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V. A. Demakov - One of the best experts on this subject based on the ideXlab platform.

  • Catalytic properties of a Nitrile Hydratase immobilized on activated chitosan
    Applied Biochemistry and Microbiology, 2012
    Co-Authors: Yu. G. Maksimova, T. A. Rogozhnikova, G. V. Ovechkina, A. Yu. Maksimov, V. A. Demakov
    Abstract:

    The catalytic properties of a Nitrile Hydratase, isolated from a strain of Rhodococcus ruber gt1 and immobilized by covalent cross-linking with chitosan activated with 0.1% benzoquinone solution, have been investigated. The kinetic parameters of acryloNitrile hydration catalyzed by immobilized Nitrile Hydratase and the enzyme in a solution have been determined. It is found that the immobilization does not lead to a decrease in the maximum reaction rate ( V _max), whereas the Michaelis constant ( K _M) is reduced by a factor of 2.4. The possibility of reusing an immobilized enzyme for 50 consecutive cycles of acryloNitrile transformation was shown, and the Nitrile Hydratase activity in the 50th cycle exceeded that in the first cycle by 3.5 times. It is shown that the effect of temperature on activity depended on the concentration of the enzyme, which confirms the dissociative nature of Nitrile Hydratase inactivation. It was found that immobilized Nitrile Hydratases remain active at pH 3.0–4.0, whereas the enzyme is inactivated in a solution under these conditions. The resulting biocatalyst can be effectively used to receive acrylamide from acryloNitrile.

  • A study of the catalytic properties of the Nitrile Hydratase immobilized on aluminum oxides and carbon-containing adsorbents
    Applied Biochemistry and Microbiology, 2010
    Co-Authors: Yu. G. Maksimova, G. V. Ovechkina, A. Yu. Maksimov, V. A. Demakov, G. A. Kovalenko
    Abstract:

    The Nitrile Hydratase isolated from Rhodococcus ruber strain gt1, displaying a high Nitrile Hydratase activity, was immobilized on unmodified aluminum oxides and carbon-containing adsorbents, including the carbon support Sibunit. The activity and operational stability of the immobilized Nitrile Hydratase were studied in the reaction of acryloNitrile transformation into acrylamide. It was demonstrated that an increase in the carbon content in the support led to an increase in the amount of adsorbed enzyme and, concurrently, to a decrease in its activity. The Nitrile Hydratase immobilized on Sibunit and carbon-containing aluminum α-oxide having a “crust” structure displayed the highest operational stability in acryloNitrile hydration. It was shown that the thermostability of adsorbed Nitrile Hydratase increased by one order of magnitude.

  • A study of the catalytic properties of the Nitrile Hydratase immobilized on aluminum oxides and carbon-containing adsorbents
    Prikladnaia biokhimiia i mikrobiologiia, 2010
    Co-Authors: Iu G Maksimova, G. V. Ovechkina, V. A. Demakov, A Iu Maksimov, G. A. Kovalenko
    Abstract:

    The Nitrile Hydratase isolated from Rhodococcus ruber strain gt1, displaying a high Nitrile Hydratase activity, was immobilized on unmodified aluminum oxides and carbon-containing adsorbents, including the carbon carrier Sibunit. The activity and operational stability of the immobilized Nitrile Hydratase were studied in the reaction of acryloNitrile transformation into acrylamide. It was demonstrated that an increase in the carbon content in the carrier led to an increase in the amount of adsorbed enzyme and, concurrently, to a decrease in its activity. The Nitrile Hydratase immobilized on Sibunit and carbon-containing aluminum alpha-oxide having a "crust" structure displayed the highest operational stability in acryloNitrile hydration. It was shown that the thermostability of adsorbed Nitrile Hydratase increased by one order of magnitude.

  • Immobilization of Rhodococcus ruber strain gt1, possessing Nitrile Hydratase activity, on carbon supports
    Applied Biochemistry and Microbiology, 2007
    Co-Authors: A. Yu. Maksimov, Yu. G. Maksimova, M. V. Kuznetsova, V. F. Olontsev, V. A. Demakov
    Abstract:

    Rhodococcus ruber strain gt1, possessing Nitrile Hydratase activity, was immobilized by adsorption on carbon supports differing in structure and porosity. The adsorption capacity of the supports towards cells, the substrate of the Nitrile Hydratase reaction (acryloNitrile), and the product (acrylamide) was studied. Also, the effect of immobilization on Nitrile Hydratase activity of bacteria was investigated, and the operational stability of the immobilized biocatalyst was determined. It was shown that crushed and granulated active coals were more appropriate for immobilization than fibrous carbon adsorbents.

  • Immobilization of Rhodococcus ruber strain gt1, possessing Nitrile Hydratase activity, on carbon sorbents
    Prikladnaia biokhimiia i mikrobiologiia, 2007
    Co-Authors: A Iu Maksimov, V. F. Olontsev, M. V. Kuznetsova, Iu G Maksimova, V. A. Demakov
    Abstract:

    Rhodococcus ruber strain gtl, possessing Nitrile Hydratase activity, was immobilized by adsorption on carbon supports differing in structure and porosity. The adsorption capacity of the supports towards cells, the substrate of the Nitrile Hydratase reaction (acryloNitrile), and the product (acrylamide) was studied. Also, the effect of immobilization and Nitrile Hydratase activity of bacteria was investigated, and the operational stability of the immobilized biocatalyst was determined. It was shown that crushed and granulated active coals were more appropriate for immobilization than fibrous carbon adsorbents.

Isao Endo - One of the best experts on this subject based on the ideXlab platform.

  • Fe-type Nitrile Hydratase.
    Journal of inorganic biochemistry, 2001
    Co-Authors: Isao Endo, Masaki Nojiri, Masafumi Yohda, Masanari Tsujimura, Masayoshi Nakasako, Shigehiro Nagashima, Masafumi Odaka
    Abstract:

    Abstract The characteristic features of Fe-type Nitrile Hydratase (NHase) from Rhodococcus sp. N-771 are described. Through the biochemical analyses, we have found that nitric oxide (NO) regulates the photoreactivity of this enzyme by association with the non-heme iron center and photoinduced dissociation from it. The regulation is realized by a unique structure of the catalytic non-heme iron center composed of post-translationally modified cysteine-sulfinic (Cys–SO 2 H) and -sulfenic acids (Cys–SOH). To understand the biogenic mechanism and the functional role of these modifications, we constructed an over-expression system of whole NHase and individual subunits in Escherichia coli . The results of the studies on several recombinant NHases have shown that the Cys–SO 2 H oxidation of αC112 is indispensable for the catalytic activity of Fe-type NHase.

  • Post-translational modification is essential for catalytic activity of Nitrile Hydratase.
    Protein science : a publication of the Protein Society, 2000
    Co-Authors: Taku Murakami, Teruyuki Nagamune, Masaki Nojiri, Masafumi Yohda, Masafumi Odaka, Naoshi Dohmae, Koji Takio, Hiroshi Nakayama, Isao Endo
    Abstract:

    Nitrile Hydratase from Rhodococcus sp. N-771 is an αβ heterodimer with a nonheme ferric iron in the catalytic center. In the catalytic center, αCys112 and αCys114 are modified to a cysteine sulfinic acid (Cys-SO2H) and a cysteine sulfenic acid (Cys-SOH), respectively. To understand the function and the biogenic mechanism of these modified residues, we reconstituted the Nitrile Hydratase from recombinant unmodified subunits. The αβ complex reconstituted under argon exhibited no activity. However, it gradually gained the enzymatic activity through aerobic incubation. ESI-LC/MS analysis showed that the anaerobically reconstituted αβ complex did not have the modification of αCys112-SO2H and aerobic incubation induced the modification. The activity of the reconstituted αβ complex correlated with the amount of αCys112-SO2H. Furthermore, ESI-LC/MS analyses of the tryptic digest of the reconstituted complex, removed of ferric iron at low pH and carboxamidomethylated without reduction, suggested that αCys114 is modified to Cys-SOH together with the sulfinic acid modification of αCys112. These results suggest that αCys112 and αCys114 are spontaneously oxidized to Cys-SO2H and Cys-SOH, respectively, and αCys112-SO2H is responsible for the catalytic activity solely or in combination with αCys114-SOH.

  • functional expression of Nitrile Hydratase in escherichia coli requirement of a Nitrile Hydratase activator and post translational modification of a ligand cysteine
    Journal of Biochemistry, 1999
    Co-Authors: Masaki Nojiri, Masafumi Yohda, Masafumi Odaka, Yusuke Matsushita, Masanari Tsujimura, Takao Yoshida, Naoshi Dohmae, Koji Takio, Isao Endo
    Abstract:

    The Nitrile Hydratase (NHase) from Rhodococcus sp. N-771 is a photoreactive enzyme that is inactivated on nitrosylation of the non-heme iron center and activated on photo-dissociation of nitric oxide (NO). The Nitrile Hydratase operon consists of six genes encoding NHase regulator 2, NHase regulator 1, amidase, NHase alpha subunit, NHase beta subunit and NHase activator. We overproduced the NHase in Escherichia coli using a T7 expression system. The NHase was functionally expressed in E. coli only when the NHase activator encoded downstream of the beta subunit gene was co-expressed and the transformant was grown at 30 degrees C or less. A ligand cysteine, alphaCys112, of the recombinant NHase was also post-translationally modified to a cysteine-sulfinic acid similar to for the native NHase. Although another modification of alphaCys114 could not be identified because of the instability under acidic conditions, the recombinant NHase could be reversibly inactivated by nitric oxide.

  • An enzyme controlled by light: the molecular mechanism of photoreactivity in Nitrile Hydratase
    Trends in biotechnology, 1999
    Co-Authors: Isao Endo, Masafumi Odaka, Masafumi Yohda
    Abstract:

    Extensive studies have revealed the molecular mechanism of the photoreactivity of Nitrile Hydratase from Rhodococcus sp. N-771. In the inactive enzyme, nitric oxide is bound to the non-heme ferric iron at the catalytic center, stabilized by a claw-like structure formed by two post-translationally modified cysteines and a serine. The inactive Nitrile Hydratase is activated by the photoinduced release of the nitric oxide. This result might provide a means of designing novel photoreactive chemical compounds or proteins that would be applicable to biochips and light-controlled metabolic systems.

  • Nitrile Hydratase: Investigation of Its Light-Induced Activation
    Biochemical Engineering for 2001, 1992
    Co-Authors: Jun Honda, Yoshitaka Teratani, Akira Hirata, Hiroyuki Sasabe, Teruyuki Nagamune, Isao Endo
    Abstract:

    The Nitrile Hydratase (NHase) hydrates various Nitrile compounds to the corresponding amides, viz.,

Yi-jun Dai - One of the best experts on this subject based on the ideXlab platform.

  • Maturation Mechanism of Nitrile Hydratase From Streptomyces canus CGMCC 13662 and Its Structural Character.
    Frontiers in microbiology, 2020
    Co-Authors: Ling Guo, Xi Cheng, Huo-yong Jiang, Yi-jun Dai
    Abstract:

    Nitrile Hydratases have received significant interest both in the large-scale industrial production of acrylamide and nicotinamide, and the remediation of environmental contamination with Nitrile-containing pollutants. Almost all known Nitrile Hydratases include an α-subunit (AnhA) and β-subunit (AnhB), and a specific activator protein is crucial for their maturation and catalytic activity. Many studies exist on Nitrile Hydratase characteristics and applications, but few have reported their metal insertion and post-translational maturation mechanism. In this study, we investigated the cobalt insertion and maturation mechanism of Nitrile Hydratase from Streptomyces canus CGMCC 13662 (ScNHase) bearing three subunits (AnhD, AnhE, and AnhA). ScNHase subunits were purified, and the cobalt content and Nitrile Hydratase activity of the ScNHase subunits were detected. We discovered that cobalt could insert into the cobalt-free AnhA of ScNHase in the absence of activator protein under reduction agent DL-dithiothreitol (DTT) environment. AnhD not only performed the function of AnhB of NHase, but also acted as a metal ion chaperone and self-subunit swapping chaperone, while AnhE did not act as similar performance. A cobalt direct-insertion under reduction condition coordinated self-subunit swapping mechanism is responsible for ScNHase post-translational maturation. Molecular docking of ScNHase and substrates suggested that the substrate specificity of ScNHase was correlated with its structure. ScNHase had a weak hydrophobic interaction with IAN through protein-ligand interaction analysis and, therefore, had no affinity with indole-3-acetoNitrile (IAN). The post-translational maturation mechanism and structure characteristics of ScNHase could help guide research on the environmental remediation of Nitrile-containing waste contamination and three-subunit Nitrile Hydratase.

  • Biodegradation of the Neonicotinoid Insecticide Acetamiprid by Actinomycetes Streptomyces canus CGMCC 13662 and Characterization of the Novel Nitrile Hydratase Involved
    2019
    Co-Authors: Ling Guo, Wen-wan Fang, Lei-lei Guo, Chuan-fei Yao, Yun-xiu Zhao, Yi-jun Dai
    Abstract:

    Neonicotinoid insecticide pollution in soil and water poses serious environmental risks. Microbial biodegradation is an important neonicotinoid insecticide degradation pathway in the environment. In this study, 70.0% of the acetamiprid in a 200 mg/L solution was degraded by actinomycetes Streptomyces canus CGMCC 13662 (isolated from soil) in 48 h, and the acetamiprid degradation half-life was 27.7 h. Acetamiprid was degraded to IM-1-2 ((E)-1-(1-(((6-chloropyridin-3-yl)­methyl)­(methyl) amino)­ethylidene)­urea) through hydrolysis of the cyanoimine moiety. Gene cloning and overexpression indicated that a novel Nitrile Hydratase with three unusual subunits (AnhD, AnhE, and AnhA) without accessory protein mediated IM-1-2 formation. The purified Nitrile Hydratase responsible for degrading acetamiprid had a Km of 5.85 mmol/L and a Vmax of 15.99 U/mg. A homology model suggested that AnhD-Glu56 and AnhE-His21 play important roles in the catalytic efficiency of the Nitrile Hydratase. S. canus CGMCC 13662 could be used to remediate environments contaminated with acetamiprid

Toru Nagasawa - One of the best experts on this subject based on the ideXlab platform.

  • Remaining acetamide in acetoNitrile degradation using Nitrile Hydratase- and amidase-producing microorganisms.
    Applied microbiology and biotechnology, 2006
    Co-Authors: Erina Kohyama, Mizuho Dohi, Akihiro Yoshimura, Toyokazu Yoshida, Toru Nagasawa
    Abstract:

    The tandem conversion process involving Nitrile Hydratase- and amidase-producing microorganisms has potential for use in the treatment of acetoNitrile-containing wastes. In that process, the acetamide hydrolysis step catalyzed by amidase is very slow compared with the acetoNitrile hydration step catalyzed by Nitrile Hydratase, and a small amount of acetamide remains in the resulting solution. This study aimed to improve the efficiency of the acetamide hydrolysis step. An amidase-producing microorganism, Rhodococcus sp. S13-4, was newly obtained, whose use enabled rapid acetamide degradation. Though residual acetamide was still detected, it was successfully reduced by the addition of cation/anion mixed ion exchange resin or calcium hydroxide after the acetamide hydrolysis reaction using Rhodococcus sp. S13-4 cells. This result implies that acetamide hydrolysis and acetamide formation are in equilibrium. The incubation of Rhodococcus sp. S13-4 cells with high concentrations of ammonium acetate produced acetamide. The purified amidase from Rhodococcus sp. S13-4 revealed the acetamide formation activity (specific activity of 30.6 U/mg protein). This suggests that the amidase-catalyzed amide formation may cause the remaining of acetamide in the acetoNitrile conversion process.

  • Low‐molecular‐mass Nitrile Hydratase from Rhodococcus rhodochrous J1: purification, substrate specificity and comparison with the analogous high‐molecular‐mass enzyme
    Fems Microbiology Letters, 1998
    Co-Authors: Marco Wieser, Koji Takeuchi, Hideaki Yamada, Yutaka Wada, Toru Nagasawa
    Abstract:

    In addition to a previously described nitrilase and a high-molecular-mass Nitrile Hydratase, Rhodococcus rhodochrous J1 contains a second, low-molecular-mass Nitrile Hydratase, which was purified, characterized and compared with the high-molecular-mass enzyme. Due to its wide substrate spectrum, the low-molecular-mass enzyme broadens the Nitrile-converting capacity of this strain. The versatility of Nitrile metabolism in this organism and the significance of the low-molecular-mass enzyme for the synthesis of valuable amides is discussed.

  • characterization of a new cobalt containing Nitrile Hydratase purified from urea induced cells of rhodococcus rhodochrous j1
    FEBS Journal, 1991
    Co-Authors: Toru Nagasawa, Koji Takeuchi, Hideaki Yamada
    Abstract:

    A new cobalt-containing Nitrile Hydratase was purified from extracts of urea-induced cells from Rhodococcus rhodochrous J1 in seven steps. At the last step, the enzyme was crystallized by adding ammonium sulfate. Nitrile Hydratase was a 500–530-kDa protein composed of two different subunits (α subunit 26kDa, β subunit 29kDa). The enzyme contained approximately 11–12 mol cobalt/mol enzyme. A concentrated solution of highly purified Nitrile Hydratase exhibited a broad absorption spectrum in the visible range, with an absorption maxima at 410 nm. The enzyme had a wide substrate specificity. Aliphatic saturated or unsaturated Nitriles as well as aromatic Nitriles, were substrates for the enzyme. The optimum pH of the Hydratase was pH 6.5–6.8. The enzyme was more stable than ferric Nitrile Hydratases. The amino-terminal sequence of each subunit of R. rhodochrous J1 enzyme was determined and compared with that of ferric Nitrile Hydratases. Prominent similarities were observed with the β subunit. However, the amino acid sequence of the α subunit from R. rhodochrous J1 was quite different from that of the ferric enzymes.

  • Optimum culture conditions for the production of cobalt-containing Nitrile Hydratase by Rhodococcus rhodochrous J1
    Applied Microbiology and Biotechnology, 1991
    Co-Authors: Toru Nagasawa, Koji Takeuchi, Vincenzo Nardi-dei, Yasuhiro Mihara, Hideaki Yamada
    Abstract:

    We sought the optimum conditions for production of Nitrile Hydratase by Rhodococcus rhodochrous J1. The addiiion of both cobalt ions and an aliphatic Nitrile or amide as an inducer was indispensable for the appearance of Nitrile Hydratase activity in R. rhodochrous J1 cells. Crotonamide was an efficient inducer and, moreover, urea was found to be the most powerful inducer for the production of Nitrile Hydratase. When R. rhodochrous J1 was cultivated under optimal conditions, the enzyme activity in the culture broth and the specific activity was approximately 32,000 and 512 times higher than the initially obtained levels, respectively. The Nitrile Hydratase formed corresponded to more than 45% of the total soluble protein in urea-induced cells, as judged by quantitative evaluation of the gel track.

Hideaki Yamada - One of the best experts on this subject based on the ideXlab platform.