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Dehydrogenation of hydrocortisone by Arthrobacter simplex in a liposomal medium
Dehydrogenation of hydrocortisone by Arthrobacter simplex in a liposomal medium Ruth Goetschel and Raphael Bar D e p a r t m e n t o f Applied Microbiology, The H e b r e w University, Jerusalem, Israel
The biotransformation of hydrocortisone to prednisolone by Arthrobacter simplex ATCC 6946 was extensively investigated in a liposomal medium, made up of multilamellar vesicles of either pure or crude egg phospholipids. The liposomal medium was found to promote faster kinetics to a higher conversion than those obtained in an aqueous medium and to be superior to water/surfactant and water/cosolvent systems, especially at high substrate loadings. The enhancing effect of the liposomal medium was partly explained by its capacity for steroid entrapment as well as by the presence of phospholipid vesicles in the bioconversion medium.
Keywords: Liposome; microbial catalysis; Arthrobacter; hydrocortisone
Introduction Microbial transformations of hydrophobic compounds are often met with two serious obstacles; (a) a limited substrate accessibility to the biocatalyst as a result of the low aqueous solubilities of most organics, and (b) inhibition or toxicity of both substrate and product exerted upon the microbe. Attempts to alleviate or o v e r c o m e these shortcomings include the use of surfactants and water-miscible or -immiscible solvents. ~-3 But again, inhibitory and toxic effects introduced by these surfactants and organic solvents may partly or fully inactivate the biocatalyst. A different type of bioconversion medium is an essentially aqueous medium into which an inherently nontoxic ingredient is added in view of eliminating the previously mentioned shortcomings. An example of such an ingredient is the biocompatible cyclodextrins used for biotransformation of toxic substrates. 4 In this study, an extensive investigation of the transformation of hydrocortisone to prednisolone by Arthrobacter simplex ATCC 6946 was undertaken in a medium consisting of another biocompatible ingredient, the liposome-forming diacyl phospholipids. Indeed, phospholipids, which are also the main constituents of natural
Address reprint requests to Dr Bar at the Department of Applied Microbiology, The Hebrew University, P.O. Box 1172, Jerusalem, Israel 91010 Received 29 March 1990; revised 11 July 1990 © 1991 Butterworth-Heinemann
cellular membranes, are generally held to be nontoxic. 5 Thus the liposomal medium, previously suggested by Bar 5 as a bioconversion medium for sterols and steroids, may be considered as a pseudobiological or a biomimetic environment. Bioconversions in a medium o f l i p o s o m e s have been primarily limited to enzymatic reactions at a microscale involving either a substrate or an e n z y m e contained in the liposome. In a recent study, 6 hydrophobic enzymes such as aminopeptidase or phosphatase were incorporated in small unilamellar vesicle (SUVs) for hydrolysing water-soluble substrates. In contrast, soluble cholesterol oxidase 7 was used to oxidize cholesterol contained in SUVs as an analytical tool in kinetic studies of cholesterol exchange. As multilamellar vesicles are one of the easiest forms of liposomes to prepare, they were employed as a medium for microbial conversions throughout this study.
Materials and methods Materials
Hydrocortisone, prednisolone, prednisone, and 2,2'dipyridyl were purchased from Sigma (St. Louis, USA). Egg-phosphatidylcholine (pure egg-PC) was obtained as a chloroform solution of I.-a-phosphatidylcholine (99%) from Sigma (Type V-E) and as a pure solid (98%) from Lipoid K G (Lipoid E-PC). Commercial-grade (or crude) egg lecithin (approx. 60% phosphatidylcholine) and pure lysolecithin were purchased from Sigma. Enzyme Microb. Technol., 1991, vol. 13, March
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Papers Egg lecithin (Sigma) and lipoid E-PC were stored at -18°C under argon as chloroform solutions. All organic solvents used were chemically pure and purchased from Frutarom (Israel) except ethylene glycol and polyethylene glycol (Riedel-deHaen, FDR), tertbutanol (BDH, England), dimethylsulfoxide (Fluka, Switzerland), and Tween 80 (polyoxyethylene sorbitan monooleate, Sigma, USA).
Cultivation of cells Arthrobacter simplex ATCC 6946 was maintained and cultivated as described elsewhere. 8 The cells were harvested by centrifugation and washed three times with a saline phosphate buffer which consisted of 8.5 g NaCI, 0.3 g KHzPO 4, and 1.13 g NazHPO4.7H20 per liter of distilled water. Finally, the cells were washed once with distilled water and then lyophilized. Each lyophilized preparation was then assayed for its specific enzymic activity (vide infra). These lyophilized cells were convenient to work with, since they retained their enzymic activity for a long time and they could be accurately weighed.
Enzyme assay Cells were assayed for 1-dehydrogenase activity at near-saturation concentration of hydrocortisone (i.e. absence of crystals). A 50-ml flask was loaded with 8 ml of a saturated solution of hydrocortisone in buffer phosphate (pH 7.8, ionic strength 0.05) and 50 tzl of a solution of 2,2'-dipyridyl (15 g !-J) in ethanol. A cell suspension containing 5 mg dry weight (DW) cells was then added to the hydrocortisone solution to bring the final volume to 10 ml and the final hydrocortisone concentration to 0.32 g 1-i. The flask was then incubated in a shaker (30°C, 150 strokes min -1) and samples (0.5 ml) were removed periodically for analysis. The initial specific activity of every batch of cells was calculated after 15 min. It is expressed in U g ~DW where 1 U is defined as 1 /xmol prednisolone formed per minute.
Preparation of liposomes Various amounts of hydrocortisone and phospholipids were dissolved in 20 ml of various organic solvents, mainly chloroform, in a 250-ml round-bottomed flask. A volume of 50 txl of an ethanolic solution of 2.2' dipyridyl (15 g 1-1) was added to the hydrocortisone and phospholipids solution, and this mixture, with or without added glass beads (diameter 3 mm), was then evaporated in a rotary evaporator at 40°C to obtain a dried lipidic film. Residual traces of organic solvents were subsequently removed by an overnight vacuum drying at room temperature and pressure of 500 tort. The lipidic film was rehydrated at ca. 40°C with 8 ml of the previously described buffer phosphate (pH 7.8) on a vortex mixer for 15 min. The resulting milky liposomal medium was transferred to a 50-ml Erlenmeyer flask, and 2 ml of a suspension of lyophilized cells was added to bring the final volume to l0 ml. The flask with the inoculated liposomal medium was incubated in a
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shaker at 30°C and 150 strokes min-J, and aliquots (0.3-0.5 ml) were periodically withdrawn for analysis. A series of control flasks with aqueous fine dispersions of hydrocortisone was incubated at identical conditions. However, unlike the homogeneous liposomai medium, the steroid slurry in the control flasks could not be properly sampled. Therefore, the whole content of one control flask was subjected to product analysis at one time.
Product analysis Aliquots (0.3-0.5 ml) from the liposomal medium were extracted by 1.5 ml ethanol and 3 ml chloroform on a vortex mixer. Following phase separation, 160 txl of the organic phase was transferred into sampling vials and mixed with 40 txl of a methanol solution of prednisone (0.5 g 1 ~) as an internal standard. Samples (I0 txl) were analyzed on a Lichrospher 5 Si 60 column (Merck, Darmstadt, FRG), employing chloroform containing 5% (v/v) methanol and 0.55% (v/v) glacial acetic acid as the mobile phase. The flow rate of the eluent was 1 ml min 1. Calibration curves of area ratios between either substrate or product and internal standard were determined at 254 nm. The aqueous medium of the control flasks was extracted by a mixtue of 30 ml ethanol and 60 ml chloroform on a vortex mixer. Then the product analysis was performed as previously mentioned.
Measurement of solubilities of steroids To a 10-ml buffer phosphate solution pH 7.8, 0.25 g of either hydrocortisone or prednisolone, or both, was added. The suspension was stirred under argon at 30°C for 72 h to saturate the aqueous solution. Then the suspension was centrifuged at 12,100 g for 30 min, and an aliquot of 0.5 ml from the clear supernatant was extracted with a mixture of 0.5 ml ethanol and 1 ml chloroform. The extract was subsequently analyzed by HPLC.
Measurement of steroid entrapment in liposomes Liposomes were prepared with various concentrations of pure egg-PC and steroid. After hydration, the liposomes were held at 30°C for 24 h and subsequently centrifuged at 12,100g for 10 rain. An aliquot of 0.5 ml of supernatant was extracted with 0.5 ml ethanol/ chloroform (2:1 v/v) and the extract analyzed by HPLC. The pellet of liposomes was washed with phosphate buffer, centrifuged at 12,100g for 10 min, and extracted with 6 ml ethanol~chloroform (I :2 v,v). A sample of this extract was analyzed by HPLC. Finally, the percent entrapped steroid was then calculated from the amount added and the amount recovered.
Tests for microbial degradation of phospholipids Tests for lipolytic activity of A. simplex ATCC 6946 were done on egg yolk agar plates by detecting the
Dehydrogenation in liposomal medium: R. Goetschel and R. Bar opalescent zone of precipitated lipid 9,1°after incubation of 24 and 48 h at 30°C. Further tests were conducted to detect lysolecithin following 24 h incubation of A. simplex cells in a phosphate buffer containing empty liposomes of pure egg-PC. The liposomes were extracted with chloroform and this was analyzed on TLC aluminum sheets (Woelm Silica Gel). The eluent consisted of chloroform, acetone, methanol, acetic acid, and water at respective volumetric ratios of 6 : 8 : 1 2 : 2 : 1 . The Rf values of pure egg-PC and lysolecithin were found to be 0.10 and 0.13, respectively.
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Results An important requirement for a microbe in a liposomal medium would be its inertness towards the constituent phospholipids. No phospholipase activity was, however, detected in A. simplex using egg yolk agar, and no lyso-PC could be detected in a liposomal medium of pure egg-PC following incubation with the microbial cells. Thus, A. simplex appears unable to degrade liposomes.
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Figure 1 Bioconversion of hydrocortisone (2 g 1-1) in liposomal media of pure egg-PC (A) Comparison of an aqueous dispersion (V) with a liposomal m e d i u m (T) of 20 g PC 1-1 (molar ratio of steroid : PC was 1 : 4.8). Concentration of cells was 6 g DW I with a specific activity of 33.9 U g 1DW. (B) Effect of phospholipid loading on the time course of bioconversion. Molar ratios of steroid : PC were (0) 1 : 0.6, (O) 1 : 2.4, (A) 1 : 3.6, (A) 1 : 4.8. Concentration of cells was 2 g DW I 1 with a specific activity of 30.3 U g-1 DW
Bioconversion in a liposomal medium A liposomal medium composed of 20 g 1-c~-phosphatidyi-choline 1-l and 2 g hydrocortisone 1-J clearly promoted the biotransformation (Figure la) at a faster rate and to a higher degree of conversion than those achieved in a fine dispersion of hydrocortisone. In the latter case, the conversion was only 63% after 8 h, in contrast to 98% in the liposomal medium. Furthermore, the biotransformation halted at a maximum conversion of 75% after 24 h in the aqueous slurry, but it proceeded to virtual completion in the liposomal medium. This remarkable enhancement effect was found to correlate with the loading of phospholipids in the medium. Figure lb shows that for liposomal media with constant substrate (2 g 1-J) and biocatalyst (2 g DW 1- l) concentrations, the bioconversion kinetics were progressively faster with increased PC to hydrocortisone molar ratio. These positive findings based on pure and expensive egg-PC prompted us to compare it with crude but much cheaper phospholipids such as egg lecithin, which contains only 60% phospholipids, mainly PC and phosphatidyl ethanolamine. An examination by a light microscope of "liposomes" made of this egg lecithin revealed indeed not only the presence of typical forms of multilamellar vesicles but also spherical oil droplets as a result of the large content of triglycerids. The effect
of lecithin on the bioconversion was investigated at hydrocortisone concentrations of 0.4, 2, 5, 10, 25, and 50 g 1-I (Table 1). At a near-saturation substrate concentration (0.4 g 1 J), lecithin offered no advantage over a regular medium. In the nonliposomal media, the biocatalytic activity was inhibited after 8 h in all other hydrocortisone Ioadings. These inhibitory effects were previously reported by other investigators, ll-13 However, inclusion of lecithin in the media enabled further progression of the biotransformation to higher degrees of conversion (Table 1). Therefore, the enhancement effect of the "liposomal medium" made of crude lecithin was prominent in high substrate loadings.
Effect of mode o f liposome preparation on the bioconversion Since the multilamellar vesicles vary widely in their sizes, it was judged important to measure the bioconversion kinetics in liposomes with two roughly different size distributions. A simple means to reduce the liposome sizes is the addition of glass beads to the chloroform solution of the lipids. Figure 2 shows indeed that Enzyme
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Papers Table 1 Degrees of bioconversions of various hydrocortisone concentrations after 12, 18, and 24 h, obtained in aqueous and liposomal media with 100 g crude egg lecithin 1-1 0.4 Substrate (g 1-1) Time (h) Conversion (%) Aqueous m e d i u m Liposomal m e d i u m
2
5
12
18
24
12
18
24
12
18
24
100 100
100 100
100 100
69 70
73 90
77 92
60 85
63 97
75 97
10
25
50
Substrate (g 1-1) Time (h)
12
18
24
24
48
72
24
48
72
Conversion (%) Aqueous m e d i u m Liposomal m e d i u m
32 54
36 81
37 83
21 50
25 59
26 64
12 20
14 38
14 74
Concentration of cells was 6 g DW 1-1 with a specific activity of 32.9 U g-~ DW
the bioconversion was significantly faster in a liposomal medium prepared with added glass beads. The influence of ultrasonic irradiation on liposome size and lamellarity is well known.14 It was interesting to investigate the effect of sonication of the tiposomal media on the subsequent bioconversion kinetics. The liposomal media were subjected to sonication for several durations of 2.5, 5, 10, or 15 min, but interestingly, no significant difference in the bioconversion time course was observed with regard to the nonsonicated liposomal medium. As the liposome preparation requires first dissolution of the steroid and phospholipids in an organic solvent, several commonly used solvents and their combinations were tried and were found to lead to different bioconversion profiles, chloroform being the
best. It is important to note that residual solvent or cosolvent, not entirely discarded, had an adverse effect on the biocatalyst. For instance, when the solvent mixture of chloroform and tert-butanol was subjected to a mere rotary evaporation, the bioconve-rsiori was smaller. When the treatment included also a vacuum drying overnight, the degree of conversion reached 95% after 8 h.
Liposomal medium vs. water/cosoluent system Figure 3 compares the performances of a liposomal medium and a water/ethylene glycol (30% v/v) system at low and high concentrations of hydrocortisone. At a concentration of 2 g 1-1, hydrocortisone dissolved
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Figure 2 Bioconversion profiles of hydrocortisone (2 g 1-1) in liposomal media of 20 g crude egg lecithin I 1 (molar ratio of steroid : lecithin was 1 : 4.8). (0) Hydration in presence of glass beads; (V) control. Concentration of cells was 6 g DW I 1 with a specific activity of 33.9 U g-1 DW
Enzyme Microb. Technol., 1991, vol. 13, March
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F i g u r e 3 Bioconversion profiles in a w a t e r / e t h y l e n e glycol (30% v/v) system (discontinuous curves) and in liposomal media of pure egg-PC (continuous curves) or hydrocortisone at a concentration of 2 g I 1 ( O a n d ©) and 1 0 g I -~ (A and A ) . T h e molar ratio steroid : PC was 1 : 4.8 (©) and 1 : 2.4 (zS). Cell concentration was 6 g DW I 1 with specific activity of 66.7 U g-1 DW
Dehydrogenation in liposomal medium: R. Goetschel and R. Bar completely in the water/cosolvent system and was bioconverted at a slightly lower rate than in the liposomal medium. However, the latter medium demonstrated a considerable advantage at the high substrate concentration of 10 g 1 ~. The high loading of hydrocortisone saturated the hydroorganic system with formation of a slurry, and at these conditions, the biocatalyst was soon inhibited. The liposomal medium, however, enabled the bioconversion to progress to a much higher extent (Figure 3).
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Figure 4 compares bioconversion profiles in a liposomal medium and in aqueous slurries with and without two surfactants. Tween 80 and polyethylene glycol at nontoxic concentrations. In these experiments, the microbial cells were very active, as evidenced by their high specific activity of 66.7 U g i and the relatively fast bioconversion in the control. Both surfactants had a marginal effect on the bioconversion, while a marked enhancement took place in the liposomal medium.
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Figure 4 Bioconversion profiles of hydrocortisone (2 g I 1) in a liposomal m e d i u m (A, 20 g pure egg-PC 1-1) and aqueous media without (V) and with Tween 80 (A, 0.1 g 1-1) and polyethylene glycol (0, 1 g I-1). Cell concentration was 4 g DW I 1 with a specific activity of 66.7 U g-1 DW
Entrapment o f steroids in liposomes The solubilities of hydrocortisone and prednisolone measured separately at 30°C were 0.45 and 0.29 g 1-j, respectively. The corresponding solubilities when measured in the presence of excessive amounts of both steroids changed, however, to 0.36 and 0.33 g 1 ~, respectively. Subsequently the entrapped steroid was determined at a near-saturation concentration that avoided precipitation of crystals. Table 2 shows the percentages of entrapped hydrocortisone and prednisolone, respectively, as a function of pure egg-PC loading following equilibration for 24 h. Comparable degrees of entrapment were obtained for the chemically similar steroids, and at a phospholipid content of 100 g 1-1, 90% of either steroid was, in fact, restained within the liposomes. The degrees of entrapment of hydrocortisone, expressed in terms of percent mole steroid per mole phospholipid, are given also in Table 2, and these did not exceed a value of 3.0% at the lowest PC concentration (20 g I J) employed.
Table 2 Entrapment of 0.4 g hydrocortisone 1-1 or 0.39 g prednisolone 1-1 at 30°C in various concentrations of pure eggPC multilamellar liposomes, expressed as a percentage of the total concentration.
Prednisolone
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Figure 5 Bioconversion profiles of hydrocortisone (2 g I 1) in aqueous media with 5 (A), 10 (A), 20 (O), and 50 g (0) pure eggPC I 1 in the form of empty liposomes. Cell concentration was 4 g DW 1-1 with a specific activity of 66.7 U g-1 DW
Effect o f empty liposomes on the bioconversion Figure 5 shows the effect of " e m p t y " liposomes, i.e.
Egg-PC (g 1-1)
Hydrocortisone
c 80 0
20
50
100
51 (2.4) 53 (2.8)
81 (2.1) 75 (1.6)
92 (0.7) 90 (1.0)
Percent steroid entrapment (mol % steroid per mol phospholipid) is given in parentheses
liposomes made up of phospholipids only, on the bioconversion. Clearly, increasing amounts of empty pure egg-PC multilamellar vesicles added to microbial slurries of hydrocortisone progressively enhanced its conversion. The " e m p t y " liposomes definitely had a positive effect on the bioconversion. A simultaneous incorporation of the steroid into the empty phospholipid vesicles, as the biotransformation took place, could partly explain the enhancing effect. Indeed,
Enzyme Microb. Technol., 1991, vol. 13, March
249
Papers Table 3
Qualitative estimation of the relative presence of prednisolone needles in various media made of either aqueous dispersions of hydrocortisone or liposomes prepared with increasing concentrations of E-PC and hydrocortisone Hydrocortisone (g 1-1) E-PC (g 1-1) 0(control) 20 50 100
2 (after 5 h) +++
(91) (100) - (100) - (100)
10 (after 24 h) ++++ + + + -
(46) (74) (82) (91)
50 (after 24 h) ++++ (12) + + + (14) + + + (15) + + + (18)
( - ) No needles; (+), ( + + ) , ( + + + ) , (++++)increasing amounts of needles. In each case the percentage of conversion is indicated. Cell concentration was 3 g DW I 1, with a specific activity of 66.7 U g-1 DW. The degrees of substrate conversion (%) are given in parentheses
equilibration of empty liposomes made of 50 g pure egg-PC 1-~ with an aqueous hydrocortisone solution (0.4 g 1-1) resulted in incorporation of 65% of the steroid after 24 h. A similar experiment with hydrocortisone-containing liposomes resulted in an entrapment of 81% (Table 2).
Crystallization phenomena in the liposomal medium Since the studied bioconversion involved sparingly water-soluble substrate and product steroids, crystallization phenomena inevitably took place in parallel with the microbial transformation. An examination of an aqueous bioconversion medium by light microscopy revealed formation of product crystals in the form of needles (length up to 50/xm) as previously reported by Kondo and Masuo. ~5 A visual estimation of the presence of prednisolone needles in media made of increasing concentrations of egg-PC at the corresponding conversion percentages is reported in Table 3. For hydrocortisone concentrations of 2 and I0 g 1-~, as the egg-PC concentrations increased, the relative amount of needles diminished, and the bioconversion almost reached completion. However, for hydrocortisone concentration of 50 g 1- ~, even a high concentration of egg-PC was insufficient to prevent formation of needles. Judging from Table 3, one can see that the appearance of product needles becomes discernible in a liposomal system with an approximate PC : hydrocortisone weight (molar) ratio of 5 : 1 (2.43 : 1). In systems with a lower ratio, the crystal needles prevailed, but at a higher ratio, they were not visible.
Discussion It appears that an enhanced microbial catalysis in a liposomal medium can in principle be explained in terms of a higher solubilization of substrate and/or product and a facilitated substrate accessibility to the biocatalyst.
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The faster kinetics and the higher conversion of the microbial transformation in either pure (Figure 1) or crude liposomal (Table 1) media could be at least partly attributed to entrapment of the reactants in the multilamellar vesicles, thus subjecting the microbes to less inhibitory concentrations of the steroids. Solubilization of water-insoluble organics can also be obtained by surfactants, as previously mentioned, and consequently, an enhanced biotransformation (Figure 4) is expected. However, the use of surfactants is limited to a concentration of ca. I g 1 1, beyond which deleterious effects on cell wall and membranes may well lead to inhibited catalysis ~6 even by nontoxic surfactants. Phospholipids can be employed at considerably higher loadings of 100 g I i (and more) with no observable inhibitory or toxic effects upon the microbe. Another simple way of increasing the solubility of sparingly soluble steroids would be by adding a water-miscible organic solvent, thus rendering the bioconversion medium more hydrophobic. Indeed, the bioconversion in question was previously investigated in the presence of various water-miscible cosolvents by Freeman and Lilly. 2 These authors, who considered diols such as ethylene glycol to be the most suitable group of solvents, showed that the solubility of hydrocortisone increased tenfold in a water/ethylene glycol (30% v/v) system, but its biotransformation was inhibited in it. The detrimental effect of this hydroorganic system with 10 g hydrocortisone 1 J upon the microbial activity was also observed by us (Figure 4), but the liposomal medium again promoted the biocatalysis with no adverse effect. Accessibility of the liposome-associated steroid to the biocatalyst is dependent upon the size and lamellarity of the liposome. In this study, three well-known means ~4were employed to grossly affect both liposome size and lamellarity. Glass beads have apparently reduced the sizes and lamellarity of multilamellar vesicles (MLVs) as a result of an increased surface area of the lipidic film and a more vigorous shaking during the hydration. The bioconversion in a liposomal medium prepared with glass beads was consequently faster (Figure 2). However, a drastic size reduction obtained by sonication, which apparently led to formation of small unilamellar and possibly oligolamellar vesicles, offered clearly no clear advantage over nonsonicated liposomal medium. This could reasonably be attributed to a reduced steroid entrapment capacity by the small vesicles. Under these conditions, a major exclusion of the steroid from the liposome would make the system behave similarly to an aqueous medium containing empty liposomes (vide infra). The third means of affecting both size and lamellarity refers to the type of solvent employed in liposome preparation. Chloroform, which is commonly employed, 14was found to lead to the fastest kinetics of biotransformation. It is important to note that a mere steroid entrapment in the liposomes cannot fully explain the enhancement of the biotransformation. The hydrocortisone loading employed in a standard bioconversion experiment (Fig-
Dehydrogenation in liposoma/ medium: R. Goetschel and R, Bar ure la) was 20.8% mole steroid per mole phospholipid, which is of course much larger than the maximum measured (Table 2) value of steroid entrapment of ca. 3%. The incorporation of hydrocortisone derivatives was previously investigated by Shaw et al. Z7 who showed that increasing the chain length of a substituted group in carbon 21 of the hydrocortisone molecule caused a marked enhancement of the steroid retention in dipalmitoyl-PC liposomes. Thus, hydrocortisone palmitate was reportedly retained longer than the octanoate ester. However, the unesterified steroid, hydrocortisone, would probably be less incorporated in the liposomes and consequently would precipitate out when initially employed at a high loading. Therefore, liposornal media with high substrate loadings definitely contained steroid crystals as well. Furthermore, the mere presence of empty liposomes in a steroid suspension exerted an enhancement effect on the bioconversion (Figure 5). Again, even though the initially empty liposomes were shown to gradually absorb hydrocortisone from the medium, the extent of this entrapment was not large enough to rationalize the enhancement effect. Other mechanisms not known to us could possibly be involved. For instance, a favorable interaction between the phospholipids of MLVs and the microbial cell wall could play a role. These mechanisms could be at the origin of the previously reported enhancing effect of oils containing phospholipids on sterol and steroid oxidations J8,19 performed in fermentation broths. As previously mentioned, crystallization phenomena accompany bioconversion of sparingly soluble substrates, and, for this reason, the microbial conversion of hydrocortisone leading to the formation of product needles was termed "pseudocrystallofermentation".J5 This crystallization would not take place, for instance, in a water/organic solvent system having the capacity to dissolve both reactant and product. It was therefore interesting to examine the various liposomal media microscopically for the appearance of these product needles. The appearance of needles (Table 3) in a bioconversion medium below a PC:hydrocortisone weight ratio of 5:1 could be attributed to product crystals formed from initially nonentrapped substrate. The absence of needles in systems with a higher ratio, wherein only a small fraction of the substrate is actually entrapped within the liposomes, testifies to the complexity of the crystallization processes taking place in a liposomal medium. These processes are not fully understood by us. Perhaps the liposomes induced formation of product microcrystals with a different habit other than needles.
Conclusion The liposomal medium was shown not only to be suitable for microbially catalysed conversions of hydrocortisone but also to offer significant advantages over aqueous media with or without cosolvents or surfactants.
Acknowledgements Support of this study from the Kay Foundation at the Hebrew University is gratefully acknowledged. The authors are thankful to Dr. L. K. Bar for useful discussions on liposome technology and to Prof. Y. Barenholz for critically reviewing the manuscript.
References 1 2 3 4 5 6 7 8 9 10
11 12 13 14 15 16 17 18 19
Ceen, E. G., Herrmann, J. P. R. and Dunnill, P. Appl. Microbiol. Biotechnol, 1987, 25, 491-494 Freeman, A. and Lilly, M. D. Appl. Microbiol. Biotechnol. 1987, 25,495-501 Silbiger, E. and Freeman, A. Appl. Microbiol. Biotechnol. 1988, 29, 413-418 Bar, R. Trends Biotechnol. 1989, 7, 2-4 Bar, R. Trends Biotechnol. 1988, 6, 1-2 Sada, E., Katoh, S., Terashima, M. and Kibushi, Y. Biotechnol. Bioeng. 1987, 30, 117-122 Moore, N. F., Patzer, E. J., Barenholz, Y. and Wagner, R. R. Biochemist~, 1977, 16, 4708-4715 Udvardy, E. N. and Hantos, G. in Overproduction of Microbial Products (Krumphanzl, V., Sikyra, B. and Vanek, Z., eds.) Academic Press, 1982, pp. 21-33 Collins, C. M. and Lyne, P. M. Microbiological Methods 5th ed, Butterworths, London, 1985, pp. 108-109 Gerhardt, P., Murray, R. G. E., Costilow, R. N., Nester, E. W., Wood, W. A., Krieg, N. R. and Phillips, G. B. in Manual of Methods for General Bacteriology American Society for Microbiology, Washington, 1981 Ohlson, S., Larsson, P. and Mosbach, K. Eur. J. Appl. Microbiol. Biotechnol. 1979, 7, 103-110 Constantinides, A. Biotechnol. Bioeng. 1980, 22, 119-136 Krysteva, M. A. and Grigorova, P. M. Enzyme Microb. Technol. 1987, 9, 538-541 Lichtenberg, D. and Barenholz, Y. in Methods" in Biochemical Analysis, Vol. 33 (Glick, D., ed.) Wiley, New York, 1988, pp. 337-462 Kondo, E. and Masuo, E. J. Gen. Appl. Microbiol. 1961, 7, 113-117 Nakamatsu, T., Beppu, T. and Arima, K. Agric. Biol. Chem. 1983, 47, 1449-1454 Shaw, I. H., Knight, C. G. and Dingle, J. T. Biochem. J. 1976. 158, 473-476 Nishikawa, D., lmada, Y., Kinoshita, M. and Takahashi, K. US Patent No. 4 101 378, 1978 Martin, C. K. in Biotechnology (Vol. 6a) (Kieslich, K., ed.) Verlag Chemie 1985, pp. 79-95
Enzyme Microb. Technol., 1991, vol. 13, March
:)51
The biotransformation of hydrocortisone to prednisolone by Arthrobacter simplex ATCC 6946 was extensively investigated in a liposomal medium, made up of multilamellar vesicles of either pure or crude egg phospholipids. The liposomal medium was found to promote faster kinetics to a higher conversion than those obtained in an aqueous medium and to be superior to water/surfactant and water/cosolvent systems, especially at high substrate loadings. The enhancing effect of the liposomal medium was partly explained by its capacity for steroid entrapment as well as by the presence of phospholipid vesicles in the bioconversion medium.
Keywords: Liposome; microbial catalysis; Arthrobacter; hydrocortisone
Introduction Microbial transformations of hydrophobic compounds are often met with two serious obstacles; (a) a limited substrate accessibility to the biocatalyst as a result of the low aqueous solubilities of most organics, and (b) inhibition or toxicity of both substrate and product exerted upon the microbe. Attempts to alleviate or o v e r c o m e these shortcomings include the use of surfactants and water-miscible or -immiscible solvents. ~-3 But again, inhibitory and toxic effects introduced by these surfactants and organic solvents may partly or fully inactivate the biocatalyst. A different type of bioconversion medium is an essentially aqueous medium into which an inherently nontoxic ingredient is added in view of eliminating the previously mentioned shortcomings. An example of such an ingredient is the biocompatible cyclodextrins used for biotransformation of toxic substrates. 4 In this study, an extensive investigation of the transformation of hydrocortisone to prednisolone by Arthrobacter simplex ATCC 6946 was undertaken in a medium consisting of another biocompatible ingredient, the liposome-forming diacyl phospholipids. Indeed, phospholipids, which are also the main constituents of natural
Address reprint requests to Dr Bar at the Department of Applied Microbiology, The Hebrew University, P.O. Box 1172, Jerusalem, Israel 91010 Received 29 March 1990; revised 11 July 1990 © 1991 Butterworth-Heinemann
cellular membranes, are generally held to be nontoxic. 5 Thus the liposomal medium, previously suggested by Bar 5 as a bioconversion medium for sterols and steroids, may be considered as a pseudobiological or a biomimetic environment. Bioconversions in a medium o f l i p o s o m e s have been primarily limited to enzymatic reactions at a microscale involving either a substrate or an e n z y m e contained in the liposome. In a recent study, 6 hydrophobic enzymes such as aminopeptidase or phosphatase were incorporated in small unilamellar vesicle (SUVs) for hydrolysing water-soluble substrates. In contrast, soluble cholesterol oxidase 7 was used to oxidize cholesterol contained in SUVs as an analytical tool in kinetic studies of cholesterol exchange. As multilamellar vesicles are one of the easiest forms of liposomes to prepare, they were employed as a medium for microbial conversions throughout this study.
Materials and methods Materials
Hydrocortisone, prednisolone, prednisone, and 2,2'dipyridyl were purchased from Sigma (St. Louis, USA). Egg-phosphatidylcholine (pure egg-PC) was obtained as a chloroform solution of I.-a-phosphatidylcholine (99%) from Sigma (Type V-E) and as a pure solid (98%) from Lipoid K G (Lipoid E-PC). Commercial-grade (or crude) egg lecithin (approx. 60% phosphatidylcholine) and pure lysolecithin were purchased from Sigma. Enzyme Microb. Technol., 1991, vol. 13, March
245
Papers Egg lecithin (Sigma) and lipoid E-PC were stored at -18°C under argon as chloroform solutions. All organic solvents used were chemically pure and purchased from Frutarom (Israel) except ethylene glycol and polyethylene glycol (Riedel-deHaen, FDR), tertbutanol (BDH, England), dimethylsulfoxide (Fluka, Switzerland), and Tween 80 (polyoxyethylene sorbitan monooleate, Sigma, USA).
Cultivation of cells Arthrobacter simplex ATCC 6946 was maintained and cultivated as described elsewhere. 8 The cells were harvested by centrifugation and washed three times with a saline phosphate buffer which consisted of 8.5 g NaCI, 0.3 g KHzPO 4, and 1.13 g NazHPO4.7H20 per liter of distilled water. Finally, the cells were washed once with distilled water and then lyophilized. Each lyophilized preparation was then assayed for its specific enzymic activity (vide infra). These lyophilized cells were convenient to work with, since they retained their enzymic activity for a long time and they could be accurately weighed.
Enzyme assay Cells were assayed for 1-dehydrogenase activity at near-saturation concentration of hydrocortisone (i.e. absence of crystals). A 50-ml flask was loaded with 8 ml of a saturated solution of hydrocortisone in buffer phosphate (pH 7.8, ionic strength 0.05) and 50 tzl of a solution of 2,2'-dipyridyl (15 g !-J) in ethanol. A cell suspension containing 5 mg dry weight (DW) cells was then added to the hydrocortisone solution to bring the final volume to 10 ml and the final hydrocortisone concentration to 0.32 g 1-i. The flask was then incubated in a shaker (30°C, 150 strokes min -1) and samples (0.5 ml) were removed periodically for analysis. The initial specific activity of every batch of cells was calculated after 15 min. It is expressed in U g ~DW where 1 U is defined as 1 /xmol prednisolone formed per minute.
Preparation of liposomes Various amounts of hydrocortisone and phospholipids were dissolved in 20 ml of various organic solvents, mainly chloroform, in a 250-ml round-bottomed flask. A volume of 50 txl of an ethanolic solution of 2.2' dipyridyl (15 g 1-1) was added to the hydrocortisone and phospholipids solution, and this mixture, with or without added glass beads (diameter 3 mm), was then evaporated in a rotary evaporator at 40°C to obtain a dried lipidic film. Residual traces of organic solvents were subsequently removed by an overnight vacuum drying at room temperature and pressure of 500 tort. The lipidic film was rehydrated at ca. 40°C with 8 ml of the previously described buffer phosphate (pH 7.8) on a vortex mixer for 15 min. The resulting milky liposomal medium was transferred to a 50-ml Erlenmeyer flask, and 2 ml of a suspension of lyophilized cells was added to bring the final volume to l0 ml. The flask with the inoculated liposomal medium was incubated in a
246
Enzyme Microb. Technol., 1991, vol. 13, March
shaker at 30°C and 150 strokes min-J, and aliquots (0.3-0.5 ml) were periodically withdrawn for analysis. A series of control flasks with aqueous fine dispersions of hydrocortisone was incubated at identical conditions. However, unlike the homogeneous liposomai medium, the steroid slurry in the control flasks could not be properly sampled. Therefore, the whole content of one control flask was subjected to product analysis at one time.
Product analysis Aliquots (0.3-0.5 ml) from the liposomal medium were extracted by 1.5 ml ethanol and 3 ml chloroform on a vortex mixer. Following phase separation, 160 txl of the organic phase was transferred into sampling vials and mixed with 40 txl of a methanol solution of prednisone (0.5 g 1 ~) as an internal standard. Samples (I0 txl) were analyzed on a Lichrospher 5 Si 60 column (Merck, Darmstadt, FRG), employing chloroform containing 5% (v/v) methanol and 0.55% (v/v) glacial acetic acid as the mobile phase. The flow rate of the eluent was 1 ml min 1. Calibration curves of area ratios between either substrate or product and internal standard were determined at 254 nm. The aqueous medium of the control flasks was extracted by a mixtue of 30 ml ethanol and 60 ml chloroform on a vortex mixer. Then the product analysis was performed as previously mentioned.
Measurement of solubilities of steroids To a 10-ml buffer phosphate solution pH 7.8, 0.25 g of either hydrocortisone or prednisolone, or both, was added. The suspension was stirred under argon at 30°C for 72 h to saturate the aqueous solution. Then the suspension was centrifuged at 12,100 g for 30 min, and an aliquot of 0.5 ml from the clear supernatant was extracted with a mixture of 0.5 ml ethanol and 1 ml chloroform. The extract was subsequently analyzed by HPLC.
Measurement of steroid entrapment in liposomes Liposomes were prepared with various concentrations of pure egg-PC and steroid. After hydration, the liposomes were held at 30°C for 24 h and subsequently centrifuged at 12,100g for 10 rain. An aliquot of 0.5 ml of supernatant was extracted with 0.5 ml ethanol/ chloroform (2:1 v/v) and the extract analyzed by HPLC. The pellet of liposomes was washed with phosphate buffer, centrifuged at 12,100g for 10 min, and extracted with 6 ml ethanol~chloroform (I :2 v,v). A sample of this extract was analyzed by HPLC. Finally, the percent entrapped steroid was then calculated from the amount added and the amount recovered.
Tests for microbial degradation of phospholipids Tests for lipolytic activity of A. simplex ATCC 6946 were done on egg yolk agar plates by detecting the
Dehydrogenation in liposomal medium: R. Goetschel and R. Bar opalescent zone of precipitated lipid 9,1°after incubation of 24 and 48 h at 30°C. Further tests were conducted to detect lysolecithin following 24 h incubation of A. simplex cells in a phosphate buffer containing empty liposomes of pure egg-PC. The liposomes were extracted with chloroform and this was analyzed on TLC aluminum sheets (Woelm Silica Gel). The eluent consisted of chloroform, acetone, methanol, acetic acid, and water at respective volumetric ratios of 6 : 8 : 1 2 : 2 : 1 . The Rf values of pure egg-PC and lysolecithin were found to be 0.10 and 0.13, respectively.
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Results An important requirement for a microbe in a liposomal medium would be its inertness towards the constituent phospholipids. No phospholipase activity was, however, detected in A. simplex using egg yolk agar, and no lyso-PC could be detected in a liposomal medium of pure egg-PC following incubation with the microbial cells. Thus, A. simplex appears unable to degrade liposomes.
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Figure 1 Bioconversion of hydrocortisone (2 g 1-1) in liposomal media of pure egg-PC (A) Comparison of an aqueous dispersion (V) with a liposomal m e d i u m (T) of 20 g PC 1-1 (molar ratio of steroid : PC was 1 : 4.8). Concentration of cells was 6 g DW I with a specific activity of 33.9 U g 1DW. (B) Effect of phospholipid loading on the time course of bioconversion. Molar ratios of steroid : PC were (0) 1 : 0.6, (O) 1 : 2.4, (A) 1 : 3.6, (A) 1 : 4.8. Concentration of cells was 2 g DW I 1 with a specific activity of 30.3 U g-1 DW
Bioconversion in a liposomal medium A liposomal medium composed of 20 g 1-c~-phosphatidyi-choline 1-l and 2 g hydrocortisone 1-J clearly promoted the biotransformation (Figure la) at a faster rate and to a higher degree of conversion than those achieved in a fine dispersion of hydrocortisone. In the latter case, the conversion was only 63% after 8 h, in contrast to 98% in the liposomal medium. Furthermore, the biotransformation halted at a maximum conversion of 75% after 24 h in the aqueous slurry, but it proceeded to virtual completion in the liposomal medium. This remarkable enhancement effect was found to correlate with the loading of phospholipids in the medium. Figure lb shows that for liposomal media with constant substrate (2 g 1-J) and biocatalyst (2 g DW 1- l) concentrations, the bioconversion kinetics were progressively faster with increased PC to hydrocortisone molar ratio. These positive findings based on pure and expensive egg-PC prompted us to compare it with crude but much cheaper phospholipids such as egg lecithin, which contains only 60% phospholipids, mainly PC and phosphatidyl ethanolamine. An examination by a light microscope of "liposomes" made of this egg lecithin revealed indeed not only the presence of typical forms of multilamellar vesicles but also spherical oil droplets as a result of the large content of triglycerids. The effect
of lecithin on the bioconversion was investigated at hydrocortisone concentrations of 0.4, 2, 5, 10, 25, and 50 g 1-I (Table 1). At a near-saturation substrate concentration (0.4 g 1 J), lecithin offered no advantage over a regular medium. In the nonliposomal media, the biocatalytic activity was inhibited after 8 h in all other hydrocortisone Ioadings. These inhibitory effects were previously reported by other investigators, ll-13 However, inclusion of lecithin in the media enabled further progression of the biotransformation to higher degrees of conversion (Table 1). Therefore, the enhancement effect of the "liposomal medium" made of crude lecithin was prominent in high substrate loadings.
Effect of mode o f liposome preparation on the bioconversion Since the multilamellar vesicles vary widely in their sizes, it was judged important to measure the bioconversion kinetics in liposomes with two roughly different size distributions. A simple means to reduce the liposome sizes is the addition of glass beads to the chloroform solution of the lipids. Figure 2 shows indeed that Enzyme
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Papers Table 1 Degrees of bioconversions of various hydrocortisone concentrations after 12, 18, and 24 h, obtained in aqueous and liposomal media with 100 g crude egg lecithin 1-1 0.4 Substrate (g 1-1) Time (h) Conversion (%) Aqueous m e d i u m Liposomal m e d i u m
2
5
12
18
24
12
18
24
12
18
24
100 100
100 100
100 100
69 70
73 90
77 92
60 85
63 97
75 97
10
25
50
Substrate (g 1-1) Time (h)
12
18
24
24
48
72
24
48
72
Conversion (%) Aqueous m e d i u m Liposomal m e d i u m
32 54
36 81
37 83
21 50
25 59
26 64
12 20
14 38
14 74
Concentration of cells was 6 g DW 1-1 with a specific activity of 32.9 U g-~ DW
the bioconversion was significantly faster in a liposomal medium prepared with added glass beads. The influence of ultrasonic irradiation on liposome size and lamellarity is well known.14 It was interesting to investigate the effect of sonication of the tiposomal media on the subsequent bioconversion kinetics. The liposomal media were subjected to sonication for several durations of 2.5, 5, 10, or 15 min, but interestingly, no significant difference in the bioconversion time course was observed with regard to the nonsonicated liposomal medium. As the liposome preparation requires first dissolution of the steroid and phospholipids in an organic solvent, several commonly used solvents and their combinations were tried and were found to lead to different bioconversion profiles, chloroform being the
best. It is important to note that residual solvent or cosolvent, not entirely discarded, had an adverse effect on the biocatalyst. For instance, when the solvent mixture of chloroform and tert-butanol was subjected to a mere rotary evaporation, the bioconve-rsiori was smaller. When the treatment included also a vacuum drying overnight, the degree of conversion reached 95% after 8 h.
Liposomal medium vs. water/cosoluent system Figure 3 compares the performances of a liposomal medium and a water/ethylene glycol (30% v/v) system at low and high concentrations of hydrocortisone. At a concentration of 2 g 1-1, hydrocortisone dissolved
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Enzyme Microb. Technol., 1991, vol. 13, March
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F i g u r e 3 Bioconversion profiles in a w a t e r / e t h y l e n e glycol (30% v/v) system (discontinuous curves) and in liposomal media of pure egg-PC (continuous curves) or hydrocortisone at a concentration of 2 g I 1 ( O a n d ©) and 1 0 g I -~ (A and A ) . T h e molar ratio steroid : PC was 1 : 4.8 (©) and 1 : 2.4 (zS). Cell concentration was 6 g DW I 1 with specific activity of 66.7 U g-1 DW
Dehydrogenation in liposomal medium: R. Goetschel and R. Bar completely in the water/cosolvent system and was bioconverted at a slightly lower rate than in the liposomal medium. However, the latter medium demonstrated a considerable advantage at the high substrate concentration of 10 g 1 ~. The high loading of hydrocortisone saturated the hydroorganic system with formation of a slurry, and at these conditions, the biocatalyst was soon inhibited. The liposomal medium, however, enabled the bioconversion to progress to a much higher extent (Figure 3).
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Figure 4 compares bioconversion profiles in a liposomal medium and in aqueous slurries with and without two surfactants. Tween 80 and polyethylene glycol at nontoxic concentrations. In these experiments, the microbial cells were very active, as evidenced by their high specific activity of 66.7 U g i and the relatively fast bioconversion in the control. Both surfactants had a marginal effect on the bioconversion, while a marked enhancement took place in the liposomal medium.
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Figure 4 Bioconversion profiles of hydrocortisone (2 g I 1) in a liposomal m e d i u m (A, 20 g pure egg-PC 1-1) and aqueous media without (V) and with Tween 80 (A, 0.1 g 1-1) and polyethylene glycol (0, 1 g I-1). Cell concentration was 4 g DW I 1 with a specific activity of 66.7 U g-1 DW
Entrapment o f steroids in liposomes The solubilities of hydrocortisone and prednisolone measured separately at 30°C were 0.45 and 0.29 g 1-j, respectively. The corresponding solubilities when measured in the presence of excessive amounts of both steroids changed, however, to 0.36 and 0.33 g 1 ~, respectively. Subsequently the entrapped steroid was determined at a near-saturation concentration that avoided precipitation of crystals. Table 2 shows the percentages of entrapped hydrocortisone and prednisolone, respectively, as a function of pure egg-PC loading following equilibration for 24 h. Comparable degrees of entrapment were obtained for the chemically similar steroids, and at a phospholipid content of 100 g 1-1, 90% of either steroid was, in fact, restained within the liposomes. The degrees of entrapment of hydrocortisone, expressed in terms of percent mole steroid per mole phospholipid, are given also in Table 2, and these did not exceed a value of 3.0% at the lowest PC concentration (20 g I J) employed.
Table 2 Entrapment of 0.4 g hydrocortisone 1-1 or 0.39 g prednisolone 1-1 at 30°C in various concentrations of pure eggPC multilamellar liposomes, expressed as a percentage of the total concentration.
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Figure 5 Bioconversion profiles of hydrocortisone (2 g I 1) in aqueous media with 5 (A), 10 (A), 20 (O), and 50 g (0) pure eggPC I 1 in the form of empty liposomes. Cell concentration was 4 g DW 1-1 with a specific activity of 66.7 U g-1 DW
Effect o f empty liposomes on the bioconversion Figure 5 shows the effect of " e m p t y " liposomes, i.e.
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Hydrocortisone
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20
50
100
51 (2.4) 53 (2.8)
81 (2.1) 75 (1.6)
92 (0.7) 90 (1.0)
Percent steroid entrapment (mol % steroid per mol phospholipid) is given in parentheses
liposomes made up of phospholipids only, on the bioconversion. Clearly, increasing amounts of empty pure egg-PC multilamellar vesicles added to microbial slurries of hydrocortisone progressively enhanced its conversion. The " e m p t y " liposomes definitely had a positive effect on the bioconversion. A simultaneous incorporation of the steroid into the empty phospholipid vesicles, as the biotransformation took place, could partly explain the enhancing effect. Indeed,
Enzyme Microb. Technol., 1991, vol. 13, March
249
Papers Table 3
Qualitative estimation of the relative presence of prednisolone needles in various media made of either aqueous dispersions of hydrocortisone or liposomes prepared with increasing concentrations of E-PC and hydrocortisone Hydrocortisone (g 1-1) E-PC (g 1-1) 0(control) 20 50 100
2 (after 5 h) +++
(91) (100) - (100) - (100)
10 (after 24 h) ++++ + + + -
(46) (74) (82) (91)
50 (after 24 h) ++++ (12) + + + (14) + + + (15) + + + (18)
( - ) No needles; (+), ( + + ) , ( + + + ) , (++++)increasing amounts of needles. In each case the percentage of conversion is indicated. Cell concentration was 3 g DW I 1, with a specific activity of 66.7 U g-1 DW. The degrees of substrate conversion (%) are given in parentheses
equilibration of empty liposomes made of 50 g pure egg-PC 1-~ with an aqueous hydrocortisone solution (0.4 g 1-1) resulted in incorporation of 65% of the steroid after 24 h. A similar experiment with hydrocortisone-containing liposomes resulted in an entrapment of 81% (Table 2).
Crystallization phenomena in the liposomal medium Since the studied bioconversion involved sparingly water-soluble substrate and product steroids, crystallization phenomena inevitably took place in parallel with the microbial transformation. An examination of an aqueous bioconversion medium by light microscopy revealed formation of product crystals in the form of needles (length up to 50/xm) as previously reported by Kondo and Masuo. ~5 A visual estimation of the presence of prednisolone needles in media made of increasing concentrations of egg-PC at the corresponding conversion percentages is reported in Table 3. For hydrocortisone concentrations of 2 and I0 g 1-~, as the egg-PC concentrations increased, the relative amount of needles diminished, and the bioconversion almost reached completion. However, for hydrocortisone concentration of 50 g 1- ~, even a high concentration of egg-PC was insufficient to prevent formation of needles. Judging from Table 3, one can see that the appearance of product needles becomes discernible in a liposomal system with an approximate PC : hydrocortisone weight (molar) ratio of 5 : 1 (2.43 : 1). In systems with a lower ratio, the crystal needles prevailed, but at a higher ratio, they were not visible.
Discussion It appears that an enhanced microbial catalysis in a liposomal medium can in principle be explained in terms of a higher solubilization of substrate and/or product and a facilitated substrate accessibility to the biocatalyst.
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The faster kinetics and the higher conversion of the microbial transformation in either pure (Figure 1) or crude liposomal (Table 1) media could be at least partly attributed to entrapment of the reactants in the multilamellar vesicles, thus subjecting the microbes to less inhibitory concentrations of the steroids. Solubilization of water-insoluble organics can also be obtained by surfactants, as previously mentioned, and consequently, an enhanced biotransformation (Figure 4) is expected. However, the use of surfactants is limited to a concentration of ca. I g 1 1, beyond which deleterious effects on cell wall and membranes may well lead to inhibited catalysis ~6 even by nontoxic surfactants. Phospholipids can be employed at considerably higher loadings of 100 g I i (and more) with no observable inhibitory or toxic effects upon the microbe. Another simple way of increasing the solubility of sparingly soluble steroids would be by adding a water-miscible organic solvent, thus rendering the bioconversion medium more hydrophobic. Indeed, the bioconversion in question was previously investigated in the presence of various water-miscible cosolvents by Freeman and Lilly. 2 These authors, who considered diols such as ethylene glycol to be the most suitable group of solvents, showed that the solubility of hydrocortisone increased tenfold in a water/ethylene glycol (30% v/v) system, but its biotransformation was inhibited in it. The detrimental effect of this hydroorganic system with 10 g hydrocortisone 1 J upon the microbial activity was also observed by us (Figure 4), but the liposomal medium again promoted the biocatalysis with no adverse effect. Accessibility of the liposome-associated steroid to the biocatalyst is dependent upon the size and lamellarity of the liposome. In this study, three well-known means ~4were employed to grossly affect both liposome size and lamellarity. Glass beads have apparently reduced the sizes and lamellarity of multilamellar vesicles (MLVs) as a result of an increased surface area of the lipidic film and a more vigorous shaking during the hydration. The bioconversion in a liposomal medium prepared with glass beads was consequently faster (Figure 2). However, a drastic size reduction obtained by sonication, which apparently led to formation of small unilamellar and possibly oligolamellar vesicles, offered clearly no clear advantage over nonsonicated liposomal medium. This could reasonably be attributed to a reduced steroid entrapment capacity by the small vesicles. Under these conditions, a major exclusion of the steroid from the liposome would make the system behave similarly to an aqueous medium containing empty liposomes (vide infra). The third means of affecting both size and lamellarity refers to the type of solvent employed in liposome preparation. Chloroform, which is commonly employed, 14was found to lead to the fastest kinetics of biotransformation. It is important to note that a mere steroid entrapment in the liposomes cannot fully explain the enhancement of the biotransformation. The hydrocortisone loading employed in a standard bioconversion experiment (Fig-
Dehydrogenation in liposoma/ medium: R. Goetschel and R, Bar ure la) was 20.8% mole steroid per mole phospholipid, which is of course much larger than the maximum measured (Table 2) value of steroid entrapment of ca. 3%. The incorporation of hydrocortisone derivatives was previously investigated by Shaw et al. Z7 who showed that increasing the chain length of a substituted group in carbon 21 of the hydrocortisone molecule caused a marked enhancement of the steroid retention in dipalmitoyl-PC liposomes. Thus, hydrocortisone palmitate was reportedly retained longer than the octanoate ester. However, the unesterified steroid, hydrocortisone, would probably be less incorporated in the liposomes and consequently would precipitate out when initially employed at a high loading. Therefore, liposornal media with high substrate loadings definitely contained steroid crystals as well. Furthermore, the mere presence of empty liposomes in a steroid suspension exerted an enhancement effect on the bioconversion (Figure 5). Again, even though the initially empty liposomes were shown to gradually absorb hydrocortisone from the medium, the extent of this entrapment was not large enough to rationalize the enhancement effect. Other mechanisms not known to us could possibly be involved. For instance, a favorable interaction between the phospholipids of MLVs and the microbial cell wall could play a role. These mechanisms could be at the origin of the previously reported enhancing effect of oils containing phospholipids on sterol and steroid oxidations J8,19 performed in fermentation broths. As previously mentioned, crystallization phenomena accompany bioconversion of sparingly soluble substrates, and, for this reason, the microbial conversion of hydrocortisone leading to the formation of product needles was termed "pseudocrystallofermentation".J5 This crystallization would not take place, for instance, in a water/organic solvent system having the capacity to dissolve both reactant and product. It was therefore interesting to examine the various liposomal media microscopically for the appearance of these product needles. The appearance of needles (Table 3) in a bioconversion medium below a PC:hydrocortisone weight ratio of 5:1 could be attributed to product crystals formed from initially nonentrapped substrate. The absence of needles in systems with a higher ratio, wherein only a small fraction of the substrate is actually entrapped within the liposomes, testifies to the complexity of the crystallization processes taking place in a liposomal medium. These processes are not fully understood by us. Perhaps the liposomes induced formation of product microcrystals with a different habit other than needles.
Conclusion The liposomal medium was shown not only to be suitable for microbially catalysed conversions of hydrocortisone but also to offer significant advantages over aqueous media with or without cosolvents or surfactants.
Acknowledgements Support of this study from the Kay Foundation at the Hebrew University is gratefully acknowledged. The authors are thankful to Dr. L. K. Bar for useful discussions on liposome technology and to Prof. Y. Barenholz for critically reviewing the manuscript.
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