Oxidative biotransformation of thioanisole by Rhodococcus rhodochrous 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.     

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.     

Sequence analysis and heterologous expression of a new cytochrome P450 monooxygenase from Rhodococcus sp. for asymmetric sulfoxidation

In this study, a 3.7-kb DNA fragment was cloned from Rhodococcus sp. ECU0066, and the sequence was analyzed. It was revealed that the largest one (2,361 bp) of this gene fragment encodes a protein consisting of 787 amino acids, with 73% identity to P450RhF (accession number AF45924) from Rhodococcus sp. NCIMB 9784. The gene of this new P450 monooxygenase (named as P450SMO) was successfully expressed in Escherichia coli BL21 (DE3), and the enzyme was also purified and characterized. In the presence of reduced nicotinamide adenine dinucleotide phosphate, the enzyme showed significant sulfoxidation activity towards several sulfides, with (S)-sulfoxides as the predominant product. The p-chlorothioanisole, p-fluorothioanisole, p-tolyl methyl sulfide, and p-methoxythioanisole showed relatively higher activities than the other sulfides, but the stereoselectivity for p-methoxythioanisole was much lower. The optimal activity of the purified enzyme toward p-chlorothioanisole occurred at pH 7.0 and 30°C. The current study is the first to report a recombinant cytochrome P450 enzyme of Rhodococcus sp. which is responsible for the asymmetric oxidation of sulfides. The new enzymatic activity of P450SMO on the above compounds makes it an attractive biocatalyst for asymmetric synthesis of enantiopure sulfoxides.     

Influence of inorganic phosphate and immobilization on Claviceps purpurea

Summary The influence of inorganic phosphate and immobilization on cells of Claviceps purpurea strain 1029/N5 producing ergot peptides in shake culture was examined. Immobilization in Ca-alginate beads resulted in a marked reduction of some metabolic activities, i.e. the periods of alkaloid formation and cell growth were prolonged. High concentrations of inorganic phosphate (1 g/l KH₂PO₄) could reduce or stop alkaloid formation both by free and immobilized cells at any time during fermentation. The optimum phosphate concentration for alkaloid production by immobilized cells (about 0.5 mM) was a quarter of that required by free cells. This optimum shift was attributed to (i) the diminished phosphate demand of immobilized cells, due to their reduced metabolic activities, and (ii) the phosphate-dependent morphological behaviour of the biocatalyst. The observed decrease in alkaloid concentrations during later periods of the fermentation supported the idea of alkaloid-degradative enzymes, activated by high phosphate concentrations. Immobilization showed an advantageous influence on this undesirable effect. Offprint requests to: H.J. Rehm     

Production of high fructose syrup from <Emphasis Type="Italic">Asparagus</Emphasis> inulin using immobilized exoinulinase from <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> YS-1

Extracellular exoinulinase from Kluyveromyces marxianus YS-1, which hydrolyzes inulin into fructose, was immobilized on Duolite A568 after partial purification by ethanol precipitation and gel exclusion chromatography on Sephadex G-100. Optimum temperature of immobilized enzyme was 55 °C, which was 5 °C higher than the free enzyme and optimal pH was 5.5. Immobilized biocatalyst retained more than 90% of its original activity after incubation at 60 °C for 3 h, whereas in free form its activity was reduced to 10% under same conditions, showing a significant improvement in the thermal stability of the biocatalyst after immobilization. Apparent K _m values for inulin, raffinose and sucrose were found to be 3.75, 28.5 and 30.7 mM, respectively. Activation energy (E _a) of the immobilized biocatalyst was found to be 46.8 kJ/mol. Metal ions like Co²⁺ and Mn²⁺ enhanced the activity, whereas Hg²⁺ and Ag²⁺ were found to be potent inhibitors even at lower concentrations of 1 mM. Immobilized biocatalyst was effectively used in batch preparation of high fructose syrup from Asparagus racemosus raw inulin and pure inulin, which yielded 39.2 and 40.2 g/L of fructose in 4 h; it was 85.5 and 92.6% of total reducing sugars produced, respectively.     

Alcoholic fermentation of D-xylose by immobilizedPichia stipitis yeast

Summary Pichia stipitis NRRL Y-7124 yeast cells were for the first time immobilized both in agar gel beads and on fine nylon net for ethanol fermentation on D-xylose, in order to investigate the possibility of using the biocatalyst for improved utilization of the biomass pentose fraction. With free cells the initial xylose level affected little ethanol production, with a maximum of 22 g/l ethanol obtained in 5 days on 5% and of 40 g/l in 8 days on 10% xylose, and an average volumetric productivity of about 0.22 g/lh. The maximum ethanol concentration of 19.5% on 5% xylose with the nylon net attached cells in a continuous packed-bed column reactor was obtained with 35 h residence time. The volumetric productivities of 0.56 g/lh at 19.5 g/l ethanol and 1.0 g/lh at 15.0 g/l ethanol were markedly higher than those obtained with free cells. The stability of the immobilized biocatalyst was excellent. The same reactor could be used for at least 80 days without significant activity loss.     

Continuous production of chiral 1,3-butanediol using immobilized biocatalysts in a packed bed reactor: promising biocatalysis method with an asymmetric hydrogen-transfer bioreduction

An asymmetric hydrogen-transfer biocatalyst consisting of mutated Rhodococcus phenylacetaldehyde reductase (PAR) or Leifsonia alcohol dehydrogenase (LSADH) was applied for some water-soluble ketone substrates. Among them, 4-hydroxy-2-butanone was reduced to (S)/(R)-1,3-butanediol, a useful intermediate for pharmaceuticals, with a high yield and stereoselectivity. Intact Escherichia coli cells overexpressing mutated PAR (Sar268) or LSADH were directly immobilized with polyethyleneimine or 1,6-diaminehexane and glutaraldehyde and evaluated in a batch reaction. This system produced (S)-1,3-butanediol [87% enantiomeric excess (e.e.)] with a space time yield (STY) of 12.5 mg h⁻¹ ml⁻¹ catalyst or (R)-1,3-butanediol (99% e.e.) with an STY of 60.3 mg h⁻¹ ml⁻¹ catalyst, respectively. The immobilized cells in a packed bed reactor continuously produced (R)-1,3-butanediol with a yield of 99% (about 49.5 g/l) from 5% (w/v) 4-hydroxy-2-butanoate over 500 h.     

Petroleum-contaminated water treatment in a fluidized-bed bioreactor with immobilized Rhodococcus cells

Biotreatment of petroleum-contaminated water in a fluidized-bed bioreactor (FBB) is a relatively new and promising technology, which efficiency is strongly affected by the biocatalyst used. Our laboratory experiments involved biotreatment of the water contaminated with a synthetic petroleum mixture consisting of aliphatic and polyaromatic hydrocarbons (PAHs) using a continuous column bioreactor with recycle. Different type biocatalysts were tested, including Rhodococcus bacteria immobilized in hydrophobized carriers such as sawdust, poly(vinyl alcohol) cryogel (cryoPVA) and poly(acrylamide) cryogel (cryoPAAG). Biocatalyst abilities to oxidize petroleum hydrocarbons were evaluated using the Columbus Micro-Oxymax^® respirometer. The hydrophobized sawdust-supported biocatalyst demonstrated substantially higher metabolic activity than C₁₂-cryoPAAG-based biocatalyst due to larger number of immobilized Rhodococcus cells and, therefore, had benefits for application in FBBs. The obtained results showed that designed FBB process is successful, providing 70–100% removal of n-alkanes (C₁₀–C₁₉) and 66–70% removal of 2–3-ring PAHs from contaminated water after 2–3 weeks.     

Ethanol production at 45°C by Kluyveromyces marxianus IMB3 immobilized in magnetically responsive alginate matrices

Summary The thermotolerant yeast, Kluyveromyces marxianus IMB3 produced 11g ethanol/l during growth at 45°C on media containing 4% (w/v) lactose when immobilized in alginate beads whereas the free cells produced 5g ethanol/l. A magnetically responsive biocatalyst, prepared by incorporating Fe₃O₄ into the alginate matrix increased ethanol production to 12g/l in batch-fed reactors. Ethanol concentrations were further increased to a maximum of 18g/l by immobilization of the endogenous K. marxianus -galactosidase to the Fe₃O₄ particles prior to inclusion into the alginate matrix. Maximum ethanol productivity by the system was 87% of the maximum theoretical yield.     

Itaconic acid production by immobilizedAspergillus terreus from xylose and glucose

Summary Aspergillus terreus NRRC 1960 spores were entrapped in calcium alginate gel beads or alternotely the fungal mycelium was immobilized either on Celite R-626 or in agar gel cubes, and the biocatalyst was employed both in repeated batch and in continuous column reactors to produce itaconic acid from D-xylose or D-glucose. The highest itaconic acid yield obtained in a submerged culture batch fermentation was 54.5% based on total initial glucose (55 g/l) with a volumetric productivity of 0.32 g/l h, and 44.8% from xylose (67 g/l) with a productivity of 0.20 g/l h. In a repeated batch fermentation mycelium immobilized in agar gel had a productivity of 0.112 g/l h, and mycelium grown from spores immobilized in calcium alginate gel 0.06 g/l h, both from xylose (60 g/l). With the best immobilized biocatalyst system used employing Celite R-626 as a carrier, volumetric productivities of 1.2 g/l h from glucose and 0.56 g/l h from xylose (both at 60 g/l) were obtained in continuous column operation for more than 2 weeks.     

Synthesis of optically pure <Emphasis Type="Italic">S</Emphasis>-sulfoxide by <Emphasis Type="Italic">Escherichia coli</Emphasis> transformant cells coexpressing the P450 monooxygenase and glucose dehydrogenase genes

A cytochrome P450 monooxygenase (P450SMO) from Rhodococcus sp. can catalyze asymmetric oxygenation of sulfides to S-sulfoxides. However, P450SMO-catalyzed biotransformations require a constant supply of NAD(P)H, the expense of which constitutes a great hindrance for this enzyme application. In this study, we investigated the asymmetric oxygenation of sulfide to S-sulfoxide using E. coli cells, which co-express both the P450SMO gene from Rhodococcus sp. and the glucose dehydrogenase (GDH) gene from Bacillus subtilis, as a catalyst. The results showed that the catalytic performance of co-expression systems was markedly improved compared to the system lacking GDH. When using recombinant E. coli BL21 (pET28a-P450-GDH) whole cell as a biocatalyst, NADPH was efficiently regenerated when glucose was supplemented in the reaction system. A total conversion of 100% was achieved within 12 h with 2 mM p-chlorothioanisole substrate, affording 317.3 mg/L S-sulfoxide obtained. When the initial sulfide concentration was increased to 5 mM, the substrate conversion was also increased nearly fivefold: S-sulfoxide amounted to 2.5 mM (396.6 mg/L) and the ee value of sulfoxide product exceeded 98%. In this system, the effects of glucose concentration and substrate concentration were further investigated for efficient biotransformation. This system is highly advantageous for the synthesis of optically pure S-sulfoxide.     

Biocatalytic Amide Reduction Using <Emphasis Type="Italic">Clostridium sporogenes</Emphasis>

Washed cells of Clostridium sporogenes reduced benzamide (up to 20 mm) to benzylamine in yields up to 73% using H₂ as electron donor with less than 10 g biocatalyst/l over 24 h. Product formation exhibited complex kinetics, with a lag before benzylamine production began. Very little substrate was hydrolysed since the maximum yield of benzoic acid was only 9% of the substrate added. Boiled cells were inactivated thus confirming that amide reduction was enzyme-catalysed.     

Production of 6-aminopenicillanic acid by immobilized Pleurotus ostreatus

Summary 6-Aminopenicillanic acid from penicillin V is produced by Pleurotus ostreatus immobilized by entrapment in a chitosan matrix. In these carriers the cell concentration increases after network formation by irreversible shrinking of the biocatalyst. Specific activity of the biocatalyst for the hydrolysis reaction is 1,31 mol.min^-1. (g wet weight of catalyst)^-1 corresponding to a relative activity of 38%. Catalytic half-life of immobilized Pl. ostreatus is 25 days compared to 2.5 days for free suspended cells.This paper is part of the Dissertation of Michael Kluge, Technical University Braunschweig 1981.     

Degradation of phenol by polymer entrapped microorganisms

Summary A Pseudomonas sp. which was isolated from phenol-containing soil was immobilized in alginate and polyacrylamide-hydrazide (PAAH) and cultivated in a special airlift fermenter.The immobilized Pseudomonas sp. was able to degrade phenol at initial concentrations up to 2 g/l in less than 2 days, although the free cells did not grow at this concentration.The immobilization materials act as a protective cover against phenol, PAAH being more effective than alginate. The degradation activity as well as the outgrowth of bacteria can be manipulated by the concentration of the immobilization material, the temperature and the nitrogen content in the medium.The cells grew predominantly in microcolonies in the outer area of the beads when nitrogen was available as 1.0g NH₄NO₃/l and 0.5g (NH₄)₂SO₄/l.Prof. Dr. A. Fiechter dedicated to his 60th birthday     

Adsorptive immobilization of rhodococcal cells in hydrophobized derivatives of wide-pore poly(acrylamide) cryogel

Adsorption of Rhodococcus ruber cells on columns with poly(acrylamide) cryogel (cryoPAAG) partially hydrophobized by different quantities (0.2, 1, and 5, mol %) of chemically grafted n-dodecane residues has been studied. The adsorption capacity (1.1 × 10⁹ cells/g) of gel carrier for rhodococcal cells and the optimal content (1 mol %) of hydrophobizing groups were determined. The respirometric method showed the high catalytic activity and functional stability of immobilized bacterial cells. Respiratory activity of immobilized rhodococci in the presence of a model mixture of oil hydrocarbons exceeded the respective parameter for free cells by 12–17%. Viability of rhodococcal cells adsorptionally fixed in hydrophobized cryoPAAG was maintained at a level of 93–95% after a half-year period of storage. The results may be used for development of immobilized biocatalyst for directed transformation of hydrocarbon compounds and biological purification of oil-polluted water.     

Ethanol production at 45°C by alginate-immobilized Kluyveromyces marxianus IMB3 during growth on lactose-containing media

The thermotolerant yeast strain Kluyveromyces marxianus IMB3 was immobilized in calcium alginate and this was used in batch-fed reactor systems to convert lactose (4?g/l) to ethanol. Production of ethanol by the free and immobilized biocatalyst in the presence and absence of Mn²⁺ was compared. In systems containing the free microorganism in the presence and absence of Mn²⁺, ethanol increased to a maximum of 8?g/l within 40 hours with no significant difference in production by both systems. Ethanol production by the immobilized system in the absence of Mn²⁺ increased to a maximum of 13?g/l within 40 hours and then decreased to 9?g/l within 80 hours. Ethanol production by the immobilized system in the presence of Mn²⁺ increased to 14?g/l within 60 hours and this decreased to 13?g/l at 80 hours. When all systems were re-fed at 80 hours, ethanol production by systems containing the free biocatalyst increased to a maximum of 3?g/l while the immobilized system in the presence of Mn²⁺ increased to a maximum of 12?g/l. Subsequent experiments involving re-feeding the system at shorter time intervals demonstrated that ethanol production by the immobilized system on lactose-containing media at 45?°C was far superior to ethanol production by the free biocatalyst.     

Enhancing the catalytic potential of nitrilase from Pseudomonas putida for stereoselective nitrile hydrolysis

(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%.     

Biosurfactant-enhanced immobilization of hydrocarbon-oxidizing Rhodococcus ruber on sawdust

Immobilization of microorganisms on/in insoluble carriers is widely used to stabilize functional activity of microbial cells in industrial biotechnology. We immobilized Rhodococcus ruber, an important hydrocarbon degrader, on biosurfactant-coated sawdust. A biosurfactant produced by R. ruber in the presence of liquid hydrocarbons was found to enhance rhodococcal adhesion to solid surfaces, and thus, it was used as a hydrophobizing agent to improve bacterial attachment to a sawdust carrier. Compared to previously used hydrophobizers (drying oil and n-hexadecane) and emulsifiers (methyl- and carboxymethyl cellulose, poly(vinyl alcohol), and Tween 80), Rhodococcus biosurfactant produced more stable and homogenous coatings on wood surfaces, thus resulting in higher sawdust affinity to hydrocarbons, uniform monolayer distribution of immobilized R. ruber cells (immobilization yield 29–30 mg dry cells/g), and twofold increase in hydrocarbon biooxidation rates compared to free rhodococcal cells. Two physical methods, i.e., high-resolution profilometry and infrared thermography, were applied to examine wood surface characteristics and distribution of immobilized R. ruber cells. Sawdust-immobilized R. ruber can be used as an efficient biocatalyst for hydrocarbon transformation and degradation.     

High solids fermentation of hydrolysates of wheat starch B in a continuous dynamic immobilized biocatalyst bioreactor

Summary A simple and efficient method of conversion of wheat starch B to ethanol was investigated. Employing a two-stage enzymatic saccharification process, 95% of the wheat starch was converted to fermentable sugars in 40 h. From 140 g/l total sugars in the feed solution, 63.6 g/l ethanol was produced continuously with a residence time of 3.3 h in a continuous dynamic immobilized biocatalyst bioreactor by immobilized cells ofSaccharomyces cerevisiae. The advantages and the application of this bioreactor to continuous alcoholic fermentation of industrial substrates are presented.     

Tyrosinase catalyzes asymmetric sulfoxidation

Mushroom tyrosinase was found to catalyze the oxidation of organic sulfides to sulfoxides in the presence of a catechol as cosubstrate, in a reaction which is unprecedented for this enzyme and resembles those performed by external monooxygenases. Only the oxy form of the enzyme is in fact capable of oxidizing the sulfide in a two-electron process, while the resulting met form can only be recycled by reduction with catechol. The cosubstrate competes with the sulfide also in the reaction with oxy-tyrosinase. For this reason, the sulfoxidation of thioanisole in the presence of l-3,4-dihydroxyphenylalanine (L-dopa) occurs with moderate yields ( approximately 20%) but high enantioselectivity ( approximately 85% e.e.), and favors ( S)-methyl phenyl sulfoxide. The enantioselectivity can be further increased to >90% when excess ascorbic acid is added to the reaction to limit enzyme inactivation by the quinones produced by L-dopa oxidation. An experiment using (18)O 2 showed that 18-O incorporation into methyl phenyl sulfoxide was above 95%, confirming that the mechanism of the sulfoxidation involves oxygen transfer from oxy-tyrosinase to the sulfide.