Bioconversion of butyronitrile to butyramide using whole cells of <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> PA-34

Butyramide is an important chemical commodity, which is used for the synthesis of hydroxamic acids and electrorheological fluids and for the preparation of β-amodoorganotin compounds. The nitrile hydratase (Nhase) of Rhodococcus rhodochrous PA-34 catalyzed the conversion of butyronitrile to butyramide. The maximum Nhase activity [18 U/mg dry cell weight (dcw)] of whole cells of R. rhodochrous PA-34 was observed at pH 7.0 with 10% (v/v) butyronitrile and 1 mg cells (dcw)/ml reaction mixture at 10°C. The cells of R. rhodochrous PA-34 retained almost 50% activity when incubated for 1 h in the presence of 85% (v/v) butyronitrile. A yield of 597 g of butyramide (6.8 M) was obtained using 60% (v/v) butyronitrile, 1 g cells (dry weight) in a 1-l batch reaction at 10°C for 6 h.     

Acrylamide synthesis using agar entrapped cells of <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> PA-34 in a partitioned fed batch reactor

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.     

Bench scale conversion of 3-cyanopyidine to nicotinamide using resting cells of <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> PA-34

The nitrile hydratase (NHase, EC 3.5.5.1) activity of Rhodococcus rhodochrous PA-34 was explored for the conversion of 3-cyanopyridine to nicotinamide. The NHase activity (∼18 U/mg dry cell weight, dcw) was observed in 0.1 M phosphate buffer, pH 8.0 containing 1M 3-cyanopyridine as substrate, and 0.75 mg of resting cells (dry cell weight) per ml reaction mixture at 40°C. However, 25°C was more suitable for prolonged batch reaction at high substrate (3-cyanopyridine) concentration. In a batch reaction (1 liter), 7M 3-cyanopyridine (729 g) was completely converted to nicotinamide (855 g) in 12h at 25°C using 9.0 g resting cells (dry cell weight) of R. rhodochrous PA-34.     

The superiority of the third-generation catalyst,Rhodococcus rhodochrous J1 nitrile hydratase,for industrial production of acrylamide

As the third-generation biocatalyst for industrial production of acrylamide, the superiority of Rhodococcus rhodochrous J1 nitrile hydratase was demonstrated in comparison with other acrylamide-producing bacteria. R. rhodochrous J1 enzyme is much more heat stable and more tolerant to a high concentration of acrylonitrile than Pseudomonas chlororaphis B23 and Brevibacterium R312 enzymes. The J1 enzyme is peculiar in its extremely high tolerance to acrylamide. The hydration reaction of acrylonitrile catalysed by J1 cells proceeded even in the presence of 50% (w/v) acrylamide. The tolerance of J1 enzyme to various organic solvents such as n-propanol and isopropanol was prominent. Using R. rhodochrous J1 resting cells, the accumulation reaction was carried out by feeding acrylonitrile to maintain a level of 6%. After 10 h incubation, the accumulation of acrylamide was approximately 65.6% (w/v) at 10°C, 56.7% (w/v) at 15°C, and 56.0 (w/v) at 20°C. The high stability, high catalytic efficiency and other outstanding features of the J1 enzyme are analysed and discussed. Correspondence to: T. Nagasawa     

Production of acrylic acid and methacrylic acid using Rhodococcus rhodochrous J1 nitrilase

Summary -Caprolactam-induced Rhodococcus rhodochrous J1 cells containing abundant nitrilase were used in the production of acrylic acid and methacrylic acid from acrylonitrile and methacrylonitrile, respectively. Under a periodic substrate feeding system, the highest accumulations, 390 g acrylic acid/l and 260 g methacrylic acid/l, were attained. Offprint requests to: T. Nagasawa     

ε-caprolactam,a new powerful inducer for the formation of Rhodococcus rhodochrous J1 nitrilase

We found that -caprolactam is a new powerful inducer for the formation of Rhodococcus rhodochrous J1 nitrilase. When Rhodococcus rhodochrous J1 cells were cultivated at 28°C for 120 h in a nutrient medium supplemented with 0.5% (w/v) -caprolactam, an enormous amount of nitrilase was formed in the cells which corresponded to approximately 30% of all soluble protein. The level of -caprolactam in the culture broth barely decreased in the course of cultivation. -Butyrolactam and -valerolactam also caused effective induction. The induction of nitrilase formation by -caprolactam was also observed in some other Rhodococcus strains.     

Oxidative biotransformation of thioanisole by <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> IEGM 66 cells

Comparative study of sulfoxidation activity of free and immobilized Rhodococcus rhodochrous IEGM 66 cells was performed. Free Rhodococcus cells (in the presence of 0.1 vol % n-hexadecane) displayed maximal oxidative activity towards thioanisole (0.5 g/l), a prochiral organic sulfide, added after 48-h cultivation of bacterial cells. Higher sulfide concentrations inhibited sulfoxidation activity of Rhodococcus. Use of immobilized cells allowed the 2-day preparatory stage to be omitted and a complete thioanisole bioconversion to be achieved in 24 h in the case that biocatalyst and 0.5 g/l thioanisole were added simultaneously. The biocatalyst immobilized on gel provides for complete thioanisole transformation into (S)-thioanisole sulfoxide (optical purity of 82.1%) at high (1.0–1.5 g/l) concentrations of sulfide substrate.     

Bioconversion of acrylonitrile to acrylamide using polyacrylamide entrapped cells of <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> PA-34

The nitrile hydratase (NHase) of Rhodococcus rhodochrous PA-34 catalyzed the conversion of acrylonitrile to acrylamide. The resting cells (having NHase activity) (8 %; 1 mL corresponds to 22 mg dry cell mass, DCM) were immobilized in polyacrylamide gel containing 12.5 % acrylamide, 0.6 % bisacrylamide, 0.2 % diammonium persulfate and 0.4 % TEMED. The polyacrylamide entrapped cells (1.12 mg DCM/mL) completely converted acrylonitrile in 3 h at 10 °C, using 0.1 mol/L potassium phosphate buffer. In a partitioned fed batch reactor, 432 g/L acrylamide was accumulated after 1 d. The polyacrylamide discs were recycled up to 3×; 405, 210 and 170 g/L acrylamide was produced in 1st, 2nd and 3rd recycling reactions. In four cycles, a total of 1217 g acrylamide was produced by recycling the same mass of entrapped cells.     

Purification and characterization of rhodobactin: a mixed ligand siderophore from Rhodococcus rhodochrous strain OFS

The siderophore produced by Rhodococcus rhodochrous strain OFS, rhodobactin, was isolated from iron-deficient cultures and purified by a combination of XAD-7 absorptive/partition resin column and semi-preparative HPLC. The siderophore structure was characterized using 1D and 2D ¹H, ¹³C and ¹⁵N NMR techniques (DQFCOSY, TOCSY, NOESY, HSQC and LR-HSQC) and was confirmed using ESI-MS and MS/MS experiments. The structural characterization revealed that the siderophore, rhodobactin, is a mixed ligand hexadentate siderophore with two catecholate and one hydroxamate moieties for iron chelation. We further investigated the effects of Fe concentrations on siderophore production and found that Fe limiting conditions (Fe concentrations from 0.1 μM to 2.0 μM) facilitated siderophore excretion. Our interests lie in the role that siderophores may have in binding metals at mixed contamination sites (containing metals/radionuclides and organics). Given the broad metabolic capacity of this microbe and its Fe scavenging ability, R. rhodochrous OFS may have a competitive advantage over other organisms employed in bioremediation. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Optimum culture conditions for the production of cobalt-containing nitrile hydratase by Rhodococcus rhodochrous J1

Summary We sought the optimum conditions for production of nitrile hydratase by Rhodococcus rhodochrous J1. The addiiion of both cobalt ions and an aliphatic nitrile or amide as an inducer was indispensable for the appearance of nitrile hydratase activity in R. rhodochrous J1 cells. Crotonamide was an efficient inducer and, moreover, urea was found to be the most powerful inducer for the production of nitrile hydratase. When R. rhodochrous J1 was cultivated under optimal conditions, the enzyme activity in the culture broth and the specific activity was approximately 32,000 and 512 times higher than the initially obtained levels, respectively. The nitrile hydratase formed corresponded to more than 45% of the total soluble protein in urea-induced cells, as judged by quantitative evaluation of the gel track.Offprint requests to: T. Nagasawa     

Nitrilase-Catalyzed Production of Nicotinic Acid from 3-Cyanopyridine in Rhodococcus rhodochrous J1

The nitrilase which occurs abundantly in cells of Rhodococcus rhodochrous J1 catalyzes the direct hydrolysis of 3-cyanopyridine to nicotinic acid without forming nicotinamide. By using resting cells, the reaction conditions for nicotinic acid production were optimized. Under the optimum conditions, 100% of the added 3-cyanopyridine could be converted to nicotinic acid, the highest yield achieved being 172 mg of nicotinic acid per 1.0 ml of reaction mixture containing 2.89 mg (dry weight) of cells in 26 h.     

A new type of muconate cycloisomerase from Rhodococcus rhodochrous strain 89

Muconate cycloisomerase (MCI) was purified from Rhodococcus rhodochrous 89 grown on phenol. The enzyme appears to contain two different type subunits with molecular masses 35.5 and 37 kD. The N-terminal amino acid sequence of both subunits showed more similarity to corresponding enzymes from gram-negative bacteria than to one from Rhodococcus opacus 1CP. MCI from R. rhodochrous 89, like analogous enzymes from gram-negative bacteria, can convert 2-chloromuconate (2-CM) with the formation of both, 2- and 5-chloromuconolactones (CML) as intermediates. Nevertheless, its unique ability to convert 5-CML to cis- but not to trans-dienelactone sets it apart from all known chloromuconate cycloisomerases from gram-negative and gram-positive bacteria.     

Effect of Medium Components on Bacteriocin Production by Lactobacillus Pentosus ST151BR,a Strain Isolated from Beer Produced by the Fermentation of Maize,Barley and Soy Flour

Lactobacillus pentosus ST151BR, isolated from home-brewed beer, produces a 3.0 kDa antibacterial peptide (bacteriocin ST151BR) active against Lactobacillus casei, Lactobacillus sakei, Pseudomonas aeruginosa, Enterococcus faecalis and Escherichia coli. Treatment with Proteinase K or Pronase resulted in loss of activity. Bacteriocin levels of 6400 AU/ml were recorded in MRSbb (De Man-Rogosa-Sharpe broth without Tween 80) at pH 5.5, 6.0 and 6.5. The same growth conditions at pH 4.5 yielded only 1600 AU/ml bacteriocin. Inclusion of Tween 80 in the growth medium reduced bacteriocin production by more than 50%. Growth in the presence of tryptone or tryptone plus meat extract stimulated bacteriocin production, whereas much lower activity was recorded when the bacteria were grown in the presence of meat extract, yeast extract, tryptone plus yeast extract, meat extract plus yeast extract, or a combination of tryptone, meat extract and yeast extract. MRSbb supplemented with maltose, lactose or mannose (2.0%, w/v) yielded bacteriocin levels of 6400 AU/ml. Sucrose or fructose at these concentrations reduced the activity by 50 and 75%, respectively. Growth in the presence of 4.0%(w/v) glucose resulted in 50% activity loss. Glycerol levels as low as 0.1%(w/v) repressed bacteriocin production. Addition of cyanocobalamin, ascorbic acid, thiamine and thioctic acid (1.0 mg/l) to the growth medium did not lead to an increase in bacteriocin production. This revised version was published online in July 2006 with corrections to the Cover Date.     

Isonicotinic acid hydrazide induced changes and inhibition in mycolic acid synthesis in Nocardia and related taxa

The mycolic acid compositions of Nocardia rubra and related bacteria grown in media containing different concentrations of antituberculous isonicotinic acid hydrazide (INH) were determined in detail by gas chromatography-mass spectrometry. On the basis of molecular species composition, average carbon numbers of mycolic acids were calculated. In Nocardia rubra, N. lutea and Rhodococcus rhodochrous IFO-13161, the ratio of mycolic to non-mycolic fatty acids and the average carbon numbers of mycolic acids were decreased at the INH concentrations of higher than 1 g/ml, paralleling with the significant inhibition of growth. In above three species the synthesis of longer chain mycolic acids (longer than C₄₄ or C₄₆) was inhibited more significantly than shorter homologues such as C₃₈ or C₄₀. In contrast, neither growth inhibition nor change in corynomycolic acid composition was observed in Corynebacteria xerosis and Rhodococcus rhodochrous IFO-13165 at the concentration region of INH up to 100 g/ml. The direct mass fragmentographic analysis of the trimethylsilylated (TMS) derivatives of mycolic acid methyl esters, monitoring [M-15] ions of individual molecular species, revealed that the chain shortening of total mycolic acid molecule by INH occurred more greatly in more highly unsaturated subclasses than in less unsaturated subclasses. Furthermore, mass fragmentographic analysis, monitoring fragment ions (A) and (B), due to straight chain and branched chain alkyl units, respectively, demonstrated the inhibition of mycolic acids was not attributed to the shortening of -alkyl chain, but to the inhibition of chain elongation of C₂₈ to C₃₂ straight chain meromycolic acids. It was also indicated the amounts of trehalose mono- and di-mycolate (cord factor) decreased significantly with the addition of INH (1 to 20 g/ml) in the above strains. From the results obtained above, INH appeared to inhibit the synthesis of mycolic acids longer than C₄₄ or C₄₆ specifically by inhibiting chain elongation or desaturation of precursor long chain fatty acids longer than C₂₈ or C₃₀.     

Enantioselective synthesis of (S)-2-cyano-2-methylpentanoic acid by nitrilase

The nitrilase gene of Rhodococcus rhodochrous J1 was expressed in Escherichia coli using the expression vector, pKK223-3. The recombinant E. coli JM109 cells hydrolyzed enantioselectively 2-methyl-2-propylmalononitrile to form (S)-2-cyano-2-methylpentanoic acid (CMPA) with 96 % e.e. Under optimized conditions, 80 g (S)-CMPA l^?1 was produced with a molar yield of 97 % at 30 °C after a 24 h without any by-products.     

Isolation and purification of methyl mercaptan oxidase fromRhodococcus rhodochrous for mercaptan detection

Methyl mercaptan oxidase was successfully induced fromRhodococcus rhodochrous IGTS8 using methyl mercaptan gas and purified to homogeneity for the detection of mecrcaptans. The purification procedure involved DEAE-Sephacel and Superose 12 column chromatography with recovery yields of 85.8 and 83.3%, and a specific activity of 92.7 and 303.4 units/mg-protein, respectively. The molecular weight of purified methyl mercaptan, oxidase was determined to be 64.5 kDa by SDS-PAGE. The extract from gel filtration chromatography oxidizes methyl mercaptan to produce formaldehyde, which can be easily detected by the purpald-coloring method. Optimum temperature for activity was achieved at 60°C. This enzyme was inhibited by both K₂SO₄ and NaCl at concentration of less than 100 mM and recovered to original activity at concentration of 200 mM. In the presence of methanol, the activity decreased by 33%.     

Simultaneous determination of rufloxacin, fenbufen and felbinac in human plasma using high-performance liquid chromatography

A simple, specific and sensitive high-performance liquid chromatographic method has been developed for the simultaneous determination of rufloxacin, fenbufen and felbinac in human plasma. Plasma, spiked with internal standard, was vortex-mixed for 1 min with a mixture of dichloromethane-diethyl ether (80:20, v/v). The evaporated extract was dissolved in 0.02 M NaOH. Drugs were resolved at room temperature on a 5 μm Zorbax SAX column (250×4.6 min I.D.) equipped with a 20×4.6 mm anion-exchange Vydac AXGU ( 10 μm particle size) precolumn. The mobile phase consisted of acetonitrile and phosphate buffer (pH 7.0), delivered at a flow-rate of 1.2 ml/min. Detection was made at 280 nm, 2-[4-(2′-Furoyl)phenyl]propionic acid was used as internal standard. The calibration curve was linear from 0.2 to 10μg/ml for rufloxacin, from 0.5 to 30 μg/ml for fenbufen and from 0.2 to 10 μg/ml for felbinac, respectively. The detection limit was 0.1 μg/ml for rufloxacin. 0.3 μg/ml for fenbufen and 0.1 μg/ml for felbinac, respectively.     

Occurrence of Amidases in the Industrial Microbe Rhodococcus rhodochrous J1

Rhodococcus rhodochrous J1, of which the high-M_r nitrile hydratase has been used for the industrial manufacture of acrylamide from acrylonitrile, produced at least two amidases differing in substrate specificity, judging from the effects of various amides on amidase activity in this strain. These amidases seemed to be inducible enzymes depending on amide compounds.     

Metabolism of lignin-related compounds by Rhodococcus rhodochrous: bioconversion of anisoin

Summary The ability of the nocardioform actinomycete Rhodococcus rhodochrous to metabolize selected lignin model compounds was studied. The compounds studied included cinnamic and ferulic acids and dimers possessing intermonomeric linkages that are characteristic of the lignin molecule. R. rhodochrous reduced the carbonyl group of anisoin, a 1,2-diarylethane (-1) structure to (1R,2R)-1,2-bis(4-methoxyphenyl)ethane-1,2-diol with an enantiomeric excess of .98%. Cleavage of 1,2-diarylethane and -O-4 structures by this strain could not be detected under our metabolic conditions. Offprint requests to: V. Andreoni     

Development of medium components for the production of glucosyl-transferring enzyme by Aureobasidium

Aureobasidium sp. ATCC 20524 produced a glucosyl-transferring enzyme which produced panose (O--D-glucopyranosyl-(1»6)-O--D-glucopyranosyl-(1»4)-d-glucose) from maltose. Optimum production for the enzyme was with maltose at 2% (w/v) and yeast extract at 1.5% (w/v). Enzymatic activity reached 0.7×10³ U/g dry cells after 48 h.