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1.
Nocardia globerula NHB-2 exhibited an intracellular acetonitrile hydrolysing activity (AHA) when cultivated in nutrient broth supplemented with glucose (10.0 g/l) and yeast extract (1.0 g/l), at pH 8.0, 30 degrees C for 21 hr. Maximum AHA was recorded in the culture containing 0.1 M of sodium phosphate buffer, (pH 8.8) at 45 degrees C for 15 min with 600 micromol of acetonitrile and resting cells of N. globerula NHB-2 equivalent to 1.0 ml culture broth. This activity was stable up to 40 degrees C and was completely inactivated at or above 60 degrees C. About five-fold increase in AHA was observed after optimization of culture and reaction conditions. Under the optimized conditions, this organism hydrolyzed various nitriles and amides such as propionitrile, benzonitrile. acetamide, and acrylamide to corresponding acids. This nitrile/amide hydrolysing activity of N. globerula NHB-2 has potential applications in enzymatic synthesis of organic acids and bioremediation of nitriles and amides contaminated soil and water system. 相似文献
2.
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
3.
Ludmila Kabaivanova Plamen Dimitrov Ivanka Boyadzhieva Stefan Engibarov Elena Dobreva Elka Emanuilova 《World journal of microbiology & biotechnology》2008,24(11):2383-2388
A moderately thermophilic Gram-positive, sporulating, rod-shaped strain of Bacillus with nitrile-degrading activity was isolated from polluted industrial waters. Whole cells and cell-free extracts from the
end of exponential growth phase expressed 7.6 nkat mg−1 and 2.0 nkat mg−1 benzonitrile-degrading activity, respectively, after cultivation in a fermentor with complex medium containing benzonitrile
as an inducer. The benzonitrile degradation took place via the nitrilase pathway directly to benzoic acid without intermediate
formation of benzamide. Samples with benzonitrilase activity of 7.6 nkat mg−1 converted 3 mg benzonitrile in 1 h at 45°C. The half-life of benzonitrilase activity for a whole cell suspension and for
cells immobilized in 2% agar was 4.5 min and 6 min at 70°C without substrate and 3 min at 90°C with substrate, respectively.
The nitrilase had a broad substrate spectrum. The active biocatalyst obtained by immobilization was used in a continuous process
and total biodegradation of 14.1 mM benzonitrile and 37.2 mM 4-cyanopyridine in a column bioreactor at 50°C for 5 h was achieved. 相似文献
4.
Mustacchi R Knowles CJ Li H Dalrymple I Sunderland G Skibar W Jackman SA 《Biotechnology and bioengineering》2005,89(1):18-23
The simultaneous enhancement of biotransformation coupled to product recovery, purification and concentration is presented. The nitrilase of Rhodococcus rhodochrous LL100-21 catalyses the single-step hydrolytic biotransformation of benzonitrile to benzoic acid and ammonia. When a direct electric current is applied across a bioreactor containing the bacterium and benzonitrile, the charged product (benzoic acid) can be removed in situ across an anion exchange membrane and recovered in a separate compartment. Over the course of a 24-hour biotransformation, benzonitrile was converted to benzoic acid which was completely removed from the bioreactor chamber and concentrated 3-fold in a separate chamber. The rate of production of benzoic acid increased by 42% when the current was applied (0.044 mmol/min/g dry cell weight in the presence of current as compared to 0.03 mmol/min/g dry cell weight in its absence). The enhanced reaction rate was achieved irrespective of product separation and therefore appears to be a direct effect upon the bacterial cells. This process has potential for enhanced productivity from biotransformations through a simultaneous increase in metabolic activity and in situ product recovery. 相似文献
5.
Shreenath Prasad Anurag Misra Ved Prakash Jangir Abhishek Awasthi Jog Raj Tek Chand Bhalla 《World journal of microbiology & biotechnology》2007,23(3):345-353
Rhodococcus sp. NDB 1165, a nitrile-transforming organism was isolated from temperate forest soil of Himalayas. The nitrilase (EC 3.5.5.2)
activity of this organism had higher substrate specificity toward aromatic nitriles (benzonitrile, 3-cyanopyridine and 4-cyanopyridine)
and unsaturated aliphatic nitrile (acrylonitrile) in comparison to saturated aliphatic nitriles (acetonitrile, propionitrile,
butyronitrile and isobutyronitrile) nitrile and arylacetonitrile (phenylacetonitrile and indole-3-acetonitrile). The nitrilase
of Rhodococcus sp. NDB 1165 was inducible in nature and propionitrile proved to be an efficient inducer. However, the salts of ferrous and
cobalt ions had an inhibitory effect. Under optimized reaction conditions (pH 8.0 and temperature 45°C) the nitrilase activity
of this organism was 2.39 ± 0.07 U/mg dry cell mass (dcm). The half-life of this enzyme was 150 min and 40 min at 45°C and
50°C respectively. However, it was quite stable at 40°C and around 58 % activity was retained even after 6 h at this temperature.
The V
max and K
m value of this nitrilase were 1.67 μmol/ml min and 0.1 M respectively using 3-cyanopyridine as substrate. However, the decrease
in V
max and K
m values (0.56 μmol/ml min and 0.02 M, respectively) were ␣observed at >0.05 M 3-cyanopyridine which revealed that this enzyme
experienced uncompetitive inhibition at higher substrate concentrations. Under optimized reaction conditions, 1.6 M 3-cyanopyridine
was successfully converted in to nicotinic acid using 2.0 mg resting cells (dcm)/ml reaction mixture in 11 h. This is the
highest production of nicotinic acid i.e. 8.95 mg/mg resting cells (dcm)/h as compared to nitrilase systems reported hitherto. 相似文献
6.
以琼脂粉为基质制备金属螯合载体,并用于固定重组腈水解酶。研究发现:制备金属螯合载体最合适的金属离子为Zn2+。当Zn2+离子浓度0.3 mol/L、给酶量15.6 mg/g、固定化pH 8.0、固定化温度40℃时,制得的固定化酶活性最高。固定化酶最适反应温度为50℃、最适反应pH为7.0。当扁桃腈浓度为10 mmol/L、反应1 h时,固定化酶最大产率为0.041 mmol/(g·h);在反应12 h时,产物e.e.值可达到99%以上。固定化酶重复使用8次以后,酶活力仍保持在45%。 相似文献
7.
A. B. Veselá M. Franc H. Pelantová D. Kubáč V. Vejvoda M. Šulc T. C. Bhalla M. Macková P. Lovecká P. Janů K. Demnerová L. Martínková 《Biodegradation》2010,21(5):761-770
The soil actinobacteria Rhodococcus rhodochrous PA-34, Rhodococcus sp. NDB 1165 and Nocardia globerula NHB-2 grown in the presence of isobutyronitrile exhibited nitrilase activities towards benzonitrile (approx. 1.1–1.9 U mg?1 dry cell weight). The resting cell suspensions eliminated benzonitrile and the benzonitrile analogues chloroxynil (3,5-dichloro-4-hydroxybenzonitrile), bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) and ioxynil (3,5-diiodo-4-hydroxybenzonitrile) (0.5 mM each) from reaction mixtures at 30°C and pH 8.0. The products were isolated and identified as the corresponding substituted benzoic acids. The reaction rates decreased in the order benzonitrile ? chloroxynil > bromoxynil > ioxynil in all strains. Depending on the strain, 92–100, 70–90 and 30–51% of chloroxynil, bromoxynil and ioxynil, respectively, was hydrolyzed after 5 h. After a 20-h incubation, almost full conversion of chloroxynil and bromoxynil was observed in all strains, while only about 60% of the added ioxynil was converted into carboxylic acid. The product of ioxynil was not metabolized any further, and those of the other two herbicides very slowly. None of the nitrilase-producing strains hydrolyzed dichlobenil (2,6-dichlorobenzonitrile). 3,5-Dibromo-4-hydroxybenzoic acid exhibited less inhibitory effect than bromoxynil both on luminescent bacteria and germinating seeds of Lactuca sativa. 3,5-Diiodo-4-hydroxybenzoic acid only exhibited lower toxicity than ioxynil in the latter test. 相似文献
8.
(R)-mandelic acid was produced from racemic mandelonitrile using free and immobilized cells of Pseudomonas putida MTCC 5110 harbouring a stereoselective nitrilase. In addition to the optimization of culture conditions and medium components, an inducer feeding approach is suggested to achieve enhanced enzyme production and therefore higher degree of conversion of mandelonitrile. The relationship between cell growth periodicity and enzyme accumulation was also studied, and the addition of the inducer was delayed by 6 h to achieve maximum nitrilase activity. The nitrilase expression was also authenticated by the sodium dodecyl phosphate-polyacrylamide gel electrophoresis analysis. P. putida MTCC 5110 cells were further immobilized in calcium alginate, and the immobilized biocatalyst preparation was used for the enantioselective hydrolysis of mandelonitrile. The immobilized system was characterized based on the Thiele modulus (ϕ). Efficient biocatalyst recycling was achieved as a result of immobilization with immobilized cells exhibiting 88% conversion even after 20 batch recycles. Finally, a fed batch reaction was set up on a preparative scale to produce 1.95 g of (R)-(-)-mandelic acid with an enantiomeric excess of 98.8%. 相似文献
9.
Liu ZQ Zhou M Zhang XH Xu JM Xue YP Zheng YG 《Journal of molecular microbiology and biotechnology》2012,22(1):35-47
Iminodiacetic acid (IDA) is widely used as an intermediate in the manufacture of chelating agents, glyphosate herbicides and surfactants. In the current work, the fragment with the length of 1,110 bp encoding the Acidovorax facilis nitrilase was obtained. The recombinant nitrilase expressed in Escherichia coli BL21 (DE3) was successfully used in the production of IDA from iminodiacetonitrile. To improve the stability of operation, the recombinant cells were entrapped in polyvinyl alcohol (PVA) and sodium alginate (SA) copolymer. The maximum relative nitrilase activity with 98.1% was further observed at 1.0% SA, 8.0% PVA, 1.0% CaCl(2), and 5.0% wet cells, under conditions of 1.0% iminodiacetonitrile in distilled water and a temperature of 40°C, respectively. The entrapped cells facilitated easy separation and good recycling compared with free cells. Moreover, the immobilized cells showed good operation and storage stability. This report is the first to describe IDA preparation using immobilized recombinant E. coli harboring nitrilase. 相似文献
10.
V. K. Nigam A. K. Khandelwal R. K. Gothwal M. K. Mohan B. Choudhury A. S. Vidyarthi P. Ghosh 《Journal of biosciences》2009,34(1):21-26
The biotransformation of acrylonitrile was investigated using thermophilic nitrilase produced from a new isolate Streptomyces sp. MTCC 7546 in both the free and immobilized state. Under optimal conditions, the enzyme converts nitriles to acids without
the formation of amides. The whole cells of the isolate were immobilized in agar-agar and the beads so formed were evaluated
for 25 cycles at 50°C. The enzyme showed a little loss of activity during reuse. Seventy-one per cent of 0.5 M acrylonitrile
was converted to acid at 6 h of incubation at a very low density of immobilized cells, while 100% conversion was observed
at 3 h by free cells. 相似文献
11.
Li-Qun Jin Dong-Jing Guo Zong-Tong Li Zhi-Qiang Liu Yu-Guo Zheng 《Journal of industrial microbiology & biotechnology》2016,43(5):585-593
In this paper, a simple and effective method using sodium metasilicate as precursor and amine as additive was first reported to immobilize recombinant nitrilase, for efficient production of 2-hydroxy-4-(methylthio) butanoic acid from 2-hydroxy-4-(methylthio) butanenitrile. High immobilization recovery of enzyme activity (above 90 %) was achieved. The immobilized enzyme displayed better thermal stability, pH stability and shelf life compared to free nitrilase. Moreover, it showed excellent reusability and could be recycled up to 16 batches without significant loss in activity. 200 mM 2-hydroxy-4-(methylthio) butanenitrile was completely converted by the immobilized enzyme within 30 min, and the accumulation amount of 2-hydroxy-4-(methylthio) butanoic acid reached 130 mmol/g of immobilized beads after 16 batches. These encouraging results demonstrated the efficiency of the new technology for nitrilase immobilization, which has great potential in preparation of 2-hydroxy-4-(methylthio) butanoic acid. 相似文献
12.
Microbial metabolism of aromatic nitriles. Enzymology of C–N cleavage by Nocardia sp. (Rhodochrous group) N.C.I.B. 11216
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David B. Harper 《The Biochemical journal》1977,165(2):309-319
1. An organism utilizing benzonitrile as sole carbon and nitrogen source was isolated by the enrichment-culture technique and identified as a Nocardia sp. of the rhodochrous group. 2. Respiration studies indicate that nitrile degradation proceeds through benzoic acid and catechol. 3. Cell-free extracts of benzonitrile-grown cells contain an enzyme that catalyses the conversion of benzonitrile directly into benzoic acid without intermediate formation of benzamide. 4. This nitrilase enzyme was purified by DEAE-cellulose chromatography and gel filtration on Sephadex G-100 in the presence and absence of substrate. The purity of the enzyme was confirmed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectric focusing on polyacrylamide gel. 5. The enzyme shows a time-dependent substrate-activation process in which the substrate catalyses the association of inactive subunits of mol.wt. 45000 to form the polymeric 12-unit active enzyme of mol.wt. 560000. The time required for complete association is highly dependent on the concentration of the enzyme, temperature and pH. 6. The associated enzyme has a pH optimum of 8.0 and K(m) with benzonitrile as substrate of 4mm. The activation energy of the reaction as deduced from the Arrhenius plot is 51.8kJ/mol. 7. Enzyme activity is inhibited by thiol-specific reagents and several metal ions. 8. Studies with different substrates indicate that the nitrilase is specific for nitrile groups directly attached to the benzene ring. Various substituents in the ring are compatible with activity, though ortho-substitution, except by fluorine, renders the nitrile invulnerable to attack. 9. The environmental implications of these findings and the possible significance of the enzyme in the regulation of metabolism are discussed. 相似文献
13.
Sookkheo B Sinchaikul S Thannan H Thongprasong O Phutrakul S Chen ST 《Proteomics》2002,2(9):1311-1315
Thermophilic bacterium Bacillus stearothermophilus TLS33 isolated from a hot spring in Chiang Mai, Thailand produces an extracellular superoxide dismutase (SOD). SOD is a free radical metabolizing enzyme that protects the cell membrane from damage by the highly reactive superoxide free radicals. To identify the secreted SOD, we used the systematically proteomic approaches of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analysis and database searching. The bacterium was grown in a medium containing 0.1% w/v yeast extract and 0.1% w/v tryptone in 100% v/v base mixture at 65 degrees C for 72 h, by assessing their growth by protein and SOD activity. The bacterium produced the highest SOD activity at 65 degrees C for 48 h and the extracellular SOD was run on 2-D PAGE using broad range pH 3-10 immobilized pH gradients (IPGs) and narrow range pH 4-7 IPGs. The isoelectric point and molecular mass of the extracellular SOD were approximately 5.8 and 28 kDa, respectively. In addition, the NH(2)-terminal amino acid sequence was found to be P-F-E-L-P-A-L-P-Y-P-Y-D-A-L-E-P-P-I-I-D, which had a homology of approximately 85% to the Mn-SOD family and 65% to the Fe-SOD family. 相似文献
14.
Mustacchi R Knowles CJ Li H Dalrymple I Sunderland G Skibar W Jackman SA 《Biotechnology and bioengineering》2005,91(4):436-440
The nitrilase of Rhodococcus rhodochrous performs a one-step biotransformation of nitriles to their corresponding carboxylic acids. Application of a direct electric current moves the charged carboxylic acid towards an anode, across an anion exchange membrane, into a separate compartment. Cells encapsulated within alginate beads (2.9 mm diameter) for protection against the current biotransformed benzonitrile to benzoic acid with a 26% reduction in the biotransformation rate, from 0.054 mmol/min/g dcw with free cells to 0.040 mmol/min/g dcw with immobilised cells. When the electric current was applied, the biotransformation rate increased to 0.047 mmol/min/g dcw and product recovery increased from 19% to 79%. 相似文献
15.
T Nagasawa M Wieser T Nakamura H Iwahara T Yoshida K Gekko 《European journal of biochemistry》2000,267(1):138-144
Nitrilase-containing resting cells of Rhodococcus rhodochrous J1 converted acrylonitrile and benzonitrile to the corresponding acids, but the purified nitrilase hydrolyzed only benzonitrile, and not acrylonitrile. The activity of the purified enzyme towards acrylonitrile was recovered by preincubation with 10 mM benzonitrile, but not by preincubation with aliphatic nitriles such as acrylonitrile. It was shown by light-scattering experiments, that preincubation with benzonitrile led to the assembly of the inactive, purified and homodimeric 80-kDa enzyme to its active 410-kDa aggregate, which was proposed to be a decamer. Furthermore, the association concomitant with the activation was reached after dialysis of the enzyme against various salts and organic solvents, with the highest recovery reached at 10% saturated ammonium sulfate and 50% (v/v) glycerol, and by preincubation at increased temperatures or enzyme concentrations. 相似文献
16.
Dias JC Rezende RP Rosa CA Lachance MA Linardi VR 《Canadian journal of microbiology》2000,46(6):525-531
Candida guilliermondii UFMG-Y65, isolated from a gold mine, was able to utilize different nitriles and the corresponding amides as sole source of nitrogen, at concentrations up to 2 M. Resting cells cultivated on YCB-acetonitrile medium showed nitrile hydrolyzing enzyme activities against acrylonitrile and benzonitrile. These enzymes were inducible and intracellular; the optimum pH was 7.0-8.0, and the optimum temperature 25 degrees C-30 degrees C. Liquid chromatographic analysis indicated that C. guilliermondii UFMG-Y65 metabolized 12 mM benzonitrile to 11 mM benzoic acid and 10 mM acrylonitrile to 7.9 mM acrylic acid. The results suggest that C. guilliermondii UFMG-Y65 may be useful for the bioproduction of amides and acids, and for the bioremediation of environments contaminated with nitriles. 相似文献
17.
Production and properties of beta-mannanase by free and immobilized cells of Aspergillus oryzae NRRL 3488 总被引:4,自引:0,他引:4
Seven fungi were tested for production of mannanases. The highest mannanase activities were produced by Aspergillus oryzae NRRL 3488 after 7 days in static cultures. Mannanases were induced by gum locust bean (1.0%). The highest mannanase activity was produced when a mixture of peptone, urea and ammonium sulphate was used as nitrogen source. Zn2+ or Co2+ favoured enzyme production. The immobilized cells on Ca-alginate and agar were able to produce beta-mannanase for four runs with a slight decrease in the activity. The optimum temperature for enzyme reaction was 50-55 degrees C at pH 6.0. In the absence of substrate the enzyme was thermostable retaining 75% activity for 1 h at 50 degrees C, and 68% activity for 1 h at 60 degrees C. 相似文献
18.
Raj J Sharma NN Prasad S Bhalla TC 《Journal of industrial microbiology & biotechnology》2008,35(1):35-40
The nitrile hydratase (Nhase) induced cells of Rhodococcus rhodochrous PA-34 catalyzed the conversion of acrylonitrile to acrylamide. The cells of R. rhodochrous PA-34 immobilized in 2% (w/v) agar (1.76 mg dcw/ml agar matrix) exhibited maximum Nhase activity (8.25 U/mg dcw) for conversion
of acrylonitrile to acrylamide at 10°C in the reaction mixture containing 0.1 M potassium phosphate buffer (pH 7.5), 8% (w/v)
acrylonitrile and immobilized cells equivalent to 1.12 mg dcw (dry cell weight) per ml. In a partitioned fed batch reaction
at 10°C, using 1.12 g dcw immobilized cells in a final volume of 1 l, a total of 372 g of acrylonitrile was completely hydrated
to acrylamide (498 g) in 24 h. From the above reaction mixture 87% acrylamide (432 g) was recovered through crystallization
at 4°C. By recycling the immobilized biocatalyst (six times), a total of 2,115 g acrylamide was produced. 相似文献
19.
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
20.
Preparation and properties of an immobilized pectinlyase for the treatment of fruit juices 总被引:5,自引:0,他引:5
Pectinlyase, present in different commercial pectinases used in juice technology, was immobilized on alginate beads. The optimal conditions were: 0.17 g alginate ml(-1), 1.2% (w/v or v/v) enzyme concentration and acetic-HCl/glycine-HCl buffer at pH 3.6 or tris-HCl/imidazole buffer at pH 6.4. Maximum percentage of immobilization (10.6%) was obtained with Rapidase C80. Kinetic parameters of free and immobilized pectinlyase were also determined. The pH and temperature at which activity of soluble and immobilized enzyme was maximum were 7.2 and 55 degrees C. Thermal stability was not significantly altered by immobilization, especially at 40 degrees C, showing two periods of different stability. Free and immobilized preparation reduced the viscosity of highly esterified pectin from 1.09 to 0.70 and 0.72 mm(2) s(-1), respectively, after 30 min at 40 degrees C. Furthermore, the immobilized enzyme could be re-used through 4 cycles and the efficiency loss in viscosity reduction was found to be only 9.2%. 相似文献