Microbial Technology || Microbial Transformation of Steroids and Sterols

Chapter 14

Microbial Transformationof Steroids and Sterols

o. K. SEBEKD. PERLMAN

I. IntroductionII. Commercial Development

III. Types of Microbial TransformationsIV. Side-Chain Degradation of Sterols

References

I. INTRODUCTION

483488492493495

Naturally occurring steroids possess remarkable hormonal propertieswhich are of critical importance to human well-being. They include hormones of the adrenal cortex (cortisone, cortisol, corticosterone), theprogestational hormone (progesterone), the androgens or male hormones (testosterone, dihydrotestosterone), and the female sex hormones(estradiol, estrone) (see Scheme 1).

They are all derived from a tetracyclic hydrocarbon (perhydrocyclopentanophenanthrene) and differ primarily in the number, type, andlocation of the substituent functional groups and in the number andposition of the double bonds (Scheme 2).

In the last 30 years, thousands of derivatives of these compounds havebeen synthesized and tested for their endocrine and other activities.Many of them were found to be active as antiinflammatory and progestational agents and also as sedatives and anabol ic and antitumor substances. Some are effective in allergic, dermatologic, and ocular diseases, in cardiovascular therapy, and in veterinary products, and some

483

MICROBIAL TECHNOLOGY, 2nd ed., VOL. ICopyright © 1979 by Academic Press, Inc.

All rights of reproduction in any form reserved. ISBN 0-12-551501-4

484 Ο. Κ. Sebek and D. Perlman

CH2OH ÇH2OH Ç H 3

Λ 5 * , 3 5 * Cortisone Cortisol (hydrocortisone) Progestorone

OH OH

Testosterone Estradiol

SCHEME 1 I8

i9 I 14J I5l

SCHEME 2

have been outstandingly successful as oral contraceptives (Applezweig, 1962, 1978).

The demonstration of such extraordinary activities had its origin in the discovery by Hench and his associates in 1949 of the remarkable an-tiinflammatory property of cortisone in rheumatoid arthritis (Hench et al., 1949, 1950).

In the wake of this discovery, the dramatic surge in the demand for cortisone to treat millions of arthritic sufferers went unmet since no adequate method for its manufacture existed. The chemical synthesis from deoxycholic acid (Sarett, 1946) developed at Merck and Company, was workable but complicated and uneconomical: 31 steps were needed to obtain 1 kg of cortisone acetate from 615 kg of deoxycholic acid. The oxygen shift from C-12 to C-11 alone, which is essential for the activity of cortisone (and of all adrenal corticosteroid hormones), re-quired nine steps* and is shown in Scheme 3.

Economic synthesis of cortisone, Cortisol, and other corticosteroids eventually developed from three lines of information: (1) diosgenin from

*The efficiency of this shift was subsequently improved and deoxycholic acid, obtained from beef bile, is at present a substrate in some manufacturing processes.

14. Microbial Transformation of Steroids and Sterols 485

H , C C H

2 °H

Deoxycholic acid Cort isone

SCHEME 3

the Mexican barbasco plant (Dioscorea composita) and stigmasterol from soybeans (Glycine max) were found abundant and inexpensive raw materials for chemical conversion to 16-dehydropregnenolone and pregnenolone, respectively; (2) deoxycorticosterone was hydroxylated to corticosterone by perfusion with isolated beef adrenal glands* (Hechter ei ai, 1949) (see Scheme 4); (3) yeasts and bacteria were reported to

C H 2O H CH2OH

Deoxycort icosterone Cort icosterone

SCHEME 4

carry out nuclear reduction, oxidation, isomerization, and hydrolysis of steroids (Mamoli and Vercellone, 1937a,b).

By applying this information, Peterson and Murray succeeded in 1952 in introducing the essential oxygen at C-11 by oxidizing progesterone to 11a-hydroxyprogesterone by means of first Rhizopus arrhizus and then by Rhizopus nigricans in practically quantitative yields (>85%) (Peterson and Murray, 1952; see also Murray and Peterson, 1953) (Scheme 5).

C H 3 C H 3

Progesterone 11a-Hydroxyprogesterone

SCHEME 5

Almost simultaneously, Streptomyces argenteolus was reported to oxidize progesterone to 16a-hydroxyprogesterone (Perlman et ai, 1952) (Scheme 6).

*This process was employed at one time by G. D. Searle and Company; IT produced the

first reasonably large quantities of CORTISOL for cl inical testing.


Ç H 3 Ç H 3

ç=o c=o

Progesterone 16a-Hydroxprogesterone

SCHEME 6

This reaction became of considerable importance 5 years later in the synthesis of 16a-hydroxy-9a-f luoroprednisolone (triamcinolone) which has greatly improved antiinflammatory activity.

Following these reports, the disclosures of other hydroxylations were made in a rapid sequence: Cunninghamella blakesleeana (Hanson et al., 1 9 5 3 ) and Curvularia lunata (Shull ef a/., 1 9 5 3 ; Shull and Kita, 1 9 5 5 ) were found to carry out 11 ß-hydroxylation at C -11 in good yields (about 6 0 % ) and the latter fungus is used in the manufacture of Cortisol from compound S (Scheme 7 ) .

CHpOH CH I I oo υΛ c=o

Compound S Cortisol

SCHEME 7

These observations with their highly practical applications stimulated an intensive search for additional microbial modifications of these com-pounds. In time, all available carbon atoms were found to be hy-droxylated by different organisms and by now the number of microbial hydroxylations exceeds the number of hydroxylations carried out by mammalian systems (Table I). In many cases, polyhydroxylations in different combinations were also noted with dihydroxylations (such as 6/3, 1 1 α ; 7 α , 1 4 a ; 9 a , 1 4 a ; or 11)3 ,21) prevailing. They are not, however, of practical importance. The oxygen atom involved in these reactions is derived from gaseous oxygen and not from water, and the hydroxyl group thus formed retains the same configuration as the hydrogen that has been replaced (Hayano et al., 1958a,b).

The therapeutic properties of cortisone and Cortisol were further im-proved by microbial introduction of a 1,2-double bond whereby pred-nisone (1-dehydrocortisone) and prednisolone (1-dehydrocortisol) were formed, respectively (for example, see Scheme 8 ) .

This 1-dehydrogenation was first described to be carried out by Cylin-


TABLE I. Comparison of Hydroxylation of Steroids by Microbial and Mammalian Systems

Microbial systems Mammalian systems

1α 1a

1/3 2a 2a 2/3 2/3 6a 3ß 6/3 5 a

a 7a

5ßb 11/3

6/3 12a 7a 15a 7 β 16a 9a 17a

10/3C 18

11a 19 11/3 21 12a 12/3 14a 15a 15/3 16a 16)8 17a 18 19 21

a In A-norprogesterone series.

b In cardiac glycosides.

c In 19-nortestosterone.

drocarpon radicicola and Streptomyces lavendulae (Fried ef a/., 1953), by Fusarium solani and Fusarium caucasicum (Vischer and Wettstein, 1953), and by Septomyxa affinis, which is also used in the preparation of 6a-methylprednisolone (Medrol), a valuable synthetic corticoid which does not cause significant sodium retention (Sebek and Spero, 1959; Spero ef al., 1956). These organisms also degrade the side chain,

ÇHgOH CH2OH

C O 0=0

Cortisol Prednisolone

SCHEME 8


oxidize the hydroxyl group at C-17, and bring about expansion of ring D of some steroids (such as progesterone and deoxycorticosterone) (see Scheme 9).

R I O O OH Ο

PROGESTERONE (R = CH3) 1-DEHYDROTESTOSTERONE 1,4-ANDROSTADIENEDIONE 1-DEHYDROTESTOLOLACTONE DEOXYCORTICOSTERONE

(R = CH 20H)

SCHEME 9

In contrast, Arthrobacter (Corynebacterium) simplex which was the first organism used commercially to 1-dehydrogenate C o r t i s o l to pred-nisolone (Nobile ei al., 1955) is devoid of these attendant reactions. The same is true of two other 1 -dehydrogenating bacteria (Bacillus sphaericus and Bacterium cyclooxydans) which have also been used in industrial practice.

A process called "pseudo-crystallofermentation" was also described which was reported to give high yields of the 1-dehydrogenated product. According to this procedure, A. simplex transforms finely powdered solid C o r t i s o l in 1-50% concentrations to crystalline prednisolone in high (>93%) yields directly in the fermentation beers within 5 days (Kondö and Masuo, 1961).

II. COMMERCIAL DEVELOPMENT

As these considerations indicate, microbial modifications were di-rectly responsible for the generation of new analogs, which in turn served in many cases as substrates for a still larger number of steroid products. They had also a significant effect on the cost of the desired hormones. Thus the 11 a-hydroxylation of progesterone brought the 1949 price of cortisone in a very short time from $200 to $6 per gram and, through further improvements, to less than $1 per gram at present.

To carry out these conversions on a large scale, the culture is grown in fermentation tanks with aeration and agitation. The steroid is dissolved in a suitable solvent and added at different growth stages, preferably toward the end of the growth phase, and the transformation is allowed to proceed under the same aerobic conditions to a maximum. Since ste-roids are essentially insoluble or sparingly soluble in water, it is of considerable importance that they react in aqueous suspensions and that the reactions are completed in most cases within a reasonable time.


Thus 11a-hydroxylation of 2 gm of progesterone per liter by R. nigricans proceeds satisfactorily in 4 8 hours. The product is released into the medium from which it is recovered by extraction with methylene chloride. With further development (use of surfactants, improved meth-ods of steroid addition) the substrate levels have been increased with the concomitant yield increases of the product. Solid substrate addi-tion also improved the efficiency of 11a-hydroxylat ion. Thus 2 g of finely ground aqueous suspension of progesterone with 0 . 0 1 % wetting agent (Tween 8 0 ) per 1 0 0 ml was converted by Aspergillus ochraceus to 1 1 a -hydroxyprogesterone in 9 0 % yields in 3 days with a minimum of a side-product (6/8, 11a-dihydroxyprogesterone, Weaver et al., 1 9 6 0 ) . A study of this hydroxylation indicated that 1 4 % of the substrate progesterone re-mained unconverted regardless of the concentrations at which it had been added. This was due to the formation of a crystalline structure consisting of 6 parts of the product and 1 part of progesterone, which thus became unavailable for the transformation. Continuous feeding of the substrate overcame this drawback. A similar substrate-product interaction was observed during 1-dehydrogenation by S. affinis of 11/3,21-dihydroxy-4, 17(20 ) -pregnadien -3 -one (dienediol) to 11 /3 ,21 -d ihydroxy -1 ,4 ,17(20) -pregnatrien-3-one (trienediol), an intermediary step in the synthesis of 6a-methylprednisolone (see above). This difficulty was overcome by mul-tistage feeding of the substrate (Maxon et al., 1 9 6 6 ) .

For illustration, some of the steroid transformations of commercial importance are listed in Table II. The fermentation conditions used in laboratory studies of these and related transformations are summarized in Table III (also see Perlman, 1 9 7 6 ) .

The complexity of industrial processes may be illustrated by the infor-mation in Fig. 1 , which outlines the conversion of stigmasterol to six steroids of clinical importance.

Twenty-one chemical and microbial steps are involved in the conver-sion of stigmasterol to oxylone, and 1 0 steps to prednisolone and to cortisone. By continuous refinement of the process operations as high as 1 0 mole-% conversions of the starting material to final product have been realized.

III. TYPES OF MICROBIAL TRANSFORMATIONS

In addition to the six reactions of commercial importance (see Table II), many other microbial transformations have been discovered. They are listed in Table IV and include introduction of nuclear and side-chain hydroxy groups; cleavage of carbon-to-carbon linkages in the side chain as well as in ring D; introduction of a 1,2-double bond in ring A; oxidation

~CDo

TABLE II. Some Steroid Transformations of Commercial Importance

Reaction

11a-Hydroxylation11~-Hydroxylation16a-Hydroxylation

1-Dehydrogenation

1-Dehydrogenation,side-chain cleavage,and ring 0 expansion

Side-chain cleavace

Substrate ~ product

Progesterone ~ 11a-hydroxyprogesteroneCompound S~ cortisol9a-Fluorocortisol ~ 9a-fluoro-

16a-hydroxycortisolCortisol ~ prednisoloneDienediolv-» trienediol"Progesterone ~ 1-dehydrotestololactone

~-Sitosterol~ androstadienedioneand/or and rostened ione

Microorganism

Rhizopus nigricansCurvularia lunataStreptomyces roseochromogenus

Arthrobacter simplexSeptomyxa affinisCylindrocarpon radicicola

Mycobacterium spp.

Some industrial producers

The Upjohn CompanyPfizer, Inc.; Gist-BrocadesE.R. Squibb and Sons;

Lederle LaboratoriesSchering CorporationThe Upjohn CompanyE.R. Squibb and Sons

G.D. Searle and Company

a See text for chemical names.

TABLE III. Media and Fermentation Conditions Used in Microbial Transformations of Steroids

Steroid product(approx. yields

Microorganism Steroid substrate wt. 0/0)

Alcaligenes faecalis Cholic acid Ketocholic acids(90-1000/0)

Corynebacterium 21-Acetoxy-3 f3- 21-Hydroxy-4-mediolanum hydroxy-5- pregnene-3,20-

preg nen-20-one dione (30%)Cunninghame//a Compound S Cortisone (190/0),

blakesleeana cortisol (65%)(H334)

Cylindrocarpon Progesterone 1-0ehydrotestolo-radicicola lactone (50%)(ATCC 11011)

Fusarium solani Progesterone 1,4-Androstadiene-3,17-dione (85%)

Rhizopus arrhizus 4-Androstene-3, 17- 11a-Hydroxy-4-ATCC 11145 dione androstene-3, 17-

dione (25%)Streptomyces Estradiol Estrone (90-950/0)

albus

Streptomyces Progesterone 15a-Hydroxy-4-aureus preg nene-3, 20-

dione (11%)

Compositionof medium"

A

B

C

o

E

F

G

H

Length of incubation,temperature, aeration

2 days (monoketo acid);4 days (diketo acid);6 days (triketo acid);37-39°; surface culture

6 days, 36-37°C, pureoxygen with agitation

3 days, 28°C, rotaryshaker (250 rpm)

3 days, 25°C, reciprocating shaker (120 spm)

4 days 25°C, rotaryshaker (100 rpm)

4 days, 28°C smallaerated tank (6-7 mM02/liter/min)

6 hours of substrateoxidation with restingcells, 30°C

3 days, 25°C, rotaryshaker (280 rpm)

Reference

Schmidt and Hughes(1944)

Mamoli, (1944)

O'Connell, et al.(1955)

Fried and Thoma,(1956)

Vischer and Wettstein(1953)Murray and Peterson(1953)

Heusghem and Welch(1948)

Fried et al. (1956)

a Composition of the media: (A) 4.7 gm (NH4)2S04, 0.5 gm asparagine, 5 gm NaCI, 0.65 gm NaOH, 2 gm glycerol, inorganic salts, 5 gm cholicacid, distilled water to 1 liter; (8) 60 ml yeast water, 10 ml of 0.2 M Na2HP04, 1 gm KH2PO, 0.2 gm 21-acetoxy-3-hydroxypregnan-20-one; (C) 3 gmNaN03, 0.5 gm KCI, 30 gm dextrin, 0.01 gm FeS04 • 7H20, 10 mg Tween 80,0.5 gm MgS04' 7H20, 1,3 gm K2HP04·3H 20, distilled water to 1 liter, pH7.2; (0) 3 9 corn steep solids, 3 gm NH4H2P04, 2.5 gm CaC03 , 2.2 gm soybean oil, 0.5 gm progesterone, distilled water to 1 liter, pH 7.0; (E) 15 gm

~ peptone, 6 ml corn steep liquor, 50 gm glucose, distilled water to 1 liter, pH 6.0, progesterone (0.25 gm) added after 2 days of incubation; (F) 20 gmlactalbumin digest, 5 ml corn steep liquor, 50 gm glucose, tap water to 1 liter, pH 5.5-5.9; androstenedione (0.25 gm) added after 27 hours ofincubation; (G) nutrient broth (to grow cells), phosphate buffer (pH 7.0) (to oxidize estradiol); and, (H) 2.2 gm soybean oil, 15 gm soybean meal, 10gm glucose, 2.5 gm CaC0 3, 0.25 gm progesterone, water to 1 liter


X Ç H 3

c=o I I I 4 C h e m i c al s teps . I I I

Kotr * oCCr S t i g m a s t e r ol P r o g e s t e r o ne

1 M i c r o b i o l o g i c al and 2 M i c r o b i o l o g i c al a nd 2 M i c r o b i o l o g i c al

4 c h e m i c al s t e ps 4 c h e m i c al s t e ps a nd 9 c h e m i c al s t e ps

C H 2O H C H 2 O H C H 2O H

C H 3

C o r t i s ol P r e d n i s o l o ne M e d r ol

I 1 C h e m i c al s tep I 1 C h e m i c al s tep 6

stepT'™' I

* Ç H 20 H * ÇH 2OH Φ

(JH3

0 CH3

C o r t i s o ne P r e d n i s o ne O x v l o ne

FIGURE 1 . Conversion of stigmasterol to six cl inical ly important steroids.

of hydroxyl groups to ketones; reduction of carbonyl groups; and hy-drolysis and formation of esters.

The methods generally used in screening microorganisms for ability to transform steroids are: (1) the microorganism is first grown in suitable media for 1 or 2 days; (2) the steroid, dissolved in a water-soluble solvent (e.g., dimethylformamide, acetone, propylene glycol), is then added; (3) incubation is continued for 1 or 5 days; (4) the transformation products are obtained by extraction of the beer with methylene chloride or nonpo-lar solvents (chloroform, ethyl acetate) and by purification by column chromatography and other methods; (5) the structure of the transforma-tion products is determined by classical methods of organic chemistry. Most of the fungi and streptomycetes examined have been found to transform some steroids, and sufficient information has accumu-lated to predict the type of transformation that might be expected if the genus and species of the culture is known or, conversely, to predict the identity of the microorganism knowing the transformation (Öapek ef a/., 1966, 1976; Charneyand Herzog, 1968, 1980; Vézina and Rakhit, 1974).

The transformations can be performed by cells at different stages of growth. Thus the steroid may be added to: (1) the growing culture (either at the time of inoculation or toward the end of the growth phase); (2) the

14. Microbial Transformation of Steroids and Sterols

TABLE IV. Types of Microbial Transformation of Steroids

493

A. Oxidation

1. Conversion of secondary alcohol to ketone 2. Introduction of primary hydroxyl on steroid side chain 3. Introduction of secondary hydroxyl on steroid nucleus 4. Introduction of tertiary hydroxyl on steroid nucleus 5. Dehydrogenations of ring A of steroid nucleus in positions 1(2) and 4(5) 6. Aromatization of ring A of the steroid nucleus

7. Oxidation of the methylene group to ketone group 8. Cleavage of side chain of pregnane at C-17 to form ketone 9. Cleavage of side chain of pregnane at C-17 and opening of D ring to form

testololactone 10. Cleavage of side chain of steroids to form carboxyl group 11. Cleavage of side chain of pregnane steroids at C-17 to form secondary alcohol 12. Formation of epoxides 13. Decarboxylation of acids

B. Reduction 1. Reduction of ketone to secondary alcohol

2. Reduction of aldehyde to primary alcohol 3. Hydrogénation of double bond at position 1(2) of ring A 4. Hydrogénation of double bond at positions 4(5) of ring A and at 5(6) of ring Β 5. Elimination of secondary alcohol 6. Formation of homosteroids of the androstane series from pregnane derivatives

C. Hydrolysis 1. Saponification of steroid esters 1. Acetylation

D. Esterification

nonproliferating ("resting") cell suspensions (or cell-free extracts); (3) microbial systems which carry out simultaneously other reactions as the major fermentation operations; (4) the spore suspensions; or (5) the immobilized whole cells (and cell-free enzymes). The last two methods are receiving considerable attention (Abbott, 1976; Vézina et al., 1968) but have not been used on a commercial scale.

IV. SIDE-CHAIN DEGRADATION OF STEROLS

In the last 15 years, the degradation of the side chain of cholesterol and of related plant sterols by microorganisms has been investigated extensively, and the results of these studies have become of considera-ble economic importance (Martin, 1977). The first data of this kind by Turfitt in 1948 reported on the conversion of cholestenone and bile acids to small amounts of 3-keto-4-androstene-17-carboxylic acid (Turfitt, 1948) and by Whitmarsh in 1964 on the degradation of cholesterol into a mixture of 3-ketobisnor-4-cholenic acid, 3-ketobisnor-1,4-choladienic


acid, 4-androstene-3,17-dione, and 1,4-androstadiene-3,17-dione in the presence of small amounts of 8-hydroxyquinoline (Whitmarsh, 1964). By this approach, other compounds were found which prevent the degrada-tion of the steroid nucleus while allowing the degradation of the choles-terol side chain (Nagasawa ei al., 1969, 1970): iron- or copper-chelating agents (such as quinoline-2-carboxylic acid, 2,2'-bipyridine, 1,10-phenanthroline); divalent ions that replace iron or block -SH functions (N i

2 +, Co

2 +, Pb

2 +, S e 0 3

2- , As0 2) ; or redox dyes. Sih and his collaborators

showed that the degradation of the sterol nucleus can be blocked also by the chemical modification of the substrate cholesterol at C-19 (19-hydroxy-, 19-nor-, 6/3, 19-oxidosterols). The same workers also estab-lished that the cholesterol side chain is degraded to the C-17 ketosteroid by a stepwise ß-oxidation via the intermediary C-24 and C-22 carboxylic acids (Sih ei al., 1968) as shown in Scheme 10.

γ ^ γ Y ^C

° O H YC O O H ο

C27 C24~ACID C 2 2- A C I D C

I 7

+ CH3-OH2-COOH +CH3COOH + CH3CH2COOH

SCHEME 10

When these inhibitors are not present or when the substrate choles-terol is not chemically modified, most of the cholesterol-utilizing micro-organisms degrade the nucleus as well. Mycobacteria (and pseu-domonads) metabolize it through the sequence shown in Scheme 11.

With this information, the problem of accumulating the desired prod-

Cholesterol 3/3-Hydroxy-5-androsten-l7-one Androstenedione

Androstadienedione 9a-Hydroxyandrosta- 9,10-seco-1,3,5(10)-Androstatriene-dienedione 9,17-dione

SCHEME 11


ucts 4-androstene-3,17-dione (androstenedione) and 1,4-androstadi-ene-3,17-dione (androstadienedione) from cholesterol and re-lated plant sterols in the absence of inhibitors or chemically modified substrates has been solved. Marsheck and his collaborators (1972) prepared a mutant of a soil mycobacterium which converted cholesterol, stigmasterol, and sitosterols to androstadienedione. By additional uv irradiation, a new mutant was selected which accumulated andro-stenedione as the main product. Both of these compounds (andro-stadienedione and androstenedione) are convenient substrates for the synthesis of other steroids. This discovery is of signal practical impor-tance. It has made soy sterols (especially ^-sitosterol and campesterol) commercially attractive new steroid substrates, since they are readily available in large quantities from soybeans and tall oil (waste product of the pulpwood kraft process; Conner ei a/., 1976).

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Microbial Technology || Microbial Transformation of Steroids and Sterols