Preparation and application of polymer-entrapped enzymes and microorganisms in microbial transformation processes with special reference to steroid 11-beta-hydroxylation and delta-dehydrogenation

Fungal cells from Curvularia lunata were entrapped in a crosslinked polyacrylamide gel. The gel-cells obtained as granules were applied in the microbial transformation of Reichstein compound S leading to cortisol through an 11-β-hydroxylation step. Some kinetic studies of this conversion using gel-cells were carried out. In addition, it was shown that gel-cell granules which had lost part of their 11-β-hydroxylase activity on storage could be reactivated yielding preparations with increased activity. From Corynebacterium simplex a steroid dehydrogenase catalyzing the Δ¹- dehydrogenation of cortisol leading to prednisolone was isolated and partially purified. The preparation was entrapped in a crosslinked polyacrylamide gel and the gel-enzyme granules obtained used in steroid dehydrogenation processes.     

Application of Urethane Prepolymers to Immobilization of Biocatalysts: Δ1Dehydrogenation of Hydrocortisone by Arthrobacter simplex Cells Entrapped with Urethane Prepolymers

Acetone-dried cells of Arthrobacter simplex having appreciable steroid Δ¹-dehydrogenase activity were immobilized by mixing the cell suspension with water-miscible urethane prepolymers synthesized from toluene diisocyanate and polyether diols. The entrapped cell activity in the transformation of hydrocortisone to prednisolone was affected by the properties of urethane prepolymers, such as the isocyanate group content in prepolymers, the molecular weight of polyether diols and the ethylene oxide content in diols. The addition of 10% of organic solvents, such as methanol and glycols, to the aqueous reaction mixture enhanced the solubility of the substrate greatly and the reaction rate of the immobilized cells. The activity of immobilized cells remained high even in the system containing 30% of methanol, which drastically inhibited the activity of free cells. The presence of an electron acceptor, phenazine methosulfate or 2,6-dichlorophenolindophenol, significantly stimulated the steroid conversion with entrapped cells, as well as free cells. The stability of the cells over repeated reactions was greatly improved by immobilization.     

Application of photo-crosslinkable resin prepolymers to entrap microbial cells. Effects of increased cell-entrapping gel hydrophobicity on the hydrocortisone Δ1-Dehydrogenation

Summary Acetone-dried cells of Arthrobacter simplex, whose steroid ¹ activity had been previously induced, were entrapped by the use of photo-crosslinkable resin prepolymers. When the hydrophobicity of the cell-entrapping gel was increased by mixing a hydrophobic prepolymer (main chain component; polypropyleneglycol) with a hydrophilic prepolymer (main chain component; polypropyleneglycol) with a hydrophilic prepolymer (main chain component; polyethyleneglycol) (up to 30%), the hydrocortisone to prednisolone conversion rate of the immobilized cells increased significantly, attaining approximately 20% of that of the free cells. A 10% addition of organic solvents, such as methanol, to the aqueous reaction mixture enhanced the solubility of the substrate greatly and to a lesser degree the reaction rate of the immobilized cells. The presence of an electron acceptor, phenazine methosulfate or 2,6-dichlorophenolindophenol, stimulated the steroid conversion of the entrapped as well as the free cells. The stability of the entrapped cells over repeated reactions was improved by immobilization.     

Steroid transformation by living cells immobilized in calcium alginate

Summary Whole cells of Arthrobacter simplex were immobilized in a living state in calcium alginate gel. The bacteria showed steroid-¹-dehydrogenase activity and the production of prednisolone from cortisol was investigated. The ¹-dehydrogenase activity of the immobilized cells could be increased about ten-fold by incubation in nutrient media (e.g., containing 0.5% peptone abd 0.2% glucose). The reason for this activation was examined and it was found that the immobilized cells were capable of multiplying when supplied with nutrients. Furthermore, provided that an inducer, cortisol, was present, the steroid-¹-dehydrogenase activity increased in proportion to the increase in the number of cells and it was thus concluded that microbial growth was the cause of activation.Experiments on repeated, batch-wise pseudocrystallofermentation with immobilized A. simplex cells also showed that immobilized cells could be advantageously used for pseudocrystallofermentation of steroids.     

Chemical oxygen demand reduction in a whey fermentation

Summary Preparations of living Pseudomonas denitrificans cells immobilized in alginate gel were used in the denitrification of water. In the presence of an exogenous carbon source the entrapped microorganisms reduced nitrate and nitrite to gaseous products and to achieve complete reduction, carbon to nitrogen ratios of over two were required. The effects on denitrification of particle size and the number of bacteria in the gel were investigated. Apparent K_m values for nitrate and nitrite reduction were calculated for free and immobilized cells. When the immobilized cells were incubated in nutrient media, an increase in reduction rate was observed and this was shown to be caused by the growth of cells within the gel particles. Immobilized P. denitrificans cells retained 75% of their initial nitrate reduction capacity after 21 days of storage at +4°C. The operational stability of the alginate-immobilized cells was studied both in batch and in a column which was operated continuously. A column (45 g of alginate-cell fibers in 80 ml) denitrified a high nitrate drinking water (100 mg NO₃/l) with a rate of 300 ml of nitrate and nitrite free water/day/g of gel. The half life for nitrate reduction was estimated to be 30 days.     

Screening of microorganisms having high fumarase activity and their immobilization with carrageenan

Summary To develop an efficient method for continuous production of L-malic acid from fumaric acid using immobilized microbial cells, screening of microorganisms having high fumarase activity was carried out and cultural conditions of selected microorganisms were investigated. As a result of screening microorganisms belonging to the genera Brevibacterium, Proteus, Pseudomonas, and Sarcina were found to produce fumarase in high levels. Among these microorganisms Brevibacterium ammoniagenes, B. flavum, Proteus vulgaris, and Pseudomonas fluorescens were further selected for their high fumarase levels in the cultivation on several media. These 4 microorganisms were entrapped into a k-carrageenan gel lattice, and the resultant immobilized B. flavum showed the highest fumarase activity and operational stability.Cultural conditions for the fumarase formation and the operational stability of fumarase activity of immobilized B. flavum are detailed. Productivity for L-malic acid using immobilized B. flavum with k-carrageenan was 2.3 fold of that using immobilized B. ammoniagenes with polyacrylamide.Presented at the Annual Meeting of the Agricultural Chemical Society of Japan, Nagoya, April 3, 1978     

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.     

Production of androsta-1,4-diene-3,17-dione from cholesterol using two-step microbial transformation

A novel two-step transformation process for the production of androsta-l by microorganisms-diene-3,17-dione (ADD) from a high concetration of cholesterol by microorganisms is proposed. Cholesterol (20 g/l) was initially converted to cholest-4-en-3-one (cholestenone) by an inducible cholesterol oxidase-producing bacterium, Arthrobacter simplex U-S-A-18. The maximum productivity of cholestenone was 8 g/l per day and the molar conversion rate was 80%. Subsequently, a fine suspension of cholestenone (50 g/l), which was prepared directly from the fermentation broth of A. simplex, was converted to ADD by Mycobacterium sp. NRRL B-3683 in the presence of an androstenone adsorbent, Amberlite XAD-7. The maximum productivity of ADD was 0.91 g/l per day and the molar conversion rate was 35%. Correspondence to: W.-H. Liu     

Entrapment of microbial cells and organelles with hydrophilic urethane prepolymers

Summary Microbial cells and cellular organelles were immobilized by mixing aqueous suspensions of the biocatalysts with water-miscible urethane prepolymers. Thus immobilized preparations of acetone-dried cells of Arthrobacter simplex and thawed cells of Nocardia rhodocrous showed appreciable {ie351-1} activities in the transformation of hydrocortisone into prednisolone and 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione, respectively. The activities of catalase and alcohol oxidase were observed in the immobilized peroxisomes (microbodies) of a methanol-grown yeast Kloeckera sp. No. 2201. Yeast mitochondria entrapped with the prepolymer showed adenylate kinase activity. These results indicate the usefulness of the urethane prepolymers as convenient materials for entrapment of not only enzymes, but also organelles and microbial cells.     

Transformation of cortisol to prednisolone by viable cells of Arthrobacter simplex covalently immobilized on cellulose granules

Arthrobacter simplex cells have been covalently immobilized to granules of microcrystallized regenerated cellulose by means of N-hydroxymethyl, N′-glucosylurea groups at pH 8.5, 18°C and cell suspension concentration of 60 mg/ml. The immobilization yield was found to exceed 100%. The maximum initial rate of Cortisol transformation to Prednisolone remained almost constant after 20-fold transformation in a nutrient medium containing 0.5% peptone at pH 8.0, 32°C and aeration with oxygen. The effect of the substrate concentration on the activity of the immobilized cells, as well as of the ratio between substrate and immobilized cells on the degree of transformation, was investigated. The immobilized cells were characterized by means of electronmicroscopic studies. Microbiological observations have shown that immobilized cells can proliferate and the free cells obtained are accumulated in the nutrient medium. The immobilized cells preserve their viability for a long time when they are stored at 4°C.     

Glutamic acid production with gel-entrapped Corynebacterium glutamicum

A glutamic acid producing microorganism (Corynebacterium glutamicum) is entrapped in a polyacrylamide gel. These immobilized microorganisms were used to produce glutamic acid in successive batches of fresh medium. Free microorganisms similarly used produced much less glutamic acid under similar conditions.     

Sequential conversion of cortexolone to prednisolone by immobilized mycelia of Curvularia lunata and immobilized cells of Arthrobacter simplex

Summary Two-step bioconversion of cortexolone (Reichstein's Compound S) to its ¹-dehaydro-11-hydroxy derivative, prednisolone, was successfully performed by the combined use of immobilized Curvularia lunata mycelia and immobilized Arthrobacter simplex cells. Immobilized living mycelia of C. lunata having a high 11-hydroxylation activity were prepared by in situ germination of spores entrapped in photo-crosslinked resin gels of a suitable net-work structure. Acetone-dried cells of A. simplex having an induced steroid ¹-dehydrogenase activity were also entrapped with photo-crosslinkable resin prepolymers and used for - dehydrogenation of hydrocortisone to prednisolone. For the production of prednisolone from cortexolone, the combination of sequential steps, 11-hydroxylation and subsequent ¹-dehydrogenation, was found to be suitable. Each immobilized microbial cell system was stable and could be used for the sequential reactions repeatedly (operational period, 25 days).     

Hydroxylation of indolyl-3-acetic acid by immobilized mycelium of Aspergillus niger

Summary The immobilization in polyacrylamide gel (PAAG) of the Aspergillus niger mycelium, which has the activity of hydroxylating indolyl-3-acetic acid (IAA) at 4-, 5-, and 6-positions of the indole nucleus, was studied. To preserve the hydroxylating activity, the immobilization should be performed at 5°C–10°C for 5–10 min. The hydroxylating activity of the A. niger mycelium entrapped in PAAG attained 70%–80% of that of free cells. The IAA transformation in the presence of sodium desoxycholate, polyethylene-glycol-400 (PEG-400), Span-60 or preincubation of granules entrapping mycelia in the presence of Tween-80 or PEG-400 not only double the hydroxylation rate but stabilize the activity as well. Gels entrapping mycelia may be used five or six times without altering activity. Incubation of gels with mycelium in the nutrient medium also increases and stabilizes the hydroxylating activity. In aerated columns, it is possible to obtain continuous hydroxylation of IAA, at a concentration of 0.5 g/l, by the immobilized mycelium of A. niger. The yield of hydroxy derivatives reached 70%, the activity remaining unaltered during 15 days' operation of the column.     

Immobilization and characterization of Saccharomyces cerevisiae in crosslinked,prepolymerized polyacrylamide-hydrazide

Summary Baker's yeast (Saccharomyces cerevisiae) was immobilized in gels made of prepolymerized, linear, water soluble polyacrylamide, partially substituted with acylhydrazide groups. Gelation was effected by the addition of controlled amounts of dialdehydes (e.g. glyoxal). The immobilized yeasts retained full glycolytic activity. Moreover, the entrapped cells were able to grow inside the chemically corsslinked gel during continuous alcohol production. Glyoxal was found to be the most favourable crosslinking agent for this system. the system employed allowed for the free exchange of substrate and products. The gel surrounding the entrapped cells had no effect on temperature stability profile. On the other hand, substantial enhancement in survival of cells in presence of high ethanol concentrations was recorded for the entrapped yeast. The capability of the immobilized yeast to carry out continuous conversion of glucose to ethanol was demonstrated.     

Activation of protease by sol-gel entrapment into organically modified hybrid silicates

To prepare an immobilized protease with a high activity for transesterification of vinyl n-butyrate with 3-methyl-1-butanol (isoamyl alcohol) in organic media, a protease was entrapped into organic–inorganic hybrid silica gel on Celite 545 by the sol-gel method. When propyltrimethoxysilane was used as the organic silane precursor mixed with tetramethoxysilane at a molar ratio of 16:1, the hybrid gel-entrapped protease on Celite 545 had 8 times the activity of the protease deposited on Celite 545 from 35 to 85°C.     

Immobilization ofArthrobacter simplex cells in thermally reversible hydrogels: Comparative effects of organic solvent and polymeric surfactant on steroid conversion

Summary Organic solvents have sometimes been used to increase the solubility of water insoluble substrates for steroid transformation using immobilized whole cells, even though the cell viability is often damaged. Polymeric surfactants which form micelles in aqueous solutions could be used instead of organic solvents to solubilize the steroid. We have successfully utilized this approach by employing a poly(dimethyl siloxane)-poly(ethyleneoxide) (PDMS-PEO) block copolymer surfactant to enhance conversion of hydrocortisone to prednisolone by immobilizedArthrobacter simplex cells, without deactivation of the immobilized cells.     

Studies on immobilized fungal spores for microbial transformation of steroids: 11 alpha-Hydroxylation of progesterone with immobilized spores of Aspergillus ochraceus G8 on polyacrylamide gel and other matrices

Hydroxylation in the 11 alpha-position in the progesterone molecule employing immobilized spores of Aspergillus ochraceus strain No. G8 (CDRI catalogue No.) was achieved. For immobilization the activity of the spores was evaluated on a variety of matrices such as alginate beads, epoxy resin beads, polyacrylamide gel, and collagen. Spores entrapped in polyacrylamide gel were found to be the most active. Studies of various parameters, e.g. monomer content, cell loading capacity, optimum pH, temperature, and substrate concentration, were carried out on polyacrylamide gel. In polyacrylamide, the entrapped spores normal decay pattern, as indicated by loss of activity, was observed after four uses. At the end of 15 cycles, the residual activity was found to be 18% of the original. It was possible to regenerate the activity by incubating the preparation in a nutrient medium. The regenerated spores showed increasing rate of loss of activity upon recycling.     

Determination Method for l-Ascorbic Acid in Foods with Immobilized Ascorbate Oxidase

Pumpkin (Cucurbita moschata) ascorbate oxidase was entrapped within 6% (w/v) Ca-alginate gel beads, and then the beads were treated with 1% (w/v) glutaraldehyde for 20 hr at 4°C. The immobilized ascorbate oxidase was much more stable than the free form. Under the optimum conditions, the immobilized enzyme remained fully active for 3 months and after 50 assays. A linear relationship was found between immobilized ascorbate oxidase activity and l-ascorbic acid concentration in the range of 2 ~ 20 μg/ml. The immobilized preparation could be employed for the simple and rapid determination of l-ascorbic acid in foods.     

Acid proteinases production by humicola lutea cells immobilized in polyhydroxyethylmethacrylate gel

The spores of Humicola lutea entrapped in polyhydroxyethylmethacrylate gel were precultivated in production medium for mycelial formation. The immobilized mycelium was reused in batch mode for acid proteinases production. The influence of precultivation time, initial inoculum gel volume, and gel particle size on the enzyme activity and proteinases production half-life were studied. After 70 h precultivation of the entrapped spores (10 ml initial inoculum volume, 12–27 mm³ gel particle size) maximum proteinases activity of 100–140% (compared with free cells) was registered in 15 reaction cycles. Under the same condition the half-life time was 18 cycles, while for the free cells it was 5 cycles. The main advantage of the polyhydroxyethylmethacylate immobilized H. lutea was the long acid proteinases production half-life at a low concentration of outgrowing cells in the medium.     

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.