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1.
Summary Forty yeast strains were screened for nitrile-hydrolysing activity. Among them Kluyveromyces thermotolerans MGBY 37 exhibited highest nitrile-hydrolysing activity (0.030 μmol/h/mg dry cell weight). This yeast contained a two-enzyme system i.e. nitrile hydratase (NHase, EC 4.2.1.84) and amidase (EC 3.5.1.4) for the hydrolysis of nitriles/amides to corresponding acids and ammonia. However, these enzymes had more affinity for N-heterocyclic aromatic and aromatic nitriles/amides rather than unsaturated and saturated aliphatic nitriles/amides. The NHase–amidase activity was constitutively produced by K. thermotolerence MGBY 37. Addition of acetonitrile in the medium enhanced the production of this activity while other nitriles and amides lowered the production of NHase–amidase activity. This organism thus exhibited two types of amidase i.e. a constitutive amidase having affinity for N-heterocyclic aromatic, unsaturated and saturated aliphatic amides and another inducible amidase with affinity for aromatic amides. Formamide proved to be the best inducer of the latter amidase activity. This is the first report on nitrile- and amide-hydrolysing activity in Kluyveromyces.  相似文献   

2.
 We investigated the optimum conditions for the formation of nitrile hydratase (NHase), which acts on indole-3-acetonitrile, in Agrobacterium tumefaciens. Good inducers for enzyme formation have been found to be roughly classified into three representative types of amides such as pivalamide, crotonamide and ɛ-caprolactam. When the strain was cultivated in the optimum culture medium containing ɛ-caprolactam as an inducer, in particular, the specific activity of NHase in the culture was 13 000 times higher than that without addition of amides, nitriles or acids. In this case, NHase formed accounted for 12% of the total cellular soluble protein. The purified NHase did not act on ɛ-caprolactam, and ɛ-caprolactam was not degraded during the cultivation by the strain, suggesting that ɛ-caprolactam seems to keep driving the NHase induction mechanism. Received: 3 March 1995/Received revision: 13 July 1995/Accepted: 7 September 1995  相似文献   

3.
A versatile nitrile-degrading bacterium was isolated by enrichment culture from the soil of a forest near Manali, Himachal Pradesh, India, and was identified as Nocardia globerula. This organism contains 3 enzymes with nitrile-degrading activity: nitrilase, nitrile hydratase, and amidase. Nocardia globerula NHB-2 cells grown on nutrient broth supplemented with 1% glucose and 0.1% yeast extract exhibited nitrile hydratase-amidase activity specific for saturated aliphatic nitriles or amide, while addition of acetonitrile in nutrient broth yielded cells with nitrile hydratase-amidase that in addition to saturated aliphatic nitriles-amide also hydrolyzed aromatic amide. Nocardia globerula NHB-2 cultivated on nutrient broth containing propionitrile exhibited nitrilase activity that hydrolyzed aromatic nitrile and unsaturated aliphatic nitrile. The versatility of this organism in the hydrolysis of various nitriles and amides makes it a potential bioresource for use in organic synthesis.  相似文献   

4.
Nitrile metabolising actinomycetes previously recovered from deep-sea sediments and terrestrial soils were investigated for their nitrile transforming properties. Metabolic profiling and activity assays confirmed that all strains catalysed the hydrolysis of nitriles by a nitrile hydratase/amidase system. Acetonitrile and benzonitrile, when used as growth substrates for enzyme induction experiments, had a significant influence on the biotransformation activities towards various nitriles and amides. The specific activities of selected deep-sea and terrestrial acetonitrile-grown bacteria against a suite of nitriles and amides were higher than those of the only other reported marine nitrile-hydrolysing R. erythropolis, isolated from a shallow sediment. The increase of nitrile chain length appeared to have negative influence on the nitrile hydratase activity of acetonitrile-grown bacteria, but the same was not true for benzonitrile-grown bacteria. The nitrile hydratases and amidases were constitutive in 10 of the 16 deep-sea and terrestrial actinomycetes studied, and one strain showed an inducible hydratase and a constitutive amidase. Most of the deep-sea strains had constitutive activities and showed some of the highest activities and broadest substrate specificities of organisms included in this study. This revised version was published online in August 2006 with corrections to the Cover Date.  相似文献   

5.
In this study, nitriles were used as sole sources of nitrogen in the enrichments to isolate nitrile-converting microorganisms. A novel fungus named ZJB-09150 possessing nitrile-converting enzymes was obtained with 3-cyanopyridine as sole source of nitrogen, which was identified by morphology, biology and 18S rDNA gene sequence as Fusarium proliferatum. It was found that F. proliferatum had ability to convert nitriles to corresponding acids or amides and showed wide substrate specificity to aliphatic nitriles, aromatic nitriles and ortho-substituted heterocyclic nitriles. The nitrile converting enzymes including nitrilase and nitrile hydratase in ZJB-09150 were induced by ε-caprolactam. Nitrilase obtained in this study showed high activity toward 3-cyanopyridine. It was active within pH 3.0–12.0 and temperature ranging from 25 to 65 °C with optimal at pH 9.0 and temperature 50–55 °C. The enzyme was thermostable and its half-life was 12.5 and 6 h at 45 and 55 °C, respectively. Under optimized reaction conditions, 60 mM 3-cyanopyridine was converted to nicotinic acid in 15 min, which indicated ZJB-09150 has potentials of application in large scale production of nicotinic acid.  相似文献   

6.
Biocatalytic transformations converting aromatic and arylaliphatic nitriles into the analogous related amide or acid were investigated. These studies included synthesis of the -substituted nitrile 3-hydroxy-3-phenylpropionitrile, subsequent enrichment and isolation on this substrate of nitrile-degrading microorganisms from the environment, and a comparative study of enzymatic reactions of nitriles by resting cell cultures and enzymes. Each biocatalyst exhibited a distinctive substrate selectivity profile, generally related to the length of the aliphatic chain of the arylaliphatic nitrile and the position of substituents on the aromatic ring or aliphatic chain. Cell-free nitrilases generally exhibited a narrower substrate range than resting whole cells of Rhodococcus strains. The Rhodococcus strains all exhibited nitrile hydratase activity and converted -hydroxy nitriles (but did not demonstrate enantioselectivity on this substrate). The biocatalysts also mediated the synthesis of a range of -hydroxy carboxylic acids or amides from aldehydes in the presence of cyanide. The use of an amidase inhibitor permits halting the nitrile hydratase/amidase reaction at the amide intermediate.  相似文献   

7.
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 the 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 of 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 of the Rhodococcus sp. strain gt1.  相似文献   

8.
《Process Biochemistry》2010,45(6):866-873
Strain Amycolatopsis sp. IITR215 was isolated from a sewage sample using polyacrylonitrile powder as the sole nitrogen source. Identification was performed by 16S rDNA analysis. The isolated strain harbored multiple nitrile-metabolizing enzymes having a wide range of substrate specificities. It metabolized nitrile and amide compounds with constitutive enzymes. Studies using an amidase inhibitor showed that hydrolysis of acrylonitrile and acrylamide occurred due to nitrile hydratase and amidase, respectively, while hydrolysis of hexanenitrile was due to the action of either nitrilase or a second nitrile hydratase/amidase system. The inhibitory effects of N-bromosuccinimide and N-ethylmaleimide on enzymes of this culture were also studied and this further indicated the involvement of either a nitrilase or a second nitrile hydratase/amidase system for hydrolysis of hexanenitrile. Interestingly, hexanenitrile hydrolysis exhibited an optimum temperature of 55 °C, whereas acrylonitrile and acrylamide hydrolysis showed an optimum temperature of 45 °C. The optimum pH was 5.8 for hexanenitrile hydrolysis and 7.0 for acrylonitrile and acrylamide hydrolysis. Hexanenitrile hydrolysis by enzymes of this strain showed better organic solvent tolerance in the presence of alcohols. The maximum enzyme activity of nitrile-metabolizing enzymes was found using media containing isobutyramide as the nitrogen source. This is the first report on constitutive multiple enzymes from the Amycolatopsis genus.  相似文献   

9.
Pyrazinamide has received considerable attention for its effective antibacterial action in the reappearance of tuberculosis and for its broad application in the chemical industry. In this study, a 2-cyanopyrazine-degrading bacterial strain, numbered ZJB-09104, was newly isolated and identified as Serratia marcescens, based on its physiological and biological tests, ATB system analysis, and 16S rDNA sequence analysis. The strain exhibits only nitrile hydratase (NHase) activity and this NHase belongs to the cobalt NHase family of enzymes. Thermostability tests suggested that the NHase is thermophilic with an optimum temperature of 50°C. The NHase was effective in converting nitriles to the corresponding amides under the conditions of temperature 50°C and time course 7 h, respectively.  相似文献   

10.
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   

11.
Amides of very good chemical quality have been obtained by the bioconversion of the corresponding nitriles using immobilized bacteria with nitrilase activity (nitrile hydratase). The bioreactor was polyvalent since the nitrilase used had a generalized activity and could hydrolyse a large number of nitriles. The present paper shows the polyvalence and simplicity of the process, and the quality of the amides obtained.  相似文献   

12.
Jin LQ  Li YF  Liu ZQ  Zheng YG  Shen YC 《New biotechnology》2011,28(6):610-615
2-Chloronicotinic acid is receiving much attention for its effective applications as a key precursor in the synthesis of pesticides and medicines. In this study, a strain ZJB-09149 converting 2-chloro-3-cyanopyridine to 2-chloronicotinic acid was newly isolated and identified as Rhodococcus erythropolis, based on its physiological and biological tests, and 16S rDNA sequence analysis. In addition, the effects of inducer, carbon source and nitrogen source were examined. Maximum activity was achieved when the above parameters were set as 8 g/l ?-caprolactam, 7 g/l yeast extract and 5 g/l maltose. Moreover, the biotransformation pathway of 2-chloro-3-cyanopyridine to 2-chloronicotinic acid in strain ZJB-09149 was investigated as well. This study revealed that the nitrile hydratase (NHase) and amidase expressed in R. erythropolis ZJB-09149 are responsible for the conversion of 2-chloro-3-cyanopyridine. This is the first time to report on the biotransformation preparation of 2-chloronicotinic acid.  相似文献   

13.
Aldoxime–nitrile pathway is one of the important routes of carbon and nitrogen metabolism in many life forms and a key interface for plant–microbe interactions. This pathway starts with transformation of amino acids to aldoximes, which are converted to nitriles and the later are ultimately hydrolyzed to acids and ammonia. Understanding and engineering of the enzymes involved in this pathway viz. cytochrome P450/CYP79, aldoxime dehydratase, nitrilase, nitrile hydratase, amidase and hydroxynitrile lyase, presents unprecedented opportunities in biocatalysis and green chemistry. Co-expressing these enzymes in prokaryotic and eukaryotic microbial hosts and tailoring their properties i.e. activity, specificity, stability and enantioselectivity may lead to develop sustainable bioprocesses for the synthesis of industrially important nitriles, amides and acids.  相似文献   

14.
腈化合物是一类重要的用于合成多种精细化学品的化合物,它们容易制备,并且可以合成多种化合物。传统化学水解方法将腈化合物转化为相应的羧酸或酰胺通常需要高温、强酸、强碱等相对苛刻的条件,腈转化酶(腈水解酶、腈水合酶和酰胺酶)由于其生物催化过程具有高效、高选择性、条件温和等特点,在精细化学品的合成中越来越受到人们的关注。许多腈转化酶已经被开发出来并用于精细化学品的生产。以下介绍了腈转化酶在医药及中间体、农药及中间体、食品与饲料添加剂等精细化学品生产中的应用。随着研究的不断深入,将会有更多的腈转化酶被开发出来并用于精细化学品的生产。  相似文献   

15.
A Gram-negative bacterial strain, identified as Acidovorax facilis strain 72W, has been isolated from soil by enrichment using 2-ethylsuccinonitrile as the sole nitrogen source. This strain grows on a variety of aliphatic mono- and dinitriles. Experiments using various heating regimes indicate that nitrile hydratase, amidase and nitrilase activities are present. The nitrilase is efficient at hydrolyzing aliphatic dinitriles to cyanoacid intermediates. It has a strong bias for C3–C6 dinitriles over mononitriles of the same chain length. Whole, resting cell hydrolysis of 2-methylglutaronitrile results in 4-cyanopentanoic acid and 2-methylglutaric acid as the major products. Heating, at least 20 min at 50 °C, eliminates nitrile hydratase and amidase activities, resulting in greater than 97% selectivity to 4-cyanopentanoic acid. The nitrilase activity has good heat stability, showing a half-life of 22.7 h at 50 °C and a temperature optimum of at least 65 °C for activity. The strain has been deposited as ATCC 55746. Received: 26 January 1999 / Received revision: 10 June 1999 / Accepted: 27 June 1999  相似文献   

16.
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   

17.
The reaction conditions towards the preferential action of either nitrile hydratase or amidase in the harvested whole cells of Rhodococcus rhodochrous IFO 15564 were elaborated. The amidase showed higher heat tolerance than the nitrile hydratase and, at 45 °C the amidase worked exclusively. DMSO assisted the preferential action of nitrile hydratase, however, at more than 30% (v/v) addition of DMF, the nitrile hydratase activity was completely lost and only amidase worked. A one-pot chemo-enzymatic conversion of aldehydes to amides [(1) aq. NH3, I2, DMSO; (2) Na2S2O3; (3) harvested cells of R. rhodochrous] was established. Under these reaction conditions, most of the amidase was lost, and the incubation of the firstly formed intermediates, nitriles in aq. NH3 was responsible for the selective inhibition of amidase. The freezing of harvested cells in an exhaustively deionized environment provided a long-term preservable “ready to use” for the organic chemist.  相似文献   

18.
Rapid and direct screening of nitrile-converting enzymes is of great importance in the development of industrial biocatalytic process for pharmaceuticals and fine chemicals. In this paper, a combination of ferrous and ferric ions was used to establish a novel colorimetric screening method for nitrile hydratase and amidase with α-amino nitriles and α-amino amides as substrates, respectively. Ferrous and ferric ions reacted sequentially with the cyanide dissociated spontaneously from α-amino nitrile solution, forming a characteristic deep blue precipitate. They were also sensitive to weak basicity due to the presence of amino amide, resulting in a yellow precipitate. When amino amide was further hydrolyzed to amino acid, it gave a light yellow solution. Mechanisms of color changes were further proposed. Using this method, two isolates with nitrile hydratase activity towards 2-amino-2,3-dimethyl butyronitrile, one strain capable of hydrating 2-amino-4-(hydroxymethyl phosphiny) butyronitrile and another microbe exhibiting amidase activity against 2-amino-4-methylsulfanyl butyrlamide were obtained from soil samples and culture collections of our laboratory. Versatility of this method enabled it the first direct and inexpensive high-throughput screening system for both nitrile hydratase and amidase.  相似文献   

19.
An enrichment culture from saline soda soils, using acetate as carbon and energy source and 2-phenylpropionitrile as nitrogen source (PPN) at pH 10, resulted in the isolation of strain ANL-αCH3. The strain was identified as a representative of the genus Halomonas in the Gammaproteobacteria. The bacterium was capable of PPN utilization as a nitrogen source only, while phenylacetonitrile (PAN) served both as carbon, energy and nitrogen source. This capacity was not described previously for any other haloalkaliphilic bacteria. Apart from the nitriles mentioned above, resting cells of ANL-αCH3 also hydrolyzed mandelonitrile, benzonitrile, acrylonitrile, and phenylglycinonitrile, presumably using nitrilase pathway. Neither nitrile hydratase nor amidase activity was detected. The isolate showed a capacity to grow with benzoate and salicylate as carbon and energy source and demonstrated the ability to completely mineralize PAN. These clearly indicated a potential to catabolize aromatic compounds. On the basis of unique phenotype and distinct phylogeny, strain ANL-αCH3 is proposed as a novel species of the genus HalomonasHalomonas nitrilicus sp. nov. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.  相似文献   

20.
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   

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