Bioconversion of lipophilic compounds in non-aqueous solvent. Effect of gel hydrophobicity on diverse conversions of testosterone by gel-entrapped Nocardia rhodocrous cells

Summary The bioconversion of testosterone (TS) in water-saturated benzene-n-heptane (4:1 by volume) was mediated by Nocardia rhodocrous cells whose steroid ¹-dehydrogenase and 17-hydroxysteroid dehydrogenase were induced by TS. TS was transformed into 4-androstene-3,17-dione (4-AD), dehydrotestosterone (DTS) and 1,4-androstadiene-3, 17-dione (ADD) by incubating with the cell suspensions in the presence of phenazine methosulfate (PMS). Time-courses of TS transformation revealed that DTS and 4-AD were produced initially and further oxidized to ADD. Thus, the final product, ADD; was formed via two different pathways: TS4-ADADD and TSDTSADD. In these routes, ¹-dehydrogenation required PMS, while 17-dehydrogenation could proceed without any exogenous electron acceptor. N. rhodocrous cells entrapped in hydrophilic gels (H-gel) and lipophilic gels (L-gel) prepared by photo-crosslinkable resin prepolymers and urethane prepolymers were useful for effective dehydrogenations of TS. The cells entrapped in L-gels produced 4-AD as the major product, whereas DTS was the main product by the cells in H-gel. The difference in the profiles of dehydrogenation products can be explained by low affinity of PMS for L-gel-entrapped cells and of TS for H-gel-entrapped cells. Inhibitory effect of DTS on 17-hydroxysteroid dehydrogenase also would be responsible for the accumulation of DTS in the latter case. Thus, different routes for product formation could be selected by using resin prepolymers of appropriate hydrophilicity or hydrophobicity for entrapment of biocatalysts.Abbreviations used 4-AD 4-androstene-3,17-dione - ADD ¹-dehydrotestosterone 1,4-androstadiene-3,17-dione (androst-1,4-diene-3,17-dione) - DTS ¹-dehydrotestosterone (1,4-androstadiene-17-ol-3-one) - HC hydrocortisone - TS testosterone - DPIP 2,6-dichlorophenolindophenol - PMS phenazine methosulfate - H-gel hydrophilic gel - L-gel hydrophobic (lipophilic) gel - Solvent C water-saturated benzene-n-heptane mixture (4:1 by volume)     

Stereoselective hydrolysis of dl-menthyl succinate by gel-entrapped Rhodotorula minuta var. texensis cells in organic solvent

Summary dl-Menthyl succinate was successfully hydrolyzed stereoselectively by Rhodotorula minuta var. texensis cells entrapped within photo-crosslinked or polyurethane resin gels in water-saturated n-heptane. The hydrolyzed product was found to be pure l-menthol. The catalytic activity of the immobilized cells, especially those entrapped in urethane polymers, was far more stable than that of the free cells. The half-life of the polyurethaneentrapped cells was estimated to be 55–63 days in the organic solvent.Dedicated to the 65th birthday od Professor Dr. G. Manecke     

The kinetics of cholesterol oxidase synthesis by Nocardia rhodocrous.

The production of cholesterol oxidase by 3 liter batch cultures of Nocardia rhodocrous growing on a glycerol/yeast extract medium was investigated. Cholesterol was shown to be a good inducer of the enzyme. The optimum time for cholesterol addition and the quantity to be added were determined, resulting in a 15-fold yield increase. Cholesterol oxidase synthesis was influenced by the dissolved oxygen tension. Maximum cholesterol oxidase production was obtained at 30-40% air saturation. The effect of growth conditions on the extraction of cholesterol oxidase by Triton X-100 was investigated. The scale-up of the fermentation to 800 liters in a pilot-plant fermenter is described.     

Continuous inversion of sucrose by gel-entrapped yeast cells

The feasibility of immobilizing invertase (β-d-fructofuranosidase; EC 3.2.1.26) from Saccharomyces cerevisiae cells by various methods was examined. The yeast cells were adapted for maximal invertase activity by growth in a medium containing 0.2% glucose and 1% lactate. There was no permeability barrier for the enzyme in the whole cells. Entrapment in acrylamide polymerized by gamma-rays (200 kR) was observed to be most effective, with retention of 85% of the activity. The evaluation of the properties of the immobilized invertase indicated that the kinetic values were not appreciably altered despite a broad pH optimum. The enzyme was more stable to both heat and gamma-radiation. The immobilized cells could be used repeatedly in a packed bed reactor system for inversion of sucrose without observable loss in activity for over one month.     

Transformation of steroids by fungal protoplasts

Summary Protoplasts of Cunninghamella elegans transformed cortexolone to the same products as did the mycelium. Transformation of the steroid by non-induced mycelium and by protoplasts released from it was almost completely inhibited by cycloheximide. However, hydroxylation of cortexolone was not affected by this antibiotic if mycelium grown in the presence of an enzyme inducer or protoplasts obtained from the induced mycelium were used. The transformation rate of protoplasts, on the basis of dry weight or protein units, was about four times higher than that of the mycelium, indicating that the mycelial cell wall was a serious rate-limiting factor in steroid bioconversion.     

Transformation of steroids by mixed cultures

Some results of our studios on transformation of steroids by mixed culture fermentation are presented in this paper. Arthrobacter simplex was paired in turn with each of the following: Streptomyces roseochromogenes, Curvularia lunata, Absidia coerulea, and Aspergillus ochraceus. The steroid substrates examined for multiple transformation were 16α-hydroxy-cortexolone, 16α-hydroxy-cortexolone 16,17-acetonide, 9α-fluorohydrocortisone, 9α-fluorohydrocortisone 21-acetate, and 9α-fluorohydrocortisone 21-hemisuccinate. The effects of media, steroid substrate, and microbial interaction in a mixed culture on the induction and repression of steroid transforming enzymes were unique to each case studied. The reaction mechanism of the multiple steroid transformation was also found to vary from one mixed culture system to another. Two different reaction mechanisms were observed, namely, consecutive and parallel. In the former, one of the two enzymatic reactions always preceded the other, while in the latter, two different enzyme reactions occurred simultaneously, thereby giving rise to two different intermediates. Multiple transformation of steroids by a single step mixed culture fermentation has potential economic advantages.     

Transformation of steroids by fungal spores

Summary The steroid-hydroxylating activity during spore swelling prior to germination (I), during germ tube formation (II) and during branching of the growth hyphae (III) is two to five times that of the mycelium (IV) and control spores. The increased steroid-transforming activity is not correlated with the overall degradation of free amino acid pools but only with that of alanine, glutamate, arginine and proline. A higher ratio NADPH:(NADP⁺+NADPH) was observed in development phases I, II, and III, which indicates a possible role of alanine, glutamate and other amino acids decomposed via glutamate, in NADPH generation and steroid-hydroxylase activation.     

Transformation of steroids by fungal spores

Summary The rate of cortexolone 11-, and 11-hydroxylation by the mycelium ofCunninghamella elegans was much higher than that of its sporangiospores. In the course of the transformation, the sum of the free amino acid pools was not diminished in the spores where as it was decreased by 30–40% in the mycelium. The content of the nicotinamide nucleotides varied also in both forms of the microorganism. In the mycelium the NADP(H) pool was shown to be more reduced. The role of NADPH-generating system of the amino acid degradation and other regulation mechanisms in steroid hydroxylations by spores and mycelium ofC. elegans are discussed.     

Transformation of steroids by fungal spores

Summary Treatment of Cunninghamella elegans sporangiospores with dilute KOH, EDTA or Helix pomatia digestive enzymes (HpE) followed by cortexolone transformation resulted in stimulation of cortisol and epicortisol formation. The increased ability to hydroxylate steroids was accompanied by swelling of spores and increased permeability of their envelopes to exogenous citrate.Activated spores, unlike the untreated controls, exhibited enhanced degradation of intracellular amino acids, especially alanine and glutamic acid-the main constituents of the amino acid pools. We also observed a higher NADPH: (NADP⁺+NADPH) ratio probably due to the operation of more effective NADPH-generating system(s) in HpE, KOH or EDTA pre-treated spores.     

Bioconversion of steroids in mutants of Nocardia restrictus]

Nocardia restrictus grows quickly on synthetic media containing different carbonated substrates (steroids, organic acids). The control of growth parameters of this microbial species allowed the development of the production and selection methodology for mutants unable to grow on androst-4ène-3,17-dione. A stable mutant convert androst-4-ène-3,17-dione in perhydroindan propionic acid without addition of any degradation inhibitor in the medium.     

Regeneration of NAD(H) by alcohol dehydrogenase in gel-entrapped yeast cells

Anaerobically grown cells of Saccharomyces cerevisiae entrapped in polyacrylamide gel have been shown to provide a stable source of alcohol dehydrogenase [(ADH) alcohol:NAD⁺ oxidoreductase, EC 1.1.1.1] for effective regeneration of NAD(H). This system was able to provide the coenzyme required for the operation of other dehydrogenases, such as lactate dehydrogenase [(LDH) l-lactate: NAD⁺ oxidoreductase, EC 1.1.1.27] and malate dehydrogenase [(MDH) l-malate:NAD⁺ oxidoreductase, EC 1.1.1.37]. Yeast cells coimmobilized with a dehydrogenase are capable of the reversible regeneration of the reduced or oxidized coenzyme, depending on the additions made. A two-cell system can also be constituted using the same strain of yeast, adapted differently. Cells grown anaerobically and aerobically as sources of ADH and MDH, respectively, can operate efficiently on coimmobilization. The system can be used repeatedly without measurable loss of efficiency.     

Production of adenine arabinoside by gel-entrapped cells of Enterobacter aerogenes in water-organic cosolvent system

Summary Gel-entrapped whole cells of Enterobacter aerogenes, which has a transglycosylation activity, were used to produce adenine arabinoside from uracil arabinoside and adenine, in an appropriate water-organic cosolvent system. Cells of E. aerogenes entrapped with a hydrophilic photo-crosslinkable resin prepolymer, ENT-4000, or a urethane prepolymer, PU-6, had a high and stable transglycosylation activity. To improve the poor solubility in water of the substrate (adenine) and product (adenine arabinoside), dimethyl sulfoxide was selected as the cosolvent based on the criteria of operational stability of the immobilized biocatalyst and solubility of both substrate and product. Addition of 40% dimethyl sulfoxide to the reaction mixture permitted use of a high substrate concentration range which gave high productivity under homogeneous reaction conditions. The immobilized cells of E. aerogenes exhibited a markedly improved operational stability, retaining their initial level of activity during repeated use for at least 35 days at 60°C in 40% dimethyl sulfoxide. When the reaction was carried out with 150 mM uracil arabinoside and 50 mM adenine as the substrates, the yield of adenine arabinoside was maintained at 100% based on the molar ratio of adenine, throughout the reaction.Abbreviations used AraA adenine arabinoside - AraU uracil arabinoside     

固定化细胞有机相催化不对称还原β-羰基酯

将酵母细胞用海藻酸钙包埋后用于有机相催化不对称还原4-氯乙酰乙酸乙酯制备光学活性的4-氯-3-羟基丁酸乙酯,从中筛选得到具有较高立体选择性和还原能力的菌株假丝酵母SW0401,将此菌株的细胞固定化细胞作为研究对象,系统考察了固定化条件、固定化细胞大小、反应溶剂、初始底物浓度、辅助底物、固定化细胞热处理和抑制剂对还原反应的影响。结果表明,上述因素对反应的摩尔转化率和产物（S）-CHBE光学纯度有显著影响。固定化时所用缓冲液的pH值为7．0时和固定化细胞颗粒平均直径为2.5mm较合适,以正己烷为反应介质时反应的摩尔转化率和产物光学纯度最优,初始底物浓度以54.7mmol／L为宜,辅助底物以1-己醇为佳。对固定化细胞的热处理和添加抑制剂烯丙醇均能够明显改善产物的光学纯度,但对提高摩尔转化率有负面影响。     

Long term observation of gel-entrapped microbial cells in a microscope reactor

Summary A method is described for the on-line observation of immobilized, growing microorganisms in a microreactor mounted on a light microscope to determine several physiological parameters such as cell size and colony size, growth rates, spatial and temporal distribution of cells which are entrapped within transparent gels. The results can be used for modelling growth and diffusion behaviour in biocatalysts to optimize environmental and industrial applications of immobilized cells.     

Transformation of steroids by actinobacteria: a review

Development of pharmaceutical industry is currently aimed at introducing biotechnological processes on a large-scale and thereby replacing multiple-stage chemical syntheses. Actinobacteria are efficient biocatalysts of many processes involving steroid bioconversion, which hold considerable importance for the synthesis of hormonal drugs. The potential to catalyze the conversion of a broad spectrum of steroid substrates makes it possible to expect efficient utilization of these microorganisms in development of new technologies of manufacturing steroid pharmaceutical substances. The review is a first attempt to systematize data on the potential of actinobacteria to catalyze diverse reactions of steroid transformation (such as hydroxylation, introduction and reduction of double bonds, oxidation of steroid alcohols, reduction of ketones, side chain de-esterification and degradation, etc.), with emphasis on processes of practical biotechnological importance and progress in steroid bioconversion over the last ten years.     

Transformation of steroids by actinobacteria: A review

Development of pharmaceutical industry is currently aimed at introducing biotechnological processes on a large-scale and thereby replacing multiple-stage chemical syntheses. Actinobacteria are efficient biocatalysts of many processes involving steroid bioconversion, which hold considerable importance for the synthesis of hormonal drugs. The potential to catalyze the conversion of a broad spectrum of steroid substrates makes it possible to expect efficient utilization of these microorganisms in development of new technologies of manufacturing steroid pharmaceutical substances. The review is a first attempt to systematize data on the potential of actinobacteria to catalyze diverse reactions of steroid transformation (such as hydroxylation, introduction and reduction of double bonds, oxidation of steroid alcohols, reduction of ketones, side chain de-esterification and degradation, etc.), with emphasis on processes of practical biotechnological importance and progress in steroid bioconversion over the last ten years.     

Production of indole alkaloids by gel-entrapped cells of Catharanthus roseus in a continuous flow reactor

Summary Calcium alginate gel-entrapped cells ofCatharanthus roseus were used to study the production of indole alkaloids in a flow through process. The bioreactor was functional for more than two months and product recovery was analyzed under various operating conditions.