Optimization of steroid side chain cleavage by Mycobacterium sp. in the presence of cyclodextrins

Optimization of steroid side chain deavage by Mycobacterium sp. in the presence of cydodextrins Paul G. M. Hesselink, Steven van Vliet, Harrie de Vries and Bernard Witholt Groningen Biotechnology Center, D e p a r t m e n t o f Biochemistry, University o f Groningen, Nijenborgh 16, 9747 A G Groningen, The Netherlands

The microbial side chain cleavage of sterols is a slow process because of the poor solubilities of substrates and products and their low transport rates to and from cells. In this report we show that the addition of cyclodextrins substantially enhanced the conversion of cholesterol, sitosterol, and A4cholestenone to a mixture of androstenedione and androstadienedione [AD(D)] by Mycobacterium sp. NRRL-B 3683 in a purely aqueous.fermentation system. For cholesterol, fl-cyclodextrin gave the best results, whereas y-cyclodextrin was the best clathrate for sitosterol and Aa-cholestenone biotransformation. In all cases an optimal 2 : 1 molar ratio of cyclodextrin : substrate was found, resulting in a 1.7 to 3.0-fold increase of the specific side chain cleavage activity. The cyclodextrins had no influence on cell growth. Applying optimal conditions, molar yields of AD(D) were raised from 35-40% after conversion for 175 h in control experiments to over 80% after conversion during ca 120 h in the presence of cyclodextrins.

Keywords: Side chain cleavage; sterol; AD(D); Mycobacterium sp.: NRRL-B 3683; cyclodextrin Introduction Androst-4-ene-3,17-dione (AD) and androsta-1,4-dien3,17-dione (ADD) are excellent precursors in the synthesis of nearly all expensive steroid drugs. J They can be produced by the microbial degradation of side chains of cheap sterols such as cholesterol and sitosterol. The general transformation pathway of cholesterol to AD(D) has been elucidated for a variety of microorganisms, including Mycobacterium sp. (Figure 1). Figure 1 indicates that cholesterol degradation occurs not only via side chain cleavage but alsn via degradation of the sterol nucleus. This results in loss of substrate due to the production of undesired side products and must therefore be avoided. Product inhibition must also be considered: some side chain-cleaving microorganisms are sensitive to inhibition by AD(D), thus lowering product yields. In addition, most steroids are poorly soluble in aqueous media and reaction rates are therefore rather low. One solution is to work in organic phases. However, many of the microorgan-

Address reprint requests to Dr. Hesselink at the TNO Institute of Applied Chemistry,P. O. Box 108, 3700AC Zeist, The Netherlands Received 14 December 1987; revised 15 July 1988 398

Enzyme Microb. Technol., 1989, vol. 11, July

isms used hardly transform steroids in the presence of organic phases, and expensive and laborious reactor systems have been developed to help solve these problems. 2-~ Another approach is to combine the advantages of apolar systems (higher steroid solubilities) and aqueous systems (compatible with microorganisms) by carrying out conversions in the presence of clathrating agents such as cyclodextrins. Cyclodextrins are doughnut-shaped cylic oligosaccharides of six to eight glucose units linked by a-l,4glycosidic bonds (Table 1). They dissolve easily in water and have a hydrophobic cavity able to include hydrophobic guest molecules by Van der Waals interactions and hydrogen bond formation? Among other applications, cyclodextrins are presently used as stabilizers and solubilizers of several steroid drugs such as hydrocortisone, cortisone acetate, and testosterone. 6,7 In a previous study we were able to show that the addition of fl-cyclodextrin enhanced the microbial conversion of cholesterol to AD(D). 8 A more systematic extension of these experiments to other substrates could be of interest for the applicability of cyclodextrins in microbial sterol side chain cleavage. In this report we show that cyclodextrins accelerate the conversion of cholesterol, sitosterol, and A4-cholestenone to AD(D) by M y c o b a c t e r i u m sp. NRRL-B 3683. Moreover, cyclodextrins suppress nucleus breakdown and © 1989 Butterworth Publishers

Cyclodextrins and steroid transformation: P. G. M. Hesselink et al.

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Figure 1 General pathway of microbial cholesterol degradation. Arrows indicate the route from cholesterol to AD(D). (1-2-(4-)-7-8-910) and the undesired nucleus degradations (1-2-3/4-5-6 and 10-11). Common compounds are: 1 = cholesterol, 2 = A"-cholestenone, 10 = androst-4-ene-3-one, and androsta-l,4-diene-3-one AD(D)

product inhibition in these biotransformations, resulting in increased AD(D)-production yields and reduced side product formation. Materials

and

methods

Bacterial strain, media, and growth conditions Mycobacteriurn sp. NRRL-B 3683 was purchased from the American Type Culture Collection (Rockville, MD, USA) as strain number ATCC 29472. Steroid fermentations were usually carried out in 250-ml Erlenmeyer flasks containing 50 ml L-broth (pH 7.0) on a rotary shaker (200 rev min -j) at 30°C. The series of fermentations for the optimization of AD(D) production as a function of various combinations of substrates and types of cyclodextrins was carfled out in 1-1 fermentors containing 700 ml of L-medium. These fermentors (d × h -- 9 × 20 cm) were equipped with a four-bladed rectangular impeller of 5.3 cm diameter (300 rev min -1 stirrer speed) and were operated under control of pH, temperature, and oxygen supply. The L-medium for cell culturing contained, per liter: Bacto-Yeast Extract 5 g, Bacto-Tryp-

Table 1 Some characteristics of cyclodextrins Cyclodextrin

Glucose units

e

/3

3'

6 7 8 Mol. weight 972 1135 1297 Cavity diameter (nm) 0.47-0.52 0.60-0.64 0.75-0.83 Cavity depth (nm) 0.80 0.80 0.80 Solubility in water (%w/v) 14.5 1.85 13.2

tone 10 g, NaC1 5 g. The pH was adjusted to 7.2 before sterilization. The broth was autoclaved 25 min at 121°C, which resulted in a lowering of the pH to 7.0. Cholesterol or sitosterol (usually at 1 g 1-I) and cyclodextrins or glucose were added before autoclaving. The Erlenmeyer flasks and fermentors were inoculated with 0.I ml of an overnight Mycobacteriurn sp. NRRL-B 3683 culture to start transformation. The minimal E2-medium contained E-salts with the omission of citrate as described by Vogel and Bonner. 9 Cell densities were determined by measuring the optical density at 450 nm with a Zeiss PMQ II as described earlier l° or with a Klett-Summerson (USA) 8003 colorimeter using the Klett 42 blue filter (400-465 am).

Steroid analyses Steroid transformations were followed by taking 1-ml samples from the culture broth at regular intervals. Stigmasterol was added as an internal standard before the samples were saturated with NaCI and extracted with 2 ml ethylacetate. The ethylacetate layer was removed, incubated with 20 /xl 10 M HCI (15 min at 50°C), washed with 2 ml 0.1 NaOH and twice with 2 ml demineralized water, respectively. The ethyl acetate was evaporated and the steroids were derivatized with 0.1 ml of a 10% solution of methoxamine-HC1 (MO) in pyridine (20 min at 60°C), followed by silylation with 0.1 ml of trimethylsilylimidazole (TMS) (2 h at 70°C). After evaporating to dryness, the steroid MO-TMS derivates were dissolved in 2 ml of distilled hexane and washed twice with 2 ml of demineralized water. The steroid solution was ready for injection in the gas chromatograph (GC) after removal of the water layer. GC analysis of steroids was carded out on a Pack-

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Papers ard 436 gas chromatograph equipped with a 25-m capillary fused silica CP Sil 5 CB column, 0.22 mm internal diameter (Chrompack, Middelburg, The Netherlands). Helium was used as a carrier gas. Splitless sample injections of 1 /zl were carried out by an automatic Packard LS 607 sampler/injector setup. The temperature program was as follows: 2 min initial run at 120°C, 30°C min -1 rise to 210°C min-', 2°C min 1 rise to 290°C, 5 min final run at 290°C. A Shimadzu C-R3A was connected to the GC apparatus for plotting and integration. Steroids were always analyzed as their MO-TMS derivatives. Identification of unknown peaks was performed with a Finnigan mass spectrometer at the Groningen Academic Hospital. Thin layer chromatography (TLC) was carried out on 20 x 20 cm aluminum sheets precoated with silica gel 60/Kieselguhr F T M fluorescent indicator, layer thickness 0.2 mm (type 5567, Merck, Darmstadt, FRG). Steroids were analyzed without derivatization. Elution was carried out with a 1:1 (v/v) mixture of cyclohexanol:ethylacetate. Spot detection was improved by spraying with a 1:1 : 10 (v/v/v) mixture of Ac20 : H2504 : EtOH. Steroids appeared as colored spots after 10 min heating to ca 120°C.

Chemicals All steroids, trimethylsilylimidazole (TMS) and methoxamine-HCl (MO) were purchased from Sigma (St. Louis, MO, USA). Steroids were checked for their purity by TLC and capillary GC. Cholesterol was used as a pure substrate, whereas the soybean sitosterol consisted of a 3:2 mixture of fl-sitosterol and campesterol. We use the name of sitosterol to refer to this mixture. All other reagents were of analytical grade. The cyclodextrins were gifts from TNO (Institute of Applied Chemistry, Zeist, The Netherlands) and the Netherlands Institute for Carbohydrate Research (NIKO, Groningen, The Netherlands).

Results

Effect of cyclodextrins on sitosterol conversion Previously, we showed that fl-cyclodextrin enhanced the side chain cleavage of cholesterol to AD(D) by doubling the transformation rates. 8 So far it was unknown whether cyclodextrins had the same stimulating influence on the side chain cleavage of sitosterol to AD(D) by Mycobacterium sp. NRRL-B 3683. Therefore, several fermentations were carried out in L-medium with sitosterol at 1 g 1-I and containing 1 g 1-~ of glucose, a-, fl-, or y-cyclodextrin. Table 2 shows that the addition of all three types of cyclodextrins improved the production of AD(D), whereas /3-cyclodextrin was particularly effective. Glucose had hardly any effect on AD(D) production in a control experiment. The growth rates of Mycobacterium sp. NRRL-B 3683 were only slightly affected by addition of glucose and cyclodextrins when compared to the control experiment without these compounds. 400

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T a b l e 2 Influence to several additions of (l g l 1) on the conversion of sitosterol to AD(D) by Mycobacterium sp. NRRL-B 3683 (in % m o l a r AD(D) yield) Incubation time (h)

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38 40 41 52 49

The final cell densities were about 1.1 g 1-~ in all experiments. The product AD(D) consisted of a nearly 1 : 1 mixture of AD and ADD after 100 h of incubation. This ratio shifted towards more ADD at prolonged incubation times.

Effect of cyclodextrins on cell growth The influence of cyclodextrins on cell growth was established more in detail by the determination of generation times and cellular yields on cyclodextrins. These experiments were carried out both in L-medium and in the minimal E2-medium, the latter containing a cyclodextrin or glucose as the sole source of carbon. We found that glucose and cyclodextrins had hardly any influence on cell growth rates in the rich L-medium. The generation times of Mycobacterium sp. NRRL-B 3683 were virtually unchanged and varied between 8.5 and 9.0 h in the presence as well as in the absence of cyclodextrins or glucose. These generation times corresponded to maximal specific cell growth rates (tZmax) ranging from 0.077 to 0.082 h -J. In addition, glucose and the cyclodextrins were found to be poor carbon sources for Mycobacterium sp. NRRL-B 3683. This was indicated by the Mycobacterium generation times in the minimal E2-medium of 17 and 22-30 h, respectively. This corresponded to values for/-/-max of 0.041 h -~ for glucose and ca 0.029 h -~ for a- and y-cyclodextrin. Especially/3-cyclodextrin turned out to be a poor carbon source, leading to /-/'max = 0.023 h -~. Growth yield data showed that glucose was more easily used as a carbon source by Mycobacterium sp. NRRL-B 3683 than the cyclodextrins. Cellular yields were calculated to be 0.32 and 0.03-0.07 g cell dry weight g-1 substrate consumed, respectively. Obviously, the a1,4-glycosidic binding of glucose residues to give the closed circular cyclodextrin molecule formed a serious barrier for the use of these compounds as a carbon source. Since cyclodextrins hardly affected cell growth, their stimulating effect on sterol side chain cleavage must be due to enhancement of steroid solubilities and/or transport rates.

Optimization of the cyclodextrin-substrate molar ratio The stoichiometry of a cyclodextrin inclusion complex is dependent not only on the size and structure of the

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guest molecule but also on the type of cyclodextrin involved. This was clearly demonstrated by the composition of cyclodextrin-menadione (vitamin K3) complexes: the molar ratio of the /3-cyclodextrin-menadiane complex was found to be 3 : 1, whereas a 1 : 1 inclusion complex was formed with 7-cyclodextrin. 6 In order to determine the optimal molar ratio of cyclodextrin to cholesterol, a series of fermentations was carded out with media containing 1 g 1-~ of cholesterol and increasing amounts of/3- or y-cyclodextrin. The same experiments were repeated with sitosterol as a substrate. The results of these experiments are summarized in Figure 2.

While cell growth was accelerated only slightly at higher cyclodextrin concentrations, the AD(D) production was substantially enhanced in all experiments. An optimal molar ratio of cyclodextrin : sterol of 2 : 1 was observed in all cases, being independent of the substrate and type of cylcodextrin used.

Effect of cyclodextrins on the specific cellular activity of side chain cleavage From previous experiments we knew that Mycobacterium sp. NRRL-B 3683 most actively converted sterols to AD(D) during the early exponential growth Enzyme Microb_Teehnol.,

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phase. The specific activity went down to 10-15% of this value in the stationary phase. TM The improvement of sterol side chain degradation could therefore be caused by either the prolongation of the period with high specific side chain cleavage activity or by an extra increase in transformation rates during the exponential growth phase. Therefore, the specific side chain cleavage activities of Mycobacterium cells were calculated from a series of batch fermentations containing cyciodextrins and steroids at the optimal 2 : 1 molar ratios. Figure 3 showed that the improvement of sterol side chain degradation by cyclodextrins was caused by enhancement of the specific cellular activities during the first half of the exponential growth phase. No prolongation of this active period was observed. Sitosteroltransforming Mycobacterium cells reached their maximum specific activity after ca 30 h, whereas cholesterol-degrading cells were most active after ca 20 h of incubation regardless of the presence of cyclodextrins. The effect of 7-cyclodextrin (3.0-fold increase of the highest activity) seemed to be more pronounced than that of ~-cyclodextrin (1.7-fold increase of the highest activity).

Optimization of substrate-cyclodextrin combinations Cholesterol, sitosterol, and A4-cholestenone are wellsuited substrates for the industrial production of AD(D). These compounds differ in molecular size and polarity due to the presence of different functional groups in the steroid nucleus and in the steroid side 402

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chain. The dimensions of cholesterol, sitosterol, and A4-cholestenone have been determined with X-ray crystallography and were found to be 0.52 x 0.62 × 1.89 nm, 0.45 x 0.75 x 2.06 rim, and 0.38 x 0.78 x 1.99 nm, respectively. J2,13 The cavity diameters of the three types of cyclodextrins used in this study vary from ca 0.5 to 0.8 nm (Table 1). Therefore, we expected that one specific combination of cyclodextrin and substrate would be optimal with respect to steroid solubilization and transfer to the biocatalyst. Consequently, cholesterol, sitosterol, and A4-cholestenone were used as substrates at 1 g 1-I concentrations in a series of biotransformations in 1-1 fermentors with control of pH, temperature, stirrer speed, and oxygen supply with all three types of cyclodextrins. The clathrating agents were added at the optimal 2 : 1 molar ratio of cyclodextrin : sterol. Although no optimal molar ratio had been experimentally verified for A4-cholestenone, a 2 : 1 molar ratio was assumed to be optimal. This assumption was based on the resemblance in size and structure of this substrate to the two sterols. Figure 4 shows that y-cyclodextrin was the best clathrate to improve the AD(D) yield in sitosterol and A4-cholestenone fermentations. The addition of 13-cyclodextrin was less advantageous, whereas a-cyclodextrin had no effect. For cholesterol fermentations, /3-cyclodextrin gave the best results, being slightly superior to y-cyclodextrin. The addition of a-cyclodextrin had hardly any effect. These findings suggested that both a minimum as well as a maximum size of the cyclodextrin cavity was required for optimal AD(D) production.

Discussion In order to make microbial side chain cleavage economically attractive, substrate and product end concentrations should be a few grams per liter.~ However, the solubility of sterols such as cholesterol and sitosterol in aqueous media is under 2 mg l-J. TM AD and ADD, which are the products resulting from the side chain cleavage of these sterols, are slightly more soluble, up to 50 mg 1-1 in aqueous media. T M Because of these low steroid solubilities, transport rates to and from bacterial cells are thought to be low, resulting in decreased transformation rates. Thus, improvement of AD(D) production rates could be expected by enhancement of these reaction parameters. As cyclodextrins were known to improve the solubility of steroids, microbial steroid transformations were carried out in their presence and indeed proved to be beneficial. Thus, the A~-dehydrogenation of hydroxycortisone and 17a-methyl-testosterone by Arthrobacter simplex proceeded faster and with suppression of product inhibition. The reaction rates of 21-hydroxylation of progesterone by Ophiobolus herpotrichus and the 11/3hydroxylation of 16a-methyl-Reichstein S were found to be stimulated by cyclodextrin addition as well.~V So far, the application of cyclodextrins in the microbial side chain cleavage of cholesterol was shown to be

Cyclodextrins and steroid transformation: P. G. M. Hesselink et al. 100

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Conversion of cholesterol (A), sitosterol (B), or 4 - c h o l e s t e n o n e (C) t o AD(D) in the presence of ~-,/3-, o r 7 - c y c l o d e x t r i n at t h e o p t i m a l 2 : 1 molar ratio of c y c l o d e x t r i n : s t e r o i d . Symbols used: x = control, (i) = ~ - c y c l o d e x t r i n , [ ] = / 3 - c y c l o d e x t r i n , V = 7 - c y c l o d e x t r i n

advantageous but has not been systematically investigated, s In this report, we showed that the addition of cyclodextrins strongly increased production rates and yields of the conversion of cholesterol, sitosterol, and A4cholestenone to AD(D) by Mycobacterium sp. NRRLB 3683 side chain degradation. The addition of cyclodextrins hardly influenced cell growth rates and final cell densities, as was indicated by the measurements of generation times, cellular yields, and cell growth curves. Control experiments showed that comparable amounts of glucose, which is the monomeric unit of the cyclodextrins used in this study, had no effect on cell growth, reaction rates, or steroid solubilities. Therefore, the stimulating effect was most probably due to improvement of reaction conditions. As was clearly visible, the steroid solubilities were raised substantially by cyclodextrin addition, leading to a decreased formation of (more finely dispersed) precipitates. The increased solubility and substrate surface area resulted most probably in an enhanced transport of reactants to and from Mycobacterium cells. Cyclodextrins did not influence the onset or length of the period with high cellular side chain cleaving activity. Only a 1.7-3.0-fold increase of the maximum activity during the early exponential phase could be observed. This implied that the cells were transforming steroids beneath their maximum capacity due to transport limitations in the absence of cyclodextrins. These findings strengthened our view that cyclodextrins merely act as steroid solubilizers and carriers in biotransformations. For cholesterol transformation, /3-cyclodextrin turned out to be the most effective host, whereas 7cyclodextrin was optimal for the side chain cleavage of sitosterol and A4-cholestenone. These substrates differ in size, their dimensions varying from 0.38-0.52 × 0.62-0.78 × 1.89-2.06 nm. This implies that the cavity of fl-cyclodextrin is just wide enough for encapsulation of these substrates and that 7-cyclodextrin could be a

more appropriate host for the two larger substrates, M-cholestenone and sitosterol (Table 1). In contrast, the cavity diameter of a-cyclodextrin is expected to be too small for encapsulation and hence reaction rate improvement of all three substrates used. This was indeed found experimentally. An apparent optimal molar ratio of 2 moles of cyclodextrin per mole of substrate was found with all substrates tested. Addition of more cyclodextrin hardly resulted in an extra enhancement of transformation rates. This 2 : 1 stoichiometry of cyclodextrins to guest molecules is often observed, as are 1 : I and 3 : 2 ratios. Host : guest ratios of 1 : 2, 2 : 2, 3 : 1,4 : 1, and 5 : 2 have also been reported but are less frequently found. 6 For cholic acid and deoxycholic acid, a 1 : 1 complex with fl-cyclodextrin has been proposed.IS The most important structural differences of these bile acids from cholesterol include a cis-fusion of rings A and B, one or two additional hydroxyl groups, and the presence of a shortened charged side chain. The length of these molecules was determined to be ca 1.4-1.5 nm, which is substantially shorter than the 1.98 nm of cholesterol. 19 Based on the (deoxy-) cholic acid structure, the clathrating fl-cyclodextrin molecule is expected to surround the rings B (partly), C, D, and ca three atoms of the side chain, the carboxylic acid function extending in the aqueous environment. This implies that the two clathrating cyclodextrin molecules in the 2:1 complexes with cholesterol, sitosterol, and A4-choles tenone are most probably surrounding the rings A, B, C, and ring D plus side chain, respectively. As the "length" of such a steroid molecule is 1.9-2.1 nm and the " d e p t h " of a cyclodextrin molecule is 0.8 nm, almost the complete steroid molecule could be encapsulated by two cyclodextrins stacked upon one other. However, the exact stoichiometry of these cyclodextrin-steroid complexes has to be revealed by future research. The enhancement of the steroid side chain cleavage

Enzyme Microb. Technol., 1989, vol. 11, July

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Papers rates was not the only effect of cyclodextrin addition to bioconversions with Mycobacterium sp. NRRL-B 3683. The addition of the appropriate cyclodextrins also prevented nucleus breakdown and product inhibition, since all steroid substrates were converted to AD(D) with molar yield of 80-95% within 140 h of incubation. When no cyclodextrins were present, product inhibition and nucleus degradation occurred, resulting in molar conversions under 50%. Apparently, AD(D) became encapsulated by the cyclodextrin molecules and was unable to inhibit Mycobacterium cell growth to the same extent as in the absence of cyclodextrins, z° The formation of such AD(D)-cyclodextrin complexes with a-, /3-, or y-cyclodextrin is possible from a structural point of view, as the AD and ADD molecules are smaller than the substrates used in this study. In conclusion, cyclodextrins seem to act as inert steroid solubilizers, reactant carriers, and protecting agents in the steroid side chain cleavage by Mycobacterium sp. NRRL-B 3683 in purely aqueous media, not affecting the biocatalyst itself. The use of two-liquid phase systems with organic solvents and cell immobilization is not necessary. Because the described reactor system is very simple, product recovery is easily carried out and scaled up and the cyclodextrins can be recovered for reuse, this process could be economically attractive.

References 1 2 3 4 5 6 7 8

9 10 11

12 13 14 15 16 17

Acknowledgements We thank Dr. Dick Janssen and Marinus Suijkerbuijk for help with gas chromatography, Gijs Nagel and Dr. Bert Wolthers of the Groningen Academic Hospital for GC-MS, and Nico Panman for drawing the illustrations. This research was financed by TNO (Institute of Applied Chemistry, Zeist, The Netherlands).

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18 19 20

Lenz, G. R. in Kirk-Othmer Encyclopaedia o f Chemical Technology, 3rd ed., Vol. 21, pp. 645-729 Steinert, H.-J., Vorlop, K. D., and Klein, J. in Biocatalysis in Organic Media (Laane, C., Tramper, J. and Lilly, M. D., eds) Elsevier Science Publishers, Amsterdam, pp. 51-63 Martin, C. K. A. Adv. Appl. Microbiol. 1977, 22, 29-58 Atrat, P. Z. Allg. Mikrobiologie 1982, 22, 723-761 Saenger, W. in Inclusion Compounds, Vol. H (Atwood, J. L., Davies, J. E. D. and MacNicol, D. D., eds) Academic Press, London, pp. 231-259 Szejtli, J. in Inclusion Compounds, Vol. 111 (Atwood, J. L., Davies, J. E. D. and MacNicol, D. D., eds) Academic Press, London, pp. 331-390 Saenger, W. Angew. Chem. Int. Ed. Engl. 1980, 19, 344-362 Hesselink, P. G. M., De Vries, H. and Witholt, B. Proc. 4th European Congress on Biotechnology, Vol. 2 (Neijssel, O. M., Van der Meer, R. R. and Luyben, K. Ch. A. M., eds) Elsevier Science Publishers, Amsterdam, 1987, pp. 299-302 Vogel, H. J. and Bonner, D. M. Microbial Genet. Bull. 1965, 13, 43-44 Witholt, B. J. Bacteriol. 1972, 109, 350-364 Hesselink, P. G. M., Koops, K., Harkes, M. and Witholt, B. Proceedings 2nd Netherlands Biotechnology Congress (Breteler, H., Van Lelyveld, P. H. and Luyben, K. Ch. A. M., eds) Netherlands Biotechnological Society, 1988, pp. 417-419 Bernal, J. B., Crowfoot, D. and Fankuchen, I. Trans. Roy. Soc. (London) 1940, 239, 135-182 Crowfoot, D. Vitamins and Hormones 1944, 2, 409-461 Haberland, M. E. and Reynolds, J. A. Proc. Natl. Acad. Sci. USA 1973, 70, 2313-2316 Boeren, S. and Laane, C. Biotechnol. Bioeng. 1987, 29, 305309 Granot, I., Aharonowitz, Y. and Freeman, A. Appl. Microbiol. Biotechnol. 1988, 27, 457-463 Richter Gideon Vegyeszeti Gyar, R. T. and Chinoin Gyogyszeres Vegyeszeti Termekek Gyara, R. T. Brevet d'invention Belgique 1981, 894.501 Miyajima, K., Yokoi, M., Komatsu, H. and Nakagaki, M. Chem. Pharm. Bull. 1986, 34, 1395-1398 Giglio, E. in Inclusion Compounds, Vol. H (Atwood, J. L., Davies, J. E. D. and MacNicol, D. D., eds) Academic Press, London, pp. 207-229 Koops. K., Hesselink. P. G. M. and Witholt, B. Proceedin,~,s 2nd Netherlands Biotechnology Congress (Breteler, H., Van Lelyveld, P. H. and Luyben, K. Ch. A. M.) Netherlands Biotechnological Society, 1988, pp. 453-456