and carboxyl-modifying compounds. The new enzyme has been isolated from a bacterial starter used in the manufacture of fermente...
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Ability of Kocuria varians LTH 1540 To Degrade Putrescine: Identification and Characterization of a Novel Amine Oxidase
2015Co-Authors: Sara Callejón, Sergi Ferrer, Ramón Sendra, Isabel PardoAbstract:This work describes the identification and characterization of an amine oxidase from Kocuria varians LTH 1540 (syn. Micrococcus varians) primarily acting on putrescine. Data from MALDI-TOF MS/MS and the identification of Δ1-pyrroline as degradation product from putrescine indicate that the enzyme is a flavin-dependent putrescine oxidase (PuO). Properties of partially purified enzyme have been determined. The enzyme oxidizes diamines, putrescine and cadaverine, and, to a lesser extent, polyamines, such as spermidine, but not monoamines. The kinetic constants (Km and Vmax) for the two major substrates were 94 ± 10 μM and 2.3 ± 0.1 μmol/min·mg for putrescine and 75 ± 5 μM and 0.15 ± 0.02 μmol/min·mg for cadaverine. Optimal temperature and pH were 45 °C and 8.5, respectively. Enzyme was stable until 50 °C. K. varians PuO is sensitive to human flavin-dependent amine oxidase inhibitors and carboxyl-modifying compounds. The new enzyme has been isolated from a bacterial starter used in the manufacture of fermented meat. One of the problems of fermented foods or beverages is the presence of toxic biogenic amines produced by bacteria. The importance of this works lies in the description of a new enzyme able to degrade two of the most abundant biogenic amines (putrescine and cadaverine), the use of which could be envisaged to diminish biogenic amines content in foods in the future
Rui Yamaguchi - One of the best experts on this subject based on the ideXlab platform.
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Distinct Characteristics of Single Starch-Binding Domain SBD1 Derived from Tandem Domains SBD1-SBD2 of Halophilic Kocuria varians Alpha-Amylase
The Protein Journal, 2012Co-Authors: Rui Yamaguchi, Hiroko Tokunaga, Matsujiro Ishibashi, Tsutomu Arakawa, Masao TokunagaAbstract:Kocuria varians alpha-amylase contains tandem starch-binding domains SBD1-SBD2 (SBD12) that possess typical halophilic characteristics. Recombinant tandem domains SBD12 and single domain SBD1, both with amino-terminal hexa-His tag, were expressed in and purified to homogeneity from Escherichia coli . The circular dichroism (CD) spectrum of His-SBD12 was characterized by a positive peak at 233 nm ascribed to the aromatic stacking. Although the signal occurred in the far UV region, it is an indication of tertiary structure folding. CD spectrum of single domain His-SBD1 exhibited the same peak position, signal intensity and spectral shape as those of His-SBD12, suggesting that the aromatic stacking must occur within the domain, and that two SBD domains in SBD12 and SBD1 has a similar folded structure. This structural observation was consistent with the biological activity that His-SBD1 showed binding activity against raw starch granules and amylose resin with 70–80% efficiency compared with binding of equimolar His-SBD12. Although the thermal unfolding rate of SBD12 and SBD1 were similar, the refolding rates of SBD12 and SBD1 from thermal melting were greatly different: His-SBD12 refolded slowly (T_1/2 = ~84 min), while refolding of single domain His-SBD1 was found to be 20-fold faster (T_1/2 = 4.2 min). The possible mechanism of this large difference in refolding rate was discussed. Maltose at 20 mM showed 5–6 °C increase in thermal melting of both His-SBD12 and His-SBD1, while its effects on the time course of unfolding and refolding were insignificant.
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halophilic characterization of starch binding domain from Kocuria varians α amylase
International Journal of Biological Macromolecules, 2012Co-Authors: Rui Yamaguchi, Yasuhiro Inoue, Hiroko Tokunaga, Matsujiro Ishibashi, Tsutomu Arakawa, Junichi Sumitani, Takashi Kawaguchi, Masao TokunagaAbstract:The tandem starch-binding domains (KvSBD) located at carboxy-terminal region of halophilic α-amylase from moderate halophile, Kocuria varians, were expressed in E. coli with amino-terminal hexa-His-tag and purified to homogeneity. The recombinant KvSBD showed binding activity to raw starch granules at low to high salt concentrations. The binding activity of KvSBD to starch was fully reversible after heat-treatment at 85°C. Circular dichroism and thermal scanning experiments indicated that KvSBD showed fully reversible refolding upon cooling after complete melting at 70°C in the presence of 0.2-2.0M NaCl. The refolding rate was enhanced with higher salt concentration.
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Salt-dependent thermo-reversible α-amylase: cloning and characterization of halophilic α-amylase from moderately halophilic bacterium, Kocuria varians
Applied Microbiology and Biotechnology, 2011Co-Authors: Rui Yamaguchi, Hiroko Tokunaga, Matsujiro Ishibashi, Tsutomu Arakawa, Masao TokunagaAbstract:A moderately halophilic bacterium, Kocuria varians , was found to produce active α-amylase ( K. varians α-amylase (KVA)). We have observed at least six different forms of α-amylase secreted by this bacterium into the culture medium. Characterization of these KVA forms and cloning of the corresponding gene revealed that KVA comprises pre-pro-precursor form of α-amylase catalytic domain followed by the tandem repeats, which show high similarity to each other and to the starch binding domain (SBD) of other α-amylases. The observed six forms were most likely derived by various processing of the protein product. Recombinant KVA protein was successfully expressed in Escherichia coli as a fusion protein and was purified with affinity chromatography after cleavage from fusion partner. The highly acidic amino acid composition of KVA and the highly negative electrostatic potential surface map of the modeled structure strongly suggested its halophilic nature. Indeed, KVA showed distinct salt- and time-dependent thermal reversibility: when α-amylase was heat denatured at 85°C for 3 min in the presence of 2 M NaCl, the activity was recovered upon incubation on ice (50% recovery after 15 min incubation). Conversely, KVA denatured in 0.1 M NaCl was not refolded at all, even after prolonged incubation. KVA activity was inhibited by proteinaceous α-amylase inhibitor from Streptomyces nitrosporeus , which had been implicated to inhibit only animal α-amylases. KVA with putative SBD regions was found to digest raw starch.
Peter Schumann - One of the best experts on this subject based on the ideXlab platform.
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reclassification of strain ccm 132 previously classified as Kocuria varians as Kocuria carniphila sp nov
International Journal of Systematic and Evolutionary Microbiology, 2005Co-Authors: Ludmila Tvrzova, Peter Schumann, Ivo Sedlacek, Zdena Pacova, Cathrin Sproer, Susanne Verbarg, Reiner M KroppenstedtAbstract:A Gram-positive actinobacterium, previously classified as Kocuria varians, was subjected to a polyphasic taxonomic study. The bacterium showed the peptidoglycan type Lys-Ala3 (variation A3alpha), MK-7(H2) was the major menaquinone and anteiso-C(15 : 0) and anteiso-C(17 : 0) were the major fatty acids. On the basis of the phylogenetic and phenotypic characteristics of the actinobacterium, a novel species, Kocuria carniphila sp. nov. (type strain, CCM 132T=DSM 16004T), is proposed.
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Kocuria palustris sp nov and Kocuria rhizophila sp nov isolated from the rhizoplane of the narrow leaved cattail typha angustifolia
International Journal of Systematic and Evolutionary Microbiology, 1999Co-Authors: Gabor M Kovacs, Erko Stackebrandt, Peter Schumann, Jutta Burghardt, Silke Pradella, Karoly MarialigetiAbstract:Two Gram-positive, aerobic spherical actinobacteria were isolated from the rhizoplane of narrow-leaved cattail (Typha angustifolia) collected from a floating mat in the Soroksar tributary of the Danube river, Hungary. Sequence comparisons of the 16S rDNA indicated these isolates to be phylogenetic neighbours of members of the genus Kocuria, family Micrococcaceae, in which they represent two novel lineages. The phylogenetic distinctness of the two organisms TA68Tand TAGA27Twas supported by DNA-DNA similarity values of less than 55% between each other and with the type strains of Kocuria rosea, Kocuria kristinae and Kocuria varians. Chemotaxonomic properties supported the placement of the two isolates in the genus Kocuria. The diagnostic diamino acid of the cell-wall peptidoglycan is lysine, the interpeptide bridge is composed of three alanine residues. Predominant menaquinone was MK-7(H2). The fatty acid pattern represents the straight-chain saturated iso-anteiso type. Main fatty acid was anteiso-C15:0. The phospholipids are diphosphatidylglycerol, phosphatidylglycerol and an unknown component. The DNA base composition of strains TA68Tand TAGA27Tis 69·4 and 69·6 mol% G+C, respectively. Genotypic, morphological and physiological characteristics are used to describe two new species of Kocuria, for which we propose the names Kocuria palustris, type strain DSM 11925Tand Kocuria rhizophila, type strain DSM 11926T.
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Taxonomic Dissection of the Genus Micrococcus: Kocuria gen. nov., Nesterenkonia gen. nov., Kytococcus gen. nov., Dermacoccus gen. nov., and Micrococcus Cohn 1872 gen. emend.
International journal of systematic bacteriology, 1995Co-Authors: Erko Stackebrandt, Cathrin Koch, Oxana R. Gvozdiak, Peter SchumannAbstract:The results of a phylogenetic and chemotaxonomic analysis of the genus Micrococcus indicated that it is significantly heterogeneous. Except for Micrococcus lylae, no species groups phylogenetically with the type species of the genus, Micrococcus luteus. The other members of the genus form three separate phylogenetic lines which on the basis of chemotaxonomic properties can be assigned to four genera. These genera are the genus Kocuria gen. nov. for Micrococcus roseus, Micrococcus varians, and Micrococcus kristinae, described as Kocuria rosea comb. nov., Kocuria varians comb. nov., and Kocuria kristinae comb. nov., respectively; the genus Nesterenkonia gen. nov. for Micrococcus halobius, described as Nesterenkonia halobia comb. nov.; the genus Kytococcus gen. nov. for Micrococcus nishinomiyaensis, described as Kytococcus nishinomiyaensis comb, nov.; and the genus Dermacoccus gen. nov. for Micrococcus sedentarius, described as Dermacoccus sedentarius comb. nov. M. luteus and M. lylae, which are closely related phylogenetically but differ in some chemotaxonomic properties, are the only species that remain in the genus Micrococcus Cohn 1872. An emended description of the genus Micrococcus is given.
Tsutomu Arakawa - One of the best experts on this subject based on the ideXlab platform.
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Distinct Characteristics of Single Starch-Binding Domain SBD1 Derived from Tandem Domains SBD1-SBD2 of Halophilic Kocuria varians Alpha-Amylase
The Protein Journal, 2012Co-Authors: Rui Yamaguchi, Hiroko Tokunaga, Matsujiro Ishibashi, Tsutomu Arakawa, Masao TokunagaAbstract:Kocuria varians alpha-amylase contains tandem starch-binding domains SBD1-SBD2 (SBD12) that possess typical halophilic characteristics. Recombinant tandem domains SBD12 and single domain SBD1, both with amino-terminal hexa-His tag, were expressed in and purified to homogeneity from Escherichia coli . The circular dichroism (CD) spectrum of His-SBD12 was characterized by a positive peak at 233 nm ascribed to the aromatic stacking. Although the signal occurred in the far UV region, it is an indication of tertiary structure folding. CD spectrum of single domain His-SBD1 exhibited the same peak position, signal intensity and spectral shape as those of His-SBD12, suggesting that the aromatic stacking must occur within the domain, and that two SBD domains in SBD12 and SBD1 has a similar folded structure. This structural observation was consistent with the biological activity that His-SBD1 showed binding activity against raw starch granules and amylose resin with 70–80% efficiency compared with binding of equimolar His-SBD12. Although the thermal unfolding rate of SBD12 and SBD1 were similar, the refolding rates of SBD12 and SBD1 from thermal melting were greatly different: His-SBD12 refolded slowly (T_1/2 = ~84 min), while refolding of single domain His-SBD1 was found to be 20-fold faster (T_1/2 = 4.2 min). The possible mechanism of this large difference in refolding rate was discussed. Maltose at 20 mM showed 5–6 °C increase in thermal melting of both His-SBD12 and His-SBD1, while its effects on the time course of unfolding and refolding were insignificant.
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halophilic characterization of starch binding domain from Kocuria varians α amylase
International Journal of Biological Macromolecules, 2012Co-Authors: Rui Yamaguchi, Yasuhiro Inoue, Hiroko Tokunaga, Matsujiro Ishibashi, Tsutomu Arakawa, Junichi Sumitani, Takashi Kawaguchi, Masao TokunagaAbstract:The tandem starch-binding domains (KvSBD) located at carboxy-terminal region of halophilic α-amylase from moderate halophile, Kocuria varians, were expressed in E. coli with amino-terminal hexa-His-tag and purified to homogeneity. The recombinant KvSBD showed binding activity to raw starch granules at low to high salt concentrations. The binding activity of KvSBD to starch was fully reversible after heat-treatment at 85°C. Circular dichroism and thermal scanning experiments indicated that KvSBD showed fully reversible refolding upon cooling after complete melting at 70°C in the presence of 0.2-2.0M NaCl. The refolding rate was enhanced with higher salt concentration.
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Salt-dependent thermo-reversible α-amylase: cloning and characterization of halophilic α-amylase from moderately halophilic bacterium, Kocuria varians
Applied Microbiology and Biotechnology, 2011Co-Authors: Rui Yamaguchi, Hiroko Tokunaga, Matsujiro Ishibashi, Tsutomu Arakawa, Masao TokunagaAbstract:A moderately halophilic bacterium, Kocuria varians , was found to produce active α-amylase ( K. varians α-amylase (KVA)). We have observed at least six different forms of α-amylase secreted by this bacterium into the culture medium. Characterization of these KVA forms and cloning of the corresponding gene revealed that KVA comprises pre-pro-precursor form of α-amylase catalytic domain followed by the tandem repeats, which show high similarity to each other and to the starch binding domain (SBD) of other α-amylases. The observed six forms were most likely derived by various processing of the protein product. Recombinant KVA protein was successfully expressed in Escherichia coli as a fusion protein and was purified with affinity chromatography after cleavage from fusion partner. The highly acidic amino acid composition of KVA and the highly negative electrostatic potential surface map of the modeled structure strongly suggested its halophilic nature. Indeed, KVA showed distinct salt- and time-dependent thermal reversibility: when α-amylase was heat denatured at 85°C for 3 min in the presence of 2 M NaCl, the activity was recovered upon incubation on ice (50% recovery after 15 min incubation). Conversely, KVA denatured in 0.1 M NaCl was not refolded at all, even after prolonged incubation. KVA activity was inhibited by proteinaceous α-amylase inhibitor from Streptomyces nitrosporeus , which had been implicated to inhibit only animal α-amylases. KVA with putative SBD regions was found to digest raw starch.
