Bioconversion potential of plant enzymes for the production of pharmaceuticals

Plant enzymes are able to catalyze regio- and stereospecific reactions. Freely suspended and immobilized plant cells as well as enzyme preparations can therefore be applied for the production of pharmaceuticals by bioconversion, as such or in combination with chemical syntheses. This review paper deals with bioconversions of added precursors from natural or synthetic origin by several biocatalytic systems.     

Δ′-Dehydrogenation of steroids byArthrobacter simplex immobilized in calcium polygalacturonate beads

Summary Arthrobacter simplex ATCC 6946 (viable cells) was immobilized in a calcium polygalacturonate gel. The trapped cells were used for repeated batchwise bioconversion of steroids. Reichstein's compound S and hydrocortisone were dehydrogenated introducing a double bond between C₁ and C₂ of ring A. The products 1-dehydro S and prednisolone, respectively, were identified by high pressure liquid chromatography. Steroid dehydrogenase activity increased in the system when an artificial electron acceptor, such as menadione (vitamin K₃) was present in the reaction mixture. An airlift-type reactor was used to bioconvert up to 90% of substrate in 15 min, under optimal conditions. The gel entrapped cell preparations were used for repeated batch bioconversion during 30 days; 69 batch bioconversions for Reichstein's compound S were performed during 15 days of operation of the reactor. The operational stability of the process and the feasibility of repeated batch bioconversions was shown to be comparable to similar processes.     

Bioconversion of hydrophobic compounds in a continuous closed-gas-loop bioreactor: feasibility assessment and epoxide production

Microorganisms can be used as catalysts to produce organic compounds in a highly chemo-, regio- and enantioselective manner, and whole cells do not require the costly addition of cofactors for redox reactions. However, bioconversions are slow compared to alternative chemical reactions, and the biocatalyst works at its best in an aqueous medium, while the transformations of interest frequently involve compounds with a low-aqueous solubility and that are toxic to microorganisms. This results in low-volumetric productivity in classical bioreactors. The Continuous Closed-Gas-Loop Bioreactor is described here-a reactor system with high productivity, but without the problems associated with two-phase systems, such as an emulsified product stream and phase toxicity. Its working principle is to recirculate a gas phase through a bioreaction compartment and a saturator/absorber module where the product accumulates as a clear organic solution. A wide range of bioconversions should be possible in this set-up, and proof of concept was established for the epoxidation of 1,7-octadiene to (R)-1,2-epoxyoct-7-ene by a native strain of Pseudomonas oleovorans. This reaction represents a group of terminal alkene epoxidations where the bioconversion substrate does not support growth of the microorganism. Practical results at a 5l-scale are presented for this bioconversion for both batch and continuous operation with respect to the aqueous phase, showing continuous stable epoxidation at productivities >14 micromol min(-1) L(-1) (U L(-1)). The results confirm that the metabolism does not allow a simple optimization strategy, because growth and biotransformation substrates compete for the same enzyme sites, and conversely growth on a substrate using this very enzyme system is necessary for longterm bioconversion. Integrated removal of the CO(2) formed via the liquid overflow was estimated from theory and verified in experimental work.     

Whole-cell biocatalysis in organic media

The use of water-immiscible organic solvents in whole-cell biocatalysis has been exploited for biotransformations involving sparingly water-soluble or toxic compounds. These systems can overcome the problem of low productivity levels in conventional media due to poor substrate solubility, integrate bioconversion and product recovery in a single reactor, and shift chemical equilibria enhancing yields and selectivities; nevertheless, the selection of a solvent combining adequate physicochemical properties with biocompatibility is a difficult task. The cell membrane seems to be the primary target of solvent action and the modification of its characteristics the more relevant cellular adaptation mechanism to organic solvent-caused stress. Correlations between the cellular toxicity or the extractive capacities of different solvents and some of their physical properties have been proposed in order to minimize preliminary, solvent-selection experimental work but also to help in the understanding of the molecular mechanisms of toxicity and extraction. The use of whole cells in organic-media biocatalysis provides a way to regenerate cofactors and carry out bioconversions or fermentations requiring multi-step metabolic pathways; some processes already are commercially exploited. Immobilization can further protect cells from solvent toxicity, and has thus been effectively used in organic solvent-based systems. Several examples of extractive fermentations and other whole-cell bioconversions in organic media are presented.     

Developments toward large-scale bacterial bioprocesses in the presence of bulk amounts of organic solvents

Many pseudomonads and other bacteria can grow on aliphatic and aromatic hydrocarbons that occur in the environment. We are examining the potential of such organisms as biocatalysts for the oxidation of a variety of substituted aliphatic and aromatic compounds. To attain a high production rate of oxidation products via such biotransformations, we have focused on two-liquid phase culture systems. In these systems, cells are grown in liquid media consisting of an aqueous phase containing water-soluble growth substrates and droplets of a water-immiscible organic solvent containing bioconversion substrates and products. For industrial applications of such two-liquid phase processes, several questions remain. What are the maximum rates at which apolar compounds can be transferred from the apolar phase to cells growing in the aqueous phase, i.e., what are the maximum space-time yields attainable in two-liquid phase fermentations under practical conditions? What does an efficient downstream processing of two-liquid phase medium involve? What safety regimes should be considered in working with flammable organic solvents? Can elevated pressure be used to increase oxygen transfer? Based on answers to these questions, we have recently developed a high-pressure, explosion-proof bioreactor system with Bioengineering AG (Wald, Switzerland), which will be installed in our pilot plant and used to explore two-liquid phase bioconversions at a pilot scale.     

Food Bioconversions and Metabolite Production Using Immobilized Cell Technology

Abstract

This review explores recent advances in the use of immobilized cells for the production of metabolites used in the food industry, such as enzymes, amino acids, organic acids, alcohols, aroma compounds, polysaccharides, and pigments. Some food bioconversions such as fermentation of soy sauce and various hydrolysis are also considered. Special emphasis was placed on existing or potential industrial processes. This article also reports the effects of the reactor (configuration and working conditions), the immobilized cell physiological status (growing, nongrowing, or permeabilized), and of the carrier type, configuration, and size on the performance of immobilized cell systems. Compared with free cell fermentation, the main advantage of using immobilized cells is an increase in productivity, particularly in the case of continuous fermentation. For monoenzymatic reactions, nongrowing immobilized cells are often reported to exhibit a higher stability than free or immobilized enzymes.     

Factors affecting the glucosylation capacity of cell cultures of Datura innoxia and Scopolia carniolica for monophenolic compounds

Suspension grown cells of Datura innoxia and Scopolia carniolica were tested for their glucosylation capacity and some factors affecting the efficiency of the reaction were studied.Cells at the end of the exponential growth phase showed a high glucosylation capacity. Light conditions had little effect on the bioconversion reaction. For the substrates hydroquinone and p-hydroxybenzoic acid the bioconversions were concentration-dependent. Permeabilization with propanol diminished the bioconversion capacity. Depending on the substrate used, relatively large amounts of substrate and product could not be recovered. Tannic acid could partly prevent decomposition of the compounds. The bioconversion capacity of cultures with a low glucosylation capacity could be enhanced by addition of uridine diphosphate-glucose, indicating that the sugar donor is a critical factor. From six substrates the natural compounds hydroquinone, p-hydroxybenzoic acid and vanillin were glucosylated. No glucosides were detected from tyramin and two synthetic aminotetralines.Abbreviations 5HAT 5-hydroxyaminotetralin - NO437 2-(N-propyl-N-2-thienylamino)-5-hydroxytetraline - pHBA p-hydroxybenzoic acid - UDP-glucose uridine diphosphate-glucose     

Advances in bioconversion of anthracycline antibiotics

During the last decade new anthracycline-type structures with potential usefulness in cancer treatment have been supplied both by new microbial strains and by bioconversions of precursor molecules employing cells or enzymes. We highlight recent advances in bioconversion of anthracycline structures with the main focus on late transformations such as are carried out by oxidoreductases.     

Further kinetic characterization of alginate-entrapped cells of Mucuna pruriens L

Alginate-entrapped cells of Mucuna pruriens L. hydroxylate L -tyrosine, tyramine, para-hydroxyphenylpropionic acid, and para-hydroxyphenylacetic acid to their corresponding catechols, which were released into the incubation medium. Michaëlis-Menten kinetics was applied for each bioconversion. The apparent affinity constants were comparable with the affinity constants obtained with a homogenate directly prepared from the cells used for entrapment and with a derived partly purified phenoloxidase. The values found for the apparent maximum rates of bioconversion of the entrapped cells were ca. 50% of the values of the maximum rates of bioconversion of the cell homogenate, indicating that the entrapped cell system was not operating optimally. The effective diffusivities of the substrates and products were measured with alginate-entrapped, inactivated cells. From the five inactivation methods tested, glutaric aldehyde treatment was chosen as the general procedure. Calculated effective diffusivities for the monophenols and catechols demonstrated that these compounds could diffuse freely into and out of the beads. For each bioconversion, the observable modulus was calculated from the initial rate of bioconversion and the effective diffusivity of the substrate. The resulting values indicated that the diffusional supply rate of the substrates was not the limiting factor, except for the conversion of tyramine for which a modulus higher than one was obtained. Analogously, the observable moduli were calculated for oxygen, which was utilized for bioconversion and cell respiration, and these values pointed towards strong oxygen limitation in all cases. The bioconversion rates of the entrapped cells increased with decreasing cell aggregate size. Therefore, it was concluded that direct cell-matrix contact determined the amount of phenoloxidase involved in the bioconversions. The bioconversion rate on a protein basis was constant with enhancement of the bead charge and thus, in spite of limitations, the mixing conditions as such were relatively optimal. In conclusion, the nonoptimal efficiency of the plant cell system studied was caused by oxygen limitation and a partial phenoloxidase participation, but not by mass transfer limitations for substrates and products with the exception of the conversion of tyramine into dopamine.     

A microwell platform for the scale-up of a multistep bioconversion to bench-scale reactors: Sitosterol side-chain cleavage

The microwell-scale approach is widely used for screening purposes and one-pot biotransformations, but it has seldom been applied to complex whole cell multistep bioconversions, requiring prolonged incubation periods. The present study aims to contribute to filling this gap. The side-chain cleavage of sitosterol to androstenedione (AD) with Mycobacterium sp. NRRL B-3805 cells was used as a model system, and focus was given to the screening of suitable bioconversion media with 24-well microwell plates. Results show that to perform this particular bioconversion growing cells are preferred over resting cells due to higher conversion yields obtained in aqueous medium. The use of resting cells may nevertheless present an interesting approach provided catalytic activity is retained throughout successive runs. Maintaining suitable aeration levels (air flow of 1 mL/min) allowed minimizing the decay of catalytic activity typically observed alongside consecutive bioconversion runs with resting cells. Microwell plates with dedicated oxygen and pH monitoring capabilities proved effective in media development for complex multistep bioconversions using relatively slow-growing bacteria. Under constant k_La (0.044/s) similar AD production and dissolved oxygen profiles were observed in microwell plates and in a bench-scale reactor. Selection of a suitable k_La value proved critical, since under lower k_La values scale-up proved unsuccessful. The same pattern was observed when other scale-up criteria were evaluated to perform the scale-up of this particular bioconversion. Results gathered seem to validate the proposed approach “from microwell plate to bench-scale fermentor”.     

Limitations of in silico predictability of specificity of co-immobilised cytochromes P450 and mimics in food-bioprocessing

Cytochromes P450 (EC 1.14.14.1) are mixed function oxidases (oxygenases) that can catalyse redox bioconversions of food components. Also, efficacious removal of undesirable components can be achieved using solid-support immobilised enzyme (IME) of a selection from 2700 isoforms of cytochromes P450 (CYP). Cytochromes P450 co-immobilised with other enzymes, or protein receptors, may be used to confer a secondary order of regio- or stereo-specificity of chiral bioconversion: these can be predictable in silico by utilisation of QSARs (quantitative structure/activity relationships).     

Biotechnological production of flavours and fragrances

The biotechnological generation of natural aroma compounds is rapidly expanding. Aroma chemicals, such as vanillin, benzaldehyde (bitter almond, cherry) and 4-(R)-decanolide (fruity–fatty) are marketed on a scale of several thousand tons per year. Their possible production by single-step biotransformations, bioconversions and de novo synthesis using microorganisms, plant cells or isolated enzymes is shown. The perspectives of bioprocesses for the oxifunctionalisation of lower terpenes by genetically modified organisms and economic aspects are discussed. Received: 27 May 1997 / Received revision: 25 September 1997 / Accepted: 28 September 1997     

Replacement of immobilised cell bioreactors by smaller immobilised enzyme bioreactors: unique-outcome predictability for cytochromes P450 isoforms?

Both immobilized enzymes (IME) and immobilized cells (IMC) are acceptable as the biocatalysts essential for the attainment of rapid rates of bioconversion in bioreactors. IMC can display higher than expected cellular permeability whilst IME can exhibit high catalytic constant (k_cat/K_m) despite limitations on substrate utilisation due to an unstired diffusion layer of solvent. Scale-down switching from IMC to IME involves the replacement of high-volume biotechnology by low-volume biotechnology, sometimes using IME mimics in partially non-aqueous solvent systems. Highly purified IME systems covalently immobilised to particles of, for instance, microcrystalline cellulose or porous glass, can retain both the hydrophilic and hydrophobic intermediate products in situ of the chosen sequence of enzyme reactions. These bioconversions, therefore, are as efficient as those with IMC where enzymes are often particle- or membrane-bound so that even hydrophilic intermediates are not released rapidly into solution. This mimicry of in vivo biosynthetic pathways that are compartmentalised in vivo (e.g. of lysosomes, mitochondria and endoplasmic reticulum) can replace larger IMC by IME especially in application of up to 2700 cytochromes P450 isoforms in bioprocessing. In silico investigation of appropriate model IME systems, in comparison with IMC systems, will be needed to define the optimal bioreactor configuration and parameters of operation, such as pH, T and oxygen mass transfer rate (OTR). The application solely of hazop (applied hazard and operability concepts) may, nevertheless, not be recommended to replace fully the in silico and real-lab pilot-scale and scale studies. Here, food-safe bioprocessing has to be achieved without incorporation of recognised biohazards; especially in the form of unacceptable levels of toxic metals that promote a risk-analysis uncertainty.     

Engineering the plant cell factory for secondary metabolite production

Plant secondary metabolism is very important for traits such as flower color, flavor of food, and resistance against pests and diseases. Moreover, it is the source of many fine chemicals such as drugs, dyes, flavors, and fragrances. It is thus of interest to be able to engineer the secondary metabolite production of the plant cell factory, e.g. to produce more of a fine chemical, to produce less of a toxic compound, or even to make new compounds, Engineering of plant secondary metabolism is feasible nowadays, but it requires knowledge of the biosynthetic pathways involved. To increase secondary metabolite production different strategies can be followed, such as overcoming rate limiting steps, reducing flux through competitive pathways, reducing catabolism and overexpression of regulatory genes. For this purpose genes of plant origin can be overexpressed, but also microbial genes have been used successfully. Overexpression of plant genes in microorganisms is another approach, which might be of interest for bioconversion of readily available precursors into valuable fine chemicals. Several examples will be given to illustrate these various approaches. The constraints of metabolic engineering of the plant cell factory will also be discussed. Our limited knowledge of secondary metabolite pathways and the genes involved is one of the main bottlenecks. This revised version was published online in August 2006 with corrections to the Cover Date.     

Bioconversion of lipophilic compounds by immobilized microbial cells in organic solvents

Microbial cells were gel-entrapped with photo-crosslinkable resin prepolymers or urethane prepolymers, respectively. The resulting gels have different tailor-made hydrophobic or hydrophilic character. They were used for successful bioconversion of hydrophobic steroids and terpenoids in watersaturated mixtures of organic solvents. The experiments show the influence of the hydrophobicity of the gels and the polarity of the solvent mixtures, respectively. Use of hydrophobic gels and less polar solvents is preferable for bioconversion of hydrophobic compounds. The selective formation of a desired product among diverse products from a single substrate by appropriate use of hydrophobic or hydrophilic gels is possible. In each case, tests should be made to select the appropriate gel and solvent mixture. Bioconversions tested are: dehydroepiandrosterone to 4-androstene-3,17-dione; cholesterol to cholestenone; β-sitosterol to β-sitostenone; stigmasterol to stigmastenone; pregnenolone to progesterone; testosterone to Δ¹-dehydrotestosterone or 4-androstene-3,17-dione, respectively; all with immobilized cells of Nocardia rhodocrous; and stereoselective hydrolysis of dl-menthyl-succinate to yield l-menthol with immobilized cells of Rhodotorula minuta var. texensis.     

An airlift biofilm reactor for the biodegradation of phenol by Pseudomonas stutzeri OX1

Phenol bioconversion by Pseudomonas stutzeri OX1 using either free or immobilized cells was investigated with the aim of searching for optimal operating conditions of a continuous bioconversion process. The study was developed by analyzing: (a) free-cell growth and products of phenol bioconversion by batch cultures of P. stutzeri; (b) growth of P. stutzeri cells immobilized on carrier particles; (c) bioconversion of phenol-bearing liquid streams and the establishment and growth of an active bacterial biofilm during continuous operation of an internal-loop airlift bioreactor. We have confirmed that free Pseudomonas cultures are able to transform phenol through the classical meta pathway for the degradation of aromatic molecules. Data indicate that bacterial growth is substrate-inhibited, with a limiting phenol concentration of about 600 mg/L. Immobilization tests revealed that a stable bacterial biofilm can be formed on various types of solid carriers (silica sand, tuff, and activated carbon), but not on alumina. Entrapment in alginate beads also proved to be effective for P. stutzeri immobilization. Continuous bioconversion of phenol-bearing liquid streams was successfully obtained in a biofilm reactor operated in the internal-circulation airlift mode. Phenol conversion exceeded 95%. Biofilm formation and growth during continuous operation of the airlift bioreactor were quantitatively and qualitatively assessed.     

Peptide antibiotic production through immobilized biocatalyst technology

The use of immobilized biocatalysts for producing known or new antibiotics is presented. An evaluation of the applicability of this concept in the fascinating field of peptide antibiotic bioconversions and fermentations is also given.The use of immobilized enzymes, organelles and cells to synthesize antibiotics as an alternative method to conventional fermentation is discussed. In vitro total enzymatic antibiotic synthesis is illustrated with the ‘multienzyme thiotemplate mechanism’ of Bacillus brevis, the producer of gramicidin S. Total synthesis of peptide antibiotics, based on immobilized living cells, has recently been demonstrated with penicillin, bacitracin, nisin and a few other antibiotics.As an industrial example of the use of enzymes or cells to convert peptide antibiotics into therapeutically useful derivatives, free and immobilized penicillin acylases, producing the penicillin nucleus 6-aminopenicillanic acid (6-APA), are reviewed as well as their potential to synthesize semisynthetic β-lactams (penicillins, cephalosporins).Acylases, acetylesterases and α-amino acid ester hydrolases acting on cephalosporin-compounds and yielding valuable intermediary or end products have also gained wide interest. Stereospecific enzymic side-chain preparations for semisynthetic penicillin and cephalosporin production have recently reached the industrial stage. Bioconversion possibilities with the novel β-lactam compounds are suggested.These examples of simple single-step, as well as complex multi-step, enzyme reactions point to the vast potential of immobilized biocatalyst technology in fermentation science, in organic synthesis and in biotechnological processes in general.     

Biotechnological production of vanillin

Vanillin is one of the most important aromatic flavor compounds used in foods, beverages, perfumes, and pharmaceuticals and is produced on a scale of more than 10 thousand tons per year by the industry through chemical synthesis. Alternative biotechnology-based approaches for the production are based on bioconversion of lignin, phenolic stilbenes, isoeugenol, eugenol, ferulic acid, or aromatic amino acids, and on de novo biosynthesis, applying fungi, bacteria, plant cells, or genetically engineered microorganisms. Here, the different biosynthesis routes involved in biotechnological vanillin production are discussed.     

Biotransformation of terpenes

The main application of terpenes as fragrances and flavors depends on the absolute configuration of the compounds because enantiomers present different organoleptic properties. Biotransformations allow the production of regio- and stereoselective compounds under mild conditions. These products may be labeled as "natural". Commercially useful chemical building-blocks and pharmaceutical stereo isomers can also be produced by bioconversion of terpenes. Enzymes and extracts from bacteria, cyanobacteria, yeasts, microalgae, fungi, plants, and animal cells have been used for the production and/or bioconversion of terpenes. In addition, whole cell catalysis has also been used. A variety of media and reactors have been assessed for these biotransformations and have produced encouraging results, as discussed in this review.     

Bioconversion of steroid glycosides by Nocardia restricta

The bioconversion of steroid alkaloid tomatine by Nocardia restricta yields the conjugate with lactic acid. We studied the bioconversion of some steroid glycosides without a nitrogen atom in the molecule to determine the effect of the nitrogen atom. The glycosides were of three different types: sterol glycosides, bufadienolide rhamnoside and steroid saponine. The results of bioconversions showed that Nocardia restricta converts steroid glycosides differently according to the sugar bound to the steroid aglycone. It can be concluded that in the absence of a nitrogen atom in the steroid molecule no conjugation with lactic acid by Nocardia restricta occurs.