Cobalt-dependent transcription of the nitrile hydratase gene in Rhodococcus rhodochrous M8

Abstract The effects of cobalt ions on the activities of Rhodococcus rhodochrous M8 enzymes for nitrile utilization, nitrile hydratase and amidase, were investigated. In contrast to amidase, synthesis of nitrile hydratase and its activity required cobalt ions in the growth medium. Northern blot analysis showed that in the presence of cobalt ions, the level of mRNA for nitrile hydratase genes was several times higher than that under cobalt-limited conditions. It was assumed that the low nitrile hydratase activity in cells grown in the absence of cobalt ions is connected either with the weak expression of nitrile hydratase genes or with the rapid degradation of nitrile hydratase mRNA.     

Fed-batch fermentation for production of nitrile hydratase byRhodococcus rhodochrous M33

To enhance the productivity and activity of nitrile hydratase inRhodococcus rhodochrous M33, a glucose-limited fed-batch culture was performed. In a fed-batch culture where the glucose was controlled at a limited level and cobalt was supplemented during the fermentation period, the cell mass and total activity of nitrile hydratase both increased 3.3-fold compared to that in the batch fermentation. The productivity of nitrile hydratase also increased 1.9-fold compared to that in the batch fermentation. The specific activity of nitrile hydratase in the whole cell preparation when using a fed-batch culture was 120 units/mg-DCW, which was similar to that in the batch culture.     

Optimum culture conditions for the production of cobalt-containing nitrile hydratase by Rhodococcus rhodochrous J1

Summary 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.Offprint requests to: T. Nagasawa     

Expression of nitrile hydratase gene of mutant 4D strain of Rhodococcus rhodochrous PA 34 in Pichia pastoris

The nitrile hydratase (NHase) gene of Rhodococcus rhodochrous PA-34 mutant 4D has been amplified by PCR, cloned and expressed in Pichia pastoris KM-71 using pHIL-D2 expression vector. The recombinant P. pastoris KM-71 exhibited active expression of the nitrile hydratase gene of the mutant 4D and has shown very good potential for the transformation of 3-cyanopyridine to nicotinamide. The recombinant P. pastoris KM-71 exhibited maximum NHase activity when cultivated in YPD medium was supplemented with 0.4?mM cobalt ions. The recombinant P. pastoris KM-71 showed maximum nitrile hydratase enzyme production, when incubated at 30?°C for 15?h.     

High resolution X-ray molecular structure of the nitrile hydratase from Rhodococcus erythropolis AJ270 reveals posttranslational oxidation of two cysteines into sulfinic acids and a novel biocatalytic nitrile hydration mechanism

The crystal structure of Fe-type nitrile hydratase from Rhodococcus erythropolis AJ270 was determined at 1.3A resolution. The two cysteine residues (alphaCys(112) and alphaCys(114)) equatorially coordinated to the ferric ion were post-translationally modified to cysteine sulfinic acids. A glutamine residue (alphaGln(90)) in the active center gave double conformations. Based on the interactions among the enzyme, substrate and water molecules, a new mechanism of biocatalysis of nitrile hydratase was proposed, in which the water molecule activated by the glutamine residue performed as the nucleophile to attack on the nitrile which was simultaneously interacted by another water molecule coordinated to the ferric ion.     

Characterization of a new cobalt-containing nitrile hydratase purified from urea-induced cells of Rhodococcus rhodochrous J1

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 (alpha subunit 26 kDa, beta subunit 29 kDa). 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 beta subunit. However, the amino acid sequence of the alpha subunit from R. rhodochrous J1 was quite different from that of the ferric enzymes.     

Effects of nitriles and amides on the growth and the nitrile hydratase activity of the Rhodococcus sp. strain gt1

Effects of some nitriles and amides, as well as glucose and ammonium, on the growth and the nitrile hydratase (EC 4.2.1.84) activity of the Rhodococcus sp. strain gt1 isolated from soil were studied. The activity of nitrile hydratase mainly depended on carbon and nitrogen supply to cells. The activity of nitrile hydratase was high in the presence of glucose and ammonium at medium concentrations and decreased at concentrations of glucose more than 0.3%. Saturated unsubstituted aliphatic nitriles and amides were found to be a good source of nitrogen and carbon. However, the presence of nitriles and amides in the medium was not absolutely necessary for the expression of the activity of nitrile hydratase isolated from the Rhodococcus sp. strain gt1.     

Nitrile hydratase involved in aldoxime metabolism from Rhodococcus sp. strain YH3-3 purification and characterization.

Nitrile hydratase responsible for aldoxime metabolism from the E-pyridine-3-aldoxime degrading bacterium, Rhodococcus sp. strain YH3-3 was purified and characterized. Addition of cobalt ion was necessary for the formation of enzyme. The enzyme activity was highly induced not only by nitriles and amides but also by several aldoxime compounds. The enzyme was purified approximately 108-fold with a 16% yield from the cell-free extract of the strain. The native enzyme had a Mr of approximately 130 000 and consisted of two subunits (alpha-subunit, 27 100; beta-subunit, 34 500). The enzyme contained approximately 2 mol cobalt per mol enzyme; it showed a maximum activity at 60 degrees C and at 40 degrees C under the rate assay and end-point assay conditions, respectively, and was stable over a wide range of pH (pH 2.5-11.0). The enzyme had a wide substrate specificity: it acted on aliphatic saturated and unsaturated as well as aromatic nitriles. The N-terminus of the beta-subunit showed good sequence similarities with those of other nitrile hydratases. Nitrile hydratase is part of the metabolic pathway for aldoximes in microorganisms.     

A study of the catalytic properties of the nitrile hydratase immobilized on aluminum oxides and carbon-containing adsorbents

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.     

Rhodococcus pyridinovorans MW3, a bacterium producing a nitrile hydratase

Rhodococcus pyridinovorans MW3 was isolated from an arable land of manioc from the Congo for its ability to transform acrylonitrile to acrylamide. This strain contains a cobalt nitrile hydratase (NHase) showing high sequence homology with NHases so far described. The specific NHase activity was 97 U mg(-1) dry wt. NHase production by R. pyridinovorans MW3 was urea and Co-dependent. The NHase was active for acrylamide up to 60% (w/v) indicating its potential for acrylamide production.     

Crystal structure of cobalt-containing nitrile hydratase.

The crystal structure of cobalt-containing nitrile hydratase from Pseudonocardia thermophila JCM 3095 at 1.8 A resolution revealed the structure of the noncorrin cobalt at the catalytic center. Two cysteine residues (alphaCys(111) and alphaCys(113)) coordinated to the cobalt were posttranslationally modified to cysteine-sulfinic acid and to cysteine-sulfenic acid, respectively, like in iron-containing nitrile hydratase. A tryptophan residue (betaTrp(72)), which may be involved in substrate binding, replaced the tyrosine residue of iron-containing nitrile hydratase. The difference seems to be responsible for the preference for aromatic nitriles rather than aliphatic ones of cobalt-containing nitrile hydratase.     

Adaptation of acrylamide producer Rhodococcus rhodochrous M8 to change in ammonium concentration in medium

The mechanism of adaptation of the acrylamide producing strain Rhodococcus rhodochrous M8 to changes in ammonium concentrations in the medium was studied. An increase in the content of ammonium in the medium changed the activity of glutamine synthetase (GS) (EC 6.3.1.2) and glutamine dehydrogenase (GD) (EC 1.4.1.4), the enzymes of ammonium assimilation, as well as the activities of enzymes responsible for nitrile utilization: nitrile hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4). This also caused inhibition of activation of GS induced by phosphodiesterase (EC 3.1.4.1). Increases in the activities of nitrile hydratase and amidase and resistance of these enzymes to ammonium were observed in mutant of R. rhodichrous resistant to phosphotricine, an inhibitor of GS. An important role of GS in the mechanism of adaptation is suggested.     

Immobilization of Rhodococcus ruber strain gt1, possessing nitrile hydratase activity, on carbon sorbents

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.     

Real-time monitoring of nitrile biotransformations by mid-infrared spectroscopy

In this study mid-infrared spectroscopy was used to follow the enzyme kinetics involved in nitrile biocatalysis using whole cell suspensions of the bacterium Rhodococcus rhodochrous LL100-21. The bacteria were grown on acetonitrile to induce a two-step enzymatic pathway. Acetonitrile was biotransformed to acetamide by a nitrile hydratase enzyme and subsequently to acetic acid (carboxylate ion) by an amidase enzyme. The bacteria were also grown on benzonitrile to induce a one-step enzymatic pathway. Benzonitrile was biotransformed directly to benzoic acid (carboxylate ion) by a nitrilase enzyme. These reactions were followed by React IR using a silicon probe and gave excellent quantitative and qualitative real-time data of both nitrile biocatalytic reactions. This study has shown that this novel technique has potentially useful applications in biocatalysis.     

Immobilization of <Emphasis Type="Italic">Rhodococcus ruber</Emphasis> strain gt1, possessing nitrile hydratase activity,on carbon supports

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.     

Transformation of 2- and 4-cyanopyridines by free and immobilized cells of nitrile-hydrolyzing bacteria

The transformation dynamics of 2- and 4-cyanopyridines by cells suspended and adsorbed on inorganic carriers has been studied in the Rhodococcus ruber gt1 possessing nitrile hydratase activity and the Pseudomonas fluorescens C2 containing nitrilase. It was shown that both nitrile hydratase and nitrilase activities of immobilized cells against 2-cyanopyridine were 1.5–4 times lower compared to 4-cyanopyridine and 1.6–2 times lower than the activities of free cells against 2-cyanpopyridine. The possibility of obtaining isonicotinic acid during the combined conversion of 4-cyanopyridine by a mixed suspension of R. ruber gt1 cells with a high level of nitrile hydratase activity and R. erythropolis 11-2 cells with a pronounced activity of amidase has been shown. Immobilization of Rhodococcus cells on raw coal and Pseudomonas cells on kaolin was shown to yield a heterogeneous biocatalyst for the efficient transformation of cyanopyridines into respective amides and carboxylic acids.     

An enzyme controlled by light: the molecular mechanism of photoreactivity in nitrile hydratase.

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.     

Metallochaperone function of the self-subunit swapping chaperone involved in the maturation of subunit-fused cobalt-type nitrile hydratase

The transition metal (iron or cobalt) is a mandatory part that constitutes the catalytic center of nitrile hydratase (NHase). The incorporation of the cobalt ion into cobalt-containing NHase (Co-NHase) was reported to depend on self-subunit swapping and the activator of the Co-NHase acts as a self-subunit swapping chaperone for subunit exchange. Here we discovered that the activator acting as a metallochaperone transferred the cobalt ion into subunit-fused Co-NHase. We successfully isolated two activators, P14K and NhlE, which were the activators of NHases from Pseudomonas putida NRRL-18668 and the activator of low-molecular-mass NHase from Rhodococcus rhodochrous J1, respectively. Cobalt content determination demonstrated that NhlE and P14K were two cobalt-containing proteins. Substitution of the amino acids involved in the C-terminus of the activators affected the activity of the two NHases, indicating that the potential cobalt-binding sites might be located at the flexible C-terminal region. The cobalt-free NHases could be activated by either of the two activators, and both the two activators activated their cognate NHase more efficiently than did the noncognate ones. This study provided insights into the maturation of subunit-fused NHases and confirmed the metallochaperone function of the self-subunit swapping chaperone.     

Effect of agitation and aeration on the production of nitrile hydratase by Rhodococcus erythropolis MTCC 1526 in a stirred tank reactor

Aims: To evaluate the effect of different physicochemical parameters such as agitation, aeration and pH on the growth and nitrile hydratase production by Rhodococcus erythropolis MTCC 1526 in a stirred tank reactor. Methods and Results: Rhodococcus erythropolis MTCC 1526 was grown in 7‐l reactor at different agitation, aeration and controlled pH. The optimum conditions for batch cultivation in the reactor were an agitation rate of 200 rev min^?1, aeration 0·5 v/v/m at controlled pH 8. In this condition, the increase in nitrile hydratase activity was almost threefold compared to that in the shake flask. Conclusion: Agitation and aeration rate affected the dissolved‐oxygen concentration in the reactor which in turn affected the growth and enzyme production. Significance and Impact of the Study: Cultivation of R. erythropolis MTCC 1526 in the reactor was found to have significant effect on the growth and nitrile hydratase production when compared to the shake flask.