Plasmid pRTL1 Controlling 1-Chloroalkane Degradation by Rhodococcus rhodochrous NCIMB13064

Rhodococcus rhodochrous NCIMB13064 can dehalogenate and use a wide range of 1-haloalkanes as sole carbon and energy source. The 1-chloroalkane degradation phenotype may be lost by cells spontaneously or after treatment with Mitomycin C. Two laboratory derivatives of the original strain exhibited differing degrees of stability of the chloroalkane degradation marker. Plasmids of approximately 100 kbp (pRTL1) and 80 kbp (pRTL2) have been found in R. rhodochrous NCIMB13064. pRTL1 was shown to be carrying at least some genes for the dehalogenation of 1-chloroalkanes with short chain lengths (C₃ to C₉). However, no connection was found between the utilization of 1-chloroalkanes with longer chain lengths (C₁₂ to C₁₈) and the presence of pRTL1. Three separate events were observed to lead to the inability of NCIMB13064 to dehalogenate the short-chain 1-chloroalkanes; the complete loss of pRTL1, the integration of pRTL1 into the chromosome, or the deletion of a 20-kbp fragment in pRTL1. High-frequency transfer of the 1-chloroalkane degradation marker associated with pRTL1 has been demonstrated in bacterial crosses between different derivatives of R. rhodochrous NCIMB13064.     

Control of the nitrile-hydrolyzing enzyme activity in Rhodococcus rhodochrous IFO 15564: preferential action of nitrile hydratase and amidase depending on the reaction condition factors and its application to the one-pot preparation of amides from aldehydes

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.     

Biotransformation of nitriles to amides using soluble and immobilized nitrile hydratase from Rhodococcus erythropolis A4

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.     

Preparation of (R)-3-hydroxy-3-alkylbutanolides by biocatalytic hydration of 3-alkyl-2-butenolides using Rhodococcus rhodochrous

Biocatalytic hydration of 3-methyl- or 3-ethyl-2-butenolide with resting cells of Rhodococcus rhodochrous ATCC 17895 gave the corresponding (R)-3-hydroxy-3-alkylbutanolide in moderate yield and with 95% e.e. © Rapid Science Ltd. 1998     

Biocatalytic application of nitrilases from Fusarium solani O1 and Aspergillus niger K10

The nitrilases from Fusarium solani O1 and Aspergillus niger K10 showed a broad substrate specificity for carbocyclic and nonaromatic heterocyclic amino nitriles, the preferred substrates being five-membered γ-amino nitrile (±)-1a, six-membered γ-amino nitriles (±)-3a, (±)-5a and (±)-6a, pyrrolidine-3-carbonitriles (±)-9a and (±)-10a as well as piperidine-4-carbonitriles 14a and 15a. Both enzymes showed a strong diastereopreference for cis- vs. trans-γ-amino nitriles. The electronic and steric effects of N-protecting groups affected the reactivity of the nitriles. Amides as by-products of the nitrilase-catalyzed reaction were produced from heterocyclic amino nitriles (±)-9a, (±)-10a, 14a and 15a by the A. niger enzyme but only from nitrile (±)-9a by the F. solani enzyme.     

Biotransformation of nitriles by Rhodococcus equi A4 immobilized in LentiKats

Whole cells of Rhodococcus equi A4, a producer of nitrile hydratase and amidase activities, were immobilized in lens-shaped hydrogel particles, LentiKats^®. The immobilized biocatalyst was applied to the biotransformation of benzonitrile, 3-cyanopyridine, (R,S)-3-hydroxy-2-methylenebutanenitrile and (R,S)-3-hydroxy-2-methylene-3-phenylpropanenitrile. The stability of the nitrile hydratase during the repeated use of the biocatalyst was dependent on the type of the substrate. The enzyme was most stable during the transformation of (R,S)-3-hydroxy-2-methylenebutanenitrile. No significant loss of the amidase activity was observed within the course of the biocatalytic reaction.     

Hydrophobic cell surface and bioflocculation behavior of Rhodococcus erythropolis

An alkane-biodegrading bacterium identified as Rhodococcus erythropolis (NTU-1 strain) was isolated from petroleum contaminated soil. The major purpose of the current research was to study the issues regarding biofloccules formation and cell surface hydrophobicity of NTU-1. When long-chain alkanes are supplied as the carbon source, NTU-1 tends to form biofloccules and remove significant amount of alkanes by biodegradation and physical trapping. Approximately, more than 95% of each alkane could be efficiently removed within 40–68 h. The bioremediation process was accompanied by formation of biofloccules with size ranging from 0.1 to 2 cm in diameter. The MATH test and the hydrophobic slide experiment suggested that NTU-1 might possess a hydrophobic cell surface which is one of the important factors in the formation of biofloccules. It provides the interaction of cells with hydrocarbon droplets effectively and further aggregate into larger clumps. Besides, when grown on n-hexadecane, experimental results revealed that there were at least 11 different growth-associated fatty acids produced, with carbon chain length ranging from 12 to 24, and cell surface hydrophobicity was enhanced via accumulation at the cell surface.     

Asymmetric hydrolysis of R-(−),S(+)-2-methylbutyronitrile by Rhodococcus rhodochrous NCIMB 11216

Whole cells and cell-free extracts derived from Rhodococcus rhodochrous NCIMB 11216 were shown to hydrolyse both aliphatic and aromatic nitriles, when the organism had been grown on either propionitrile or benzonitrile as the source of carbon and nitrogen. Whole cell suspensions and cell-free extracts derived from bacteria grown on either substrate were able to biotransform R-(-),S-(+)-2-methylbutyronitrile. The S-(+) enantiomer was biotransformed more rapidly than the the R-(-) enantiomer. For whole cell biotransformations at 30°C, the maximum enantiomeric excess (ee) of the remaining R-(-)-2-methylbutyronitrile was 93% when 70% of the R-(-) enantiomer had been converted to the product, 2-methylbutyric acid. For the corresponding biotransformation at 4°C, there was an ee of 93% for the residual R-(-) enantiomer of the substrate when only 60% of it had been converted to product. For biotransformations by cell-free extracts at 30°C the 2-methylbutyric acid product had an ee of 17% for the S-(+) enantiomer at the time of optimal ee for the remaining R-(-) enantiomer of the substrate. In contrast, when the reaction was carried out by whole cells, the ee for the product acid was 0.36%. This was probably due to further, non-selective metabolism of the acid, which was especially significant at the beginning of the reaction. At both temperatures, the ee for the S-(+) enantiomer of 2-methylbutyric acid was at a maximum in the early stage of the biotransformation; for example, at 4°C the maximum detectable ee was 100% when the yield was 11%.Abbreviations EDTA Ethylenediaminetetraacetic acid - ee enantiomeric excess - FID flame ionisation detector - GC gas chromatography - ¹HNMR H nuclear magnetic resonance - K_m Michaelis constant - NCIMB National Collection of Industrial and Marine Bacteria - td doubling time - V_max Maximum velocity     

A comparative molecular field analysis of the biotransformation of sulfides by Rhodococcus erythropolis

A comparative molecular field analysis (CoMFA) was used to model the efficacy with which the Rhodococcus erythropolis mono-oxygenase, DszC, catalyzes the enantioselective sulfoxidation of a broad range of substrates. Experimentally determined values of both the yield and enantiomeric excess for this reaction were employed to create these CoMFA models. A highly predictive CoMFA model was constructed for the prediction of enantiomeric excess of the sulfoxide product. The predictive ability of the model was demonstrated by both cross-validation of the training set (q² = 0.74) and for an external test set of substrates. The enantiomeric excesses of the members of the test set, which also included two amino acid sulfides that were structurally distinct from the membership of the training set, were predicted well by the CoMFA model. Product yield was not modelled well by any CoMFA model. Different models comparing the likely bioactive conformations of the substrates suggest that most compounds assume an ‘extended’ conformation upon binding. Contour diagrams illustrating significant substrate–enzyme interactions suggest that the model, which predicts the enantiomeric excess, is consistent with previous conclusions regarding the effect of various substrate substitutions on the enantiopurity of the product of the biotransformation.     

Conversion of 3-cyanopyridine to nicotinic acid by Nocardia rhodochrous LL100-21

3-Cyanopyridinase activity, i.e. the ability to convert 3-cyanopyridine to nicotinic acid plus ammonia, was induced in stationary phase cultures of Nocardia rhodochrous LL100-21 by the addition of 2-, 3-, or 4-cyanopyridine or benzonitrile; the latter nitrile gave maximum induction. Harvested bacteria possessing 3-cyanopyridinase activity could stoichiometrically convert 3-cyanopyridine at concentrations of up to 0.5 to nicotinic acid. Both 3-cyanopyridine and nicotinic acid inhibited the hydrolysis of 3-cyanopyridine by intact bacteria. Bacteria immobilized in calcium alginate beads and used in column bioreactors retained 3-cyanopyridinase activity for over 150 h when continuously supplied with 0.3 3-cyanopyridine.     

Medium improvement by orthogonal array designs for cholesterol oxidase production by Rhodococcus equi No. 23

Medium improvement for the production of cholesterol oxidase (CO, EC 1.1.3.6) by Rhodococcus equi No. 23 was investigated using an orthogonal array design in two steps. Results revealed that yeast extract, Tween 80 and zinc sulphate had positive effects on CO production, but magnesium sulphate had an inhibitory effect. In addition, interaction between cholesterol and sodium chloride also had a significant effect on enzyme production. The improved medium consisted of 2·0 g/litre cholesterol, 8·0 g/litre yeast extract, 1·0 g/litre NH₄Cl, 1·0 g/litre NaCl, 0·50 g/litre KH₂PO₄, 0·25 g/litre Na₂HPO₄, 0·10 g/litre -valine, 0·15 g/litre -tyrosine, 0·15 g/litre MgSO₄·7H₂O, 0·01 g/litre ZnSO₄·7H₂O, 0·10 g/litre FeSO₄·7H₂O and 4·0 ml/litre Tween 80. CO production at 60 h (about 0·24 units/ml) was about four-fold greater than with the control medium.     

The Role of Intercellular Contacts in the Initiation of Growth and in the Development of a Transiently Nonculturable State by Cultures of Rhodococcus rhodochrous Grown in Poor Media

It was found that the growth of Rhodococcus rhodochrous cells in a modified Saton’s medium strongly depends on the rate of culture agitation in the flask: agitation at 250 rpm in flasks with baffles stops cell multiplication, whereas slight agitation leads to pronounced culture growth. The growth retardation phenomenon was reversible and did not manifest itself in exponential-phase cultures or when the cells were grown in a rich medium; furthermore, it was not connected with the degree of culture aeration. When agitated at a moderate rate, the bacterial cells formed aggregates in the lag phase, which broke up into single cells in the exponential phase. The inhibitory effect of vigorous agitation was removed by the addition, to the medium, of the supernatant (SN) of a log-phase culture grown in the same medium with moderate agitation. Vigorous agitation is thought to interfere with cell contact, whose establishment is necessary for the development of an R. rhodochrous culture in a poor medium, which occurs in the form of (micro) cryptic growth. When grown in a modified Saton’s medium, R. rhodochrous cells were capable of transition, in the prolonged stationary phase, to a resting and transiently nonculturable state. Such cells could be resuscitated by incubation in a liquid medium with the addition of the supernatant or the Rpf secreted protein. The formation of transiently nonculturable cells was only possible under the conditions of a considerable agitation rate (250–300 rpm), which prevented secondary (cryptic) growth of the culture. This circumstance indicates the importance of intercellular contacts not only for the initiation of growth but also for the transition of the bacteria to a dormant state.__________Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 489–797.Original Russian Text Copyright © 2005 by Voloshin, Shleeva, Syroeshkin, Kaprelyants.     

Biotransformation of benzonitrile to benzohydroxamic acid by Rhodococcus rhodochrous in the presence of hydroxylamine

Whole cells of the bacterium Rhodococcus rhodochrous LL100-21, which had been grown on benzonitrile to induce the nitrilase enzyme, converted benzonitrile to benzohydroxamic acid in the presence of hydroxylamine.     

Chemo- and enantioselective hydrolysis of nitriles and acid amides, respectively, with resting cells of Rhodococcus sp. C3II and Rhodococcus erythropolis MP50

The two new bacterial strains, Rhodococcus sp. C3II and Rhodococcus erythropolis MP50, which have been especially selected for the enantioselective hydrolysis of pharmaceutically interesting 2-arylpropionitriles like naproxen nitrile, have been applied for the hydrolysis of various aliphatic and aromatic nitriles and acid amides. From the enantioselective hydrolysis of racemic ibuprofen amide 4, 2-phenylbutyronitrile 5a as well as the profen-related atrolactamide 8 we deduce the decisive role of both an alkyl and aryl substituent in the -position to the nitrile or amide function for high enantioselectivity of the hydrolysis. Strain C3II and MP50 differ in the activity of their nitrile hydratase–amidase enzyme systems. This is of interest for the regioselective hydrolysis of the dinitriles 10a–13a to diacids 10f–13f. While strain C3II is suitable to preferentially produce mononitrile monoamide derivatives, strain MP50 can be used especially to form mononitrile monoacid and monoamide monoacid derivatives.     

Biochemical characterization of Rhodococcus erythropolis N′4 nitrile hydratase acting on 4-chloro-3-hydroxybutyronitrile

The Rhodococcus erythropolis strain (N′4) possesses the ability to convert 4-chloro-3-hydroxybutyronitrile into the corresponding acid. This conversion was determined to be performed by its nitrile hydratase and amidase. Ammonium sulfate fractionation, DEAE ion exchange chromatography, and phenyl chromatography were used to partially purify nitrile hydratase from cell-free extract. A SDS-PAGE showed that the partially purified enzyme had two subunits and gel filtration chromatography showed that it consisted of four subunits of α₂β₂. The purified enzyme had a high specific activity of 860 U mg⁻¹ toward methacrylonitrile. The enzyme was found to have high activity at low temperature range, with a maximum activity occurring at 25 °C and be stable in the presence of organic acids at higher temperatures. The enzyme exhibited a preference for aliphatic saturated nitrile substrates over aliphatic unsaturated or aromatic ones. It was inhibited by sulfhydryl, oxidizing, and serine protease inhibitors, thus indicating that essential cysteine and serine residues can be found in the active site.The purified nitrile hydratase was able to convert 4-chloro-3-hydroxybutyronitrile into the corresponding amide at 15 °C. GC analysis showed that the initial conversion rate of the reaction was 215 mg substrate consumed min⁻¹ mg⁻¹. This demonstrated that this enzyme could be used in conjunction with a stereoselective amidase to synthesize ethyl (S)-4-chloro-3-hydroxybutyrate, an intermediate for a hypercholesterolemia drug, Atorvastatin.     

Biotransformation of nitriles by rhodococci

Rhodococci have been shown to be capable of a very wide range of biotransformations. Of these, the conversion of nitriles into amides or carboxylic acids has been studied in great detail because of the biotechnological potential of such activities. Initial investigations used relatively simple aliphatic nitriles. These studies were quickly followed by the examination of the regio- and stereoselective properties of the enzymes involved, which has revealed the potential synthetic utility of rhodococcal nitrile biotransforming enzymes. Physiological studies on rhodococci have shown the importance of growth medium design and bioreactor operation for the maximal conversion of nitriles. This in turn has resulted in some truly remarkable biotransformation activities being obtained, which have been successfully exploited for commercial organic syntheses (e.g. acrylamide production from acrylonitrile).The two main types of enzyme involved in nitrile biotransformations by rhodococci are nitrile hydratases (amide synthesis) and nitrilases (carboxylic acid synthesis with no amide intermediate released). It is becoming clear that many rhodococci contain both activities and multiple forms of each enzyme, often induced in a complex way by nitrogen containing molecules. The genes for many nitrile-hydrolysing enzymes have been identified and sequenced. The crystal structure of one nitrile hydratase is now available and has revealed many interesting aspects of the enzyme structure in relationship to its catalytic activity and substrate selectivity.     

Nitrilase-catalysed conversion of acrylonitrile by free and immobilized cells of <Emphasis Type="Italic">Streptomyces</Emphasis> sp.

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.     

A generalised transducing bacteriophage for Rhodococcus erythropolis

Summary Q4, a bacteriophage isolated from soil, mediated the transduction of a number of unlinked markers in Rhodococcus erythropolis. Highest numbers of transductants were obtained at multiplicities of infection of over 100, transductants only being obtained because of the temperate nature of the phage. Under optimal conditions, transduction to prototrophy of auxotrophic markers was over 50 times the spontaneous reversion rate and transduction of some antibitic resistance markers was over 10 times the spontaneous mutation rate. Segregation of unselected, but linked, markers was observed and the phage was used to order loci in a three factor cross. The virus required magnesium ions. Highest phage titres and greatest transduction frequency were obtained with stationary phase cultures.     

Purification and characterization of a nitrilase from Brassica napus

In germinating seedlings of Brassica napus glucosinolate levels decrease and are potentially degraded to nitriles by a myrosinase. Little is known about the metabolism of glucosinolate aglycone products and the objective of this work was to investigate nitrilase activity and carry out a purification of the enzyme from seedlings of B. napus . A nitrilase capable of converting phenylpropionitrile to phenylpropionic acid was purified to apparent homogeneity from seedlings of B. napus . The protein has a molecular mass of approximately 420 kDa made up of 38 kDa subunits. The pI of the native protein was found to be 4.6. Under denaturing conditions on an isoelectric focusing (IEF) gel a major and minor protein was observed with pI in the range of 5.4-5.9, suggesting the presence of isoforms. Apart from the potential role of the nitrilase in indole-3-acetic acid (IAA) synthesis a developmental study with seedlings indicates that the increase in activity observed may be linked to the in vivo degradation of glucosinolates.