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     

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.     

Isolation of Rhodococcus rhodochrous NCIMB13064 derivatives with new biodegradative abilities

Abstract Rhodococcus rhodochrous NCIMB 13064 can dehalogenate and utilise a number of halogenated aliphatic compounds as sole carbon and energy source. Mutants of NCIMB13064 can be easily isolated with an enlarged range of 1-chloroalkane utilising ability. Dehalogenation of 1-chlorononane, 1-chlorodecane and short-chain 1-chloroalkanes (C₃-C₈) is encoded by the same plasmid pRTL1. However, a different genetic element(s) is required for the dehalogenation of 3-chloropropionic acid. Two derivatives (P200 and P400) of R. rhodochrous NCIMB 13064 were isolated which had acquired the ability to utilise naphthalene as sole carbon and energy source. Both strains lost the ability to utilise short-chain 1-chloroalkanes and underwent some rearrangements associated with pRTLl plasmid.     

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     

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.     

The effect of whole cell immobilisation on the biotransformation of benzonitrile and the use of direct electric current for enhanced product removal

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%.     

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.     

宿主感染马红球菌的免疫机制

马红球菌(Rhodococcus equi)作为一种人兽共患病原菌,主要引起3～6月龄马和免疫缺陷患者肺部相关疾病,病死率为50%～80%;此外还可以感染猪、羊、猫、狗、骆驼等动物。该菌不仅对马业造成严重经济损失,还危害免疫缺陷患者的健康,因此在世界范围内受到公共卫生学的重视。马红球菌感染宿主后,其通过抑制吞噬体/自噬体与溶酶体的融合抵抗巨噬细胞杀伤从而进行免疫逃逸;此外,由于马驹固有免疫和适应性免疫存在功能缺陷使其不能有效抵抗马红球菌感染。目前关于宿主感染马红球菌的免疫机制尚未阐述清楚,从马红球菌与宿主免疫细胞相互作用的角度出发,就宿主感染马红球菌的免疫机制进行综述,以期更好地了解马红球菌感染的免疫机制,同时为该菌所引发疾病的防治策略提供参考。     

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.     

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.     

Rhodococcus ruber CGMCC3090腈水合酶纯化、酶学性质及结晶研究

Rhodococcus ruber CGMCC309菌株为酰胺酶及腈水解酶双重缺陷菌株,研究表明该菌能产宽泛底物特异性的腈水合酶。对该菌株产生的新型腈水合酶(NHase-3090)进行纯化和结晶,并研究了其酶学性质。采用疏水、离子交换及凝胶过滤3种层析方法,使该酶纯化倍数达到17.14,得率高达26.2%。电泳分析表明,全酶分子量为105 kDa,由α(24.3 k Da)和β(28.0k Da)2个亚基组成,并构成α2β2四聚体。酶的最适p H和温度分别为7.5和30℃。该酶明显受不同金属离子影响。动力学研究表明,Km为178.8 m M;Vmax为209.1μmol/L·min·mg。研究发现3种金属离子Zn~(2+),CO~(2+)和Cd~(2+)有利于酶蛋白结晶。结晶最佳条件是:采用112-34#试剂(0.05mol/L水合硫酸镉、0.1mol/L HEPS和1.0mol/L三水醋酸钠),蛋白质浓度为15 mg/ml,结晶温度为16℃,p H为7.5,结晶时间为30 d。腈水合酶蛋白单晶经X射线衍射,分辨率达到了3.7。该腈水合酶的纯化和结晶为进一步深入研究其结构和功能奠定了基础。     

响应面法优化赤红球菌HDRR2Y发酵培养参数

通过响应面法对硝化菌——赤红球菌(Rhodococcus ruber)HDRR2Y的发酵培养参数进行优化,以提高其活菌数。首先通过单因素实验筛选出赤红球菌HDRR2Y的最优碳、氮源,并采用Plackett-Burman实验得到影响活菌数的显著因素,然后进行响应面实验,经最陡爬坡及回归分析得出最佳培养参数,最后以摇瓶实验检验其合理性。结果显示,赤红球菌HDRR2Y的最优碳、氮源分别为乙酸钠和酵母膏+蛋白胨+氯化铵(1:1:1,质量比),显著影响活菌数的因素有碳、氮源及温度,经回归分析得到的最优培养参数为乙酸钠5.48 g/L、酵母膏+蛋白胨+氯化铵4.96 g/L、温度29.24℃、pH 7.0、转速200 r/min、MgSO₄ 0.2 g/L、KH₂PO₄ 0.5 g/L、NaCl 9 g/L、CaCl₂ 0.5 g/L、MnSO₄ 0.025 g/L、FeSO₄ 0.05 g/L、C₅H₉NO₄     

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.     

Enantioselective hydrolysis of β-hydroxy nitriles using the whole cell biocatalyst Rhodococcus rhodochrous ATCC BAA-870

Candida rugosa lipase was covalently immobilized onto silica gel in two different ways: via glutaraldehyde (L_GAL) and via hydrophobic spacer arm (1,6 diamino hexane) (L_SA). Free lipase, L_GAL and L_SA were used to investigate the hydrolysis of two different substrates, namely p-nitrophenyl palmytate (pNPP) and p-nitrophenyl acetate (pNPA), both in aqueous medium. In addition, these lipase samples were used to synthesize the pNPP from p-nitrophenol (pNP) and palmytic acid (PA) and pNPA from pNP and acetic acid (AA), both in hexane medium. Hydrolytic and synthetic activities of L_SA were higher than those of free lipase and L_GAL. Synthetic activities of free lipase, L_GAL and L_SA for pNPA in the presence of pNP and AA within hexane medium were higher than those of hydrolytic activities for pNPA in aqueous medium. The same tendency was also observed with pNPP. The effects of pH and temperature on hydrolytic and synthetic activities were investigated for all lipase preparations. Operational stability was the highest for L_GAL and L_SA when these enzymes were used for pNPP synthesis and in hexane medium, after 100 repeated uses, 68% and 51% of initial activities remained, respectively, at the end of 100 repeated cycles. Free lipase lost all of its activity within 15 and 20 days when stored at 25 °C and 5 °C, respectively. However, L_GAL showed 54% and 70% of initial activity at the end of 60 storage days at 25 °C and 5 °C, respectively, while these values were observed as 36% and 60% for L_SA.     

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.     

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.