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1.
The enantioselective nitrile hydratase from the bacterium Agrobacterium tumefaciens d3 was purified and completely separated from the amidase activity that is also present in cell extracts prepared from this
strain. The nitrile hydratase had an activity optimum at pH 7.0 and a temperature optimum of 40 °C. The holoenzyme had a molecular
mass of 69 kDa, the subunits a molecular mass of 27 kDa. The enzyme hydrated various 2-arylpropionitriles and other aromatic
and heterocyclic nitriles. With racemic 2-phenylpropionitrile, 2-phenylbutyronitrile, 2-(4-chlorophenyl)propionitrile, 2-(4-methoxy)propionitrile
or ketoprofen nitrile the corresponding (S)-amides were formed enantioselectively. The highest enantiomeric excesses (ee >90% until about 30% of the respective substrates
were converted) were found for the amides formed from 2-phenylpropionitrile, 2-phenylbutyronitrile and ketoprofen nitrile.
For the reaction of the purified nitrile hydratase, higher ee values were found than when whole cells were used in the presence
of an inhibitor of the amidase activity. The enantioselectivity of the whole-cell reaction was enhanced by increasing the
reaction temperature.
Received: 20 June 1997 / Received revision: 28 August 1997 / Accepted: 29 August 1997 相似文献
2.
Tauber MM Cavaco-Paulo A Robra K Gübitz GM 《Applied and environmental microbiology》2000,66(4):1634-1638
Rhodococcus rhodochrous NCIMB 11216 produced nitrile hydratase (320 nkat mg of protein(-1)) and amidase activity (38.4 nkat mg of protein(-1)) when grown on a medium containing propionitrile. These enzymes were able to hydrolyze nitrile groups of both granular polyacrylonitriles (PAN) and acrylic fibers. Nitrile groups of PAN40 (molecular mass, 40 kDa) and PAN190 (molecular mass, 190 kDa) were converted into the corresponding carbonic acids to 1.8 and 1.0%, respectively. In contrast, surfacial nitrile groups of acrylic fibers were only converted to the corresponding amides. X-ray photoelectron spectroscopy analysis showed that 16% of the surfacial nitrile groups were hydrolyzed by the R. rhodochrous enzymes. Due to the enzymatic modification, the acrylic fibers became more hydrophilic and thus, adsorption of dyes was enhanced. This was indicated by a 15% increase in the staining level (K/S value) for C. I. Basic Blue 9. 相似文献
3.
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. 相似文献
4.
5.
A petrochemical wastewater isolate, capable of utilizing high concentrations of acetonitrile and acetamide as the sole source of carbon and nitrogen was identified as Rhodococcus erythropolis A10. Cell-free extracts of acetonitrile-grown cells exhibited activities corresponding to nitrile hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4), which mediate the two-step breakdown of acetonitrile into acetic acid and ammonia. Studies indicated that both these enzymes in R. erythropolis A10 are intracellular, inducible and capable of hydrolysing a wide range of nitriles, including simple (acetonitrile, propionitrile), branched-chain (isobutyronitrile) and dinitrile (succinonitrile). The specific activity of the amidase was found to be several-fold higher than nitrile hydratase. 相似文献
6.
Characterization of a new cobalt-containing nitrile hydratase purified from urea-induced cells of Rhodococcus rhodochrous J1 总被引:2,自引:0,他引:2
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. 相似文献
7.
Yu. G. Maksimova A. Yu. Maksimov V. A. Demakov K. V. Kozlov G. V. Ovechkina V. F. Olontsev 《Applied Biochemistry and Microbiology》2011,47(7):681-687
Cells of the Pseudomonas fluorescens strain C2 containing nitrilase and Rhodococcus ruber strain gt1 with nitrile hydratase activity have been immobilized by the use of adsorption on fibrous carbon materials. It
has been shown that the maximum adsorption value of Rhodococcus cells is higher than that in pseudomonades, reaching 21 mg
of dry cells/1 g of the carrier vs. 6 mg, respectively. Cell adsorption, compared to cell suspension, gives a significant
rise in nitrilase activity (by 7.4 times, using Ural TM-4 as the carrier) and in the stability of nitrile hydratase activity
(5 reaction cycles without loss of activity, using Carbopon-B-active). Immobilized biocatalysts were also obtained by cell
growth from Ps. fluorescens strain C2 and Rhodococcus ruber strain gt1 on fibrous carbon adsorbents. Biocatalyst productivity was higher for both strains when the carbonized material
Ural TM-4 was used as the carrier. 相似文献
8.
Nitrile hydratase from Rhodococcus sp. DSM 11397 and nitrilase from Pseudomonas. DSM 11387 both retained activity in various organic/aqueous biphasic mixtures. Both enzymes were most tolerant (66–109% retained activity) of C8 and C16 alkanes with log P values greater than 4.0. Some enzyme activity (<10% nitrile hydratase, <60% nitrilase) was also retained in monophasic water-saturated C6-C11 n-alkanols. © Rapid Science Ltd. 1998 相似文献
9.
Nitrile hydratase of Pseudomonas chlororaphis B23. Purification and characterization 总被引:14,自引:0,他引:14
T Nagasawa H Nanba K Ryuno K Takeuchi H Yamada 《European journal of biochemistry》1987,162(3):691-698
Nitrile hydratase of Pseudomonas chlororaphis B23 was completely stabilized by the addition of 22 mM n-butyric acid. The enzyme was purified from extracts of methacrylamide-induced cells of P. chlororaphis B23 in eight steps. At the last step, the enzyme was crystallized by adding ammonium sulfate. The crystallized enzyme appeared to be homogeneous from analysis by polyacrylamide gel electrophoresis, analytical ultracentrifuge, and double diffusion in agarose. The enzyme has a molecular mass of about 100 kDa and consists of four subunits identical in molecular mass (approximately 25 kDa). The enzyme contained approximately 4 mol iron/mol enzyme. The concentrated solution of highly purified nitrile hydratase had a pronounced greyish green color and exhibited a broad absorption in visible range with a absorption maxima at 720 nm. A loss of enzyme activity occurred in parallel with the disappearance of the absorption in the visible range under a variety of conditions. The enzyme catalyzed stoichiometrically the hydration of nitrile to amide, and no formation of acid and ammonia were detected. The enzyme was active toward various aliphatic nitriles, particularly, nitriles with 3-6 carbon atoms, e.g. propionitrile, n-butyronitrile, acrylonitrile and cyclopropyl cyanide, served as the most suitable substrates. 相似文献
10.
David Kub
Ondej Kaplan Veronika Elikov Miroslav Ptek Vojtch Vejvoda Kristýna Slmov Andrea Tthov Marielle Lemaire Estelle Gallienne Sabine Lutz-Wahl Lutz Fischer Marek Kuzma Helena Pelantov Sander van Pelt Jean Bolte Vladimír Ken Ludmila Martínkov 《Journal of Molecular Catalysis .B, Enzymatic》2008,50(2-4):107-113
A semi-purified nitrile hydratase from Rhodococcus erythropolis A4 was applied to biotransformations of 3-oxonitriles 1a–4a, 3-hydroxy-2-methylenenitriles 5a–7a, 4-hydroxy-2-methylenenitriles 8a–9a, 3-hydroxynitriles 10a–12a and 3-acyloxynitrile 13a into amides 1b–13b. Cross-linked enzyme aggregates (CLEAs) with nitrile hydratase and amidase activities (88% and 77% of the initial activities, respectively) were prepared from cell-free extract of this microorganism and used for nitrile hydration in presence of ammonium sulfate, which selectively inhibited amidase activity. The genes nha1 and nha2 coding for and β subunits of nitrile hydratase were cloned and sequenced. 相似文献
11.
12.
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. 相似文献
13.
Maksimov AIu Kuznetsova MV Ovechkina GV Kozlov SV Maksimova IuG Demakov VA 《Prikladnaia biokhimiia i mikrobiologiia》2003,39(1):63-68
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. 相似文献
14.
Yu. G. Maksimova A. N. Gorbunova V. A. Demakov 《Doklady. Biochemistry and biophysics》2017,474(1):183-185
We studied the effect of a heterogeneous environment on the stereoselectivity of transformation of racemic phenylglycine nitrile. Immobilized biocatalysts were prepared by adhesion of Pseudomonas fluorescens C2 cells on carbon-containing supports and covalent crosslinking of nitrile hydratase and amidase of Rhodococcus rhodochrous 4–1 to activated chitosan as well as by the method of cross-linked aggregates. At a reaction duration of 20 h, the ratio of phenylglycine stereoisomers changes depending on the presence of support in medium. The highest optical purity of the product (enantiomeric excess of L-phenylglycine solution, 98%) is achieved when enzyme aggregates of nitrile hydratase and amidase cross-linked with 0.1% glutaraldehyde are used as a biocatalyst. 相似文献
15.
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. 相似文献
16.
Yu. G. Maksimova D. M. Vasilyev G. V. Ovechkina A. Yu. Maksimov V. A. Demakov 《Applied Biochemistry and Microbiology》2013,49(4):347-351
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. 相似文献
17.
Occurrence of a cobalt-induced and cobalt-containing nitrile hydratase in Rhodococcus rhodochrous J1 总被引:6,自引:0,他引:6
T Nagasawa K Takeuchi H Yamada 《Biochemical and biophysical research communications》1988,155(2):1008-1016
The formation of nitrile hydratase required cobalt ions in Rhodococcus rhodochrous J1. No other transition-metals could replace the cobalt ion. The Rhodococcus nitrile hydratase was purified to homogeneity and found to contain a cobalt atom. The occurrence of a cobalt-induced and cobalt-containing nitrile hydratase, different from the nitrile hydratases in Pseudomonas chlororaphis B23 and Brevibacterium R312 containing a ferric ion in their active center, has been demonstrated here for the first time. 相似文献
18.
Yu. G. Maksimova T. A. Rogozhnikova G. V. Ovechkina A. Yu. Maksimov V. A. Demakov 《Applied Biochemistry and Microbiology》2012,48(5):434-439
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. 相似文献
19.
Yu. G. Maksimova V. A. Demakov A. Yu. Maksimov G. V. Ovechkina G. A. Kovalenko 《Applied Biochemistry and Microbiology》2010,46(4):379-384
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. 相似文献
20.
The thermophilic soil isolate, Bacillus pallidus Dac521, expresses a constitutive nitrile hydratase. The purified enzyme was found to be a 110 kDa tetramer composed of two alpha and two beta subunits with molecular masses of 27 kDa and 29 kDa, respectively. The enzyme electrophoresed as a single protein band on native PAGE but two protein bands with isoelectric points of 4.7 and 5.5 on isoelectric focusing suggested the presence of isozymes. The purified enzyme was moderately thermostable up to 55 degrees C and the enzyme activity was stable over a broad pH range. Comparisons of the N-terminal amino acid sequences of the nitrile hydratase subunits with those of other nitrile hydratases showed up to 90% identity for the beta subunit sequence but no significant identity for the alpha subunit. The enzyme hydrolysed a narrow range of aliphatic substrates and did not hydrolyse any of the cyclic, hydroxy-, di- or aromatic nitriles tested. The activity was irreversibly inhibited by the aromatic nitrile, benzonitrile. The kinetic constants for acetonitrile, acrylonitrile and propionitrile compared favourably with those of mesophilic nitrile hydratases. 相似文献