Cloning and sequencing of a nitrile hydratase gene from Pseudonocardia thermophila JCM3095

Yamaki, Toshifumi; Oikawa, Toshihiro; Itō, Kiyoshi; Nakamura, Takeshi

doi:10.1016/s0922-338x(97)83004-8

Cited by 62 publications

(37 citation statements)

References 6 publications

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“…Because the K m was found to be independent of pH over the entire pH range studied, the D (k cat /K m ) for PtNHase is 1.7. Temperature Dependence of K m and k cat for PtNHase-It was reported previously that PtNHase is stable at 60°C and pH 7.6 (35). These data are very unusual because most enzymes undergo some denaturation at temperatures above 50°C, resulting in a decrease in V max (28).…”

Section: Catalytically Important Residues In Nitrile Hydratasesmentioning

confidence: 96%

Unraveling the Catalytic Mechanism of Nitrile Hydratases

Mitra

Holz

2007

Journal of Biological Chemistry

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To elucidate a detailed catalytic mechanism for nitrile hydratases (NHases), the pH and temperature dependence of the kinetic constants k cat and K m for the cobalt-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) were examined. PtNHase was found to exhibit a bell-shaped curve for plots of relative activity versus pH at pH 3.2-11 and was found to display maximal activity between pH 7.2 and 7.8. Fits of these data provided pK ES1 and pK ES2 values of 5.9 ؎ 0.1 and 9.2 ؎ 0.1 (k cat ‫؍‬ 130 ؎ 1 s ؊1 ), respectively, and pK E1 and pK E2 values of 5.8 ؎ 0.1 and 9.1 ؎ 0.1 (k cat /K m ‫؍‬ (6.5 ؎ 0.1) ؋ 10 3 s ؊1 mM ؊1 ), respectively. Proton inventory studies indicated that two protons are transferred in the rate-limiting step of the reaction at pH 7.6. Because PtNHase is stable at 60°C, an Arrhenius plot was constructed by plotting ln(k cat ) versus 1/T, providing E a ‫؍‬ 23.0 ؎ 1.2 kJ/mol. The thermal stability of PtNHase also allowed ⌬H 0 ionization values to be determined, thus helping to identify the ionizing groups exhibiting the pK ES1 and pK ES2 values. Based on ⌬H ion 0 data, pK ES1 is assigned to ␤Tyr 68 , whereas pK ES2 is assigned to ␤Arg 52 , ␤Arg 157 , or ␣Ser 112 (NHases are ␣ 2 ␤ 2 -heterotetramers). A combination of these data with those previously reported for NHases and synthetic model complexes, along with sequence comparisons of both iron-and cobalt-type NHases, allowed a novel catalytic mechanism for NHases to be proposed.Nitrile hydratase (NHase 2 ; EC 4.2.1.84), one of the enzymes in the nitrile degradation pathway, catalyzes the hydrolysis of nitriles to their corresponding higher value amides in a chemo-, regio-, and/or enatioselective manner at ambient pressures and temperatures at physiological pH (Scheme 1) (1-6). NHases have attracted substantial interest as biocatalysts for industrial applications such as the large-scale production of acrylamide (3, 7-9) and nicotinamide (10). Acrylamide production utilizing the bacterium Rhodococcus rhodochrous J1 has increased to Ͼ30,000 tons/year (3), whereas Ͼ3500 tons of nicotinamide are produced per year (11). Yields of Ͼ99% are achieved, and the formation of by-products such as acrylic acid, which plagues traditional methodology, is completely avoided. However, one of the most attractive features of nitrile-metabolizing enzymes is their ability to selectively hydrolyze one cyano group of a dinitrile to its corresponding amine, something that is virtually impossible using conventional chemical methods (12-14). Therefore, the potential use of nitrile-hydrolyzing enzymes for the production of several fine chemicals is increasingly recognized.NHases are metalloenzymes that contain either a non-heme Fe(III) ion (iron-type) or a non-corrin Co(III) ion (cobalt-type) in their active site and are typically ␣ 2 ␤ 2 -heterotetramers (5, 6, 15, 16). In all known NHases, each ␣-subunit has a highly homologous amino acid sequence (CXYCSCX) that forms the metal-binding site. Cobalt-type NHases contain threonine and tyrosine in the -C(T/S)YCSC(Y/T)-se...

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Section: Catalytically Important Residues In Nitrile Hydratasesmentioning

confidence: 96%

Unraveling the Catalytic Mechanism of Nitrile Hydratases

Mitra

Holz

2007

Journal of Biological Chemistry

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“…Generally, the mesophilic NHases have exhibited maximum activity near ambient temperature between 20 and 40°C [14,15]. However, thermophilic NHases of Bacillus RAPc8 [16] and Pseudonocardia thermophila [17] exhibited maximum activity at 60°C. The half-life of NHase of R. rhodochrous PA-34 was 2 h at 40°C, 0.5 h at 50°C and it was rapidly inactivated at temperature above 50°C.…”

Section: Effect Of Temperature On Activity and Stability Of Nhasementioning

confidence: 99%

Purification of a hyperactive nitrile hydratase from resting cells of Rhodococcus rhodochrous PA-34

2009

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A propionitrile-induced nitrile hydratase (NHase), a promising biocatalyst for synthesis of organic amides has been purifi ed from cell-free extract of Rhodococcus rhodochrous PA-34. About 11-fold purifi cation of NHase was achieved with 52% yield. The SDS-PAGE of the purifi ed enzyme revealed that it consisted of two subunits of 25.04 kD and 30.6 kD. However, the molecular weight of holoenzyme was speculated to be 86 kD by native-PAGE. This NHase exhibited maximum activity at pH 8.0 and temperature 40°C. Half-life was 2 h at 40°C and 0.5 h at 50°C. The Km and Vmax were 167 mM and 250 μmole/min/mg using 25 mM 3-cyanopyridine as substrate. AgNO 3 , Pb(CH 3 COO) 2 and HgCl 2 inhibited the NHase to extent of 89-100%.

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“…The NHase of the moderate thermophile Pseudonocardia thermophila JCM 3095 has been cloned and expressed in E. coli for use in the commercial production of acrylamide. [14] Comamonas testosteroni 5-MGAM-4D expresses a thermally stable NHase and amidase, and has been used for conversion of a variety of nitriles to their corresponding carboxylic acids; [15] the NHase has recently been cloned and sequenced, and active NHase has been over-produced in Escherichia coli SW132. [16] We now report an examination of this transformant catalyst for the conversion of acrylonitrile to acrylamide, where the dependence of catalyst productivity on reaction temperature and concentration of acrylonitrile and acrylamide has been evaluated.…”

Section: Introductionmentioning

confidence: 99%

Production of Acrylamide using Alginate‐Immobilized E. coli Expressing Comamonas testosteroni 5‐MGAM‐4D Nitrile Hydratase

et al. 2005

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A thermally-stable nitrile hydratase produced by Comamonas testosteroni 5-MGAM-4D has been expressed in Escherichia coli, and the alginateimmobilized transformant evaluated as catalyst for the conversion of acrylonitrile to acrylamide. In batch reactions with catalyst recycle, the catalyst productivity decreased with increasing acrylonitrile concentration or reaction temperature, but was relatively insensitive to acrylamide concentration. A total of 206 consecutive batch reactions with catalyst recycle were run at 5 8C and produced 1035 g acrylamide/g dry cell weight and 95 g acrylamide/L; the initial and final volumetric productivities of this series of recycle reactions were 197 and 96 g acrylamide/L/h, respectively. A packed column reactor yielded lower catalyst productivity than consecutive batch recycle reactions, presumably due to contact of the fixed-bed catalyst with a constant high concentration of acrylonitrile.

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Cloning and sequencing of a nitrile hydratase gene from Pseudonocardia thermophila JCM3095

Cited by 62 publications

References 6 publications

Unraveling the Catalytic Mechanism of Nitrile Hydratases

Unraveling the Catalytic Mechanism of Nitrile Hydratases

Purification of a hyperactive nitrile hydratase from resting cells of Rhodococcus rhodochrous PA-34

Production of Acrylamide using Alginate‐Immobilized E. coli Expressing Comamonas testosteroni 5‐MGAM‐4D Nitrile Hydratase

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