Entrapment of mycelial fragments in calcium alginate: A general technique for the use of immobilized filamentous fungi in biocatalysis

Article abstract of DOI:10.1016/j.steroids.2011.10.008

Transformation reactions on 3β,17β-dihydroxyandrost-5-ene using free fungal cells were compared with those carried out by macerated mycelia, immobilized in calcium alginate beads. Six fungi were utilized in this study, namely Rhizopus oryzae ATCC 11145, Mucor plumbeus ATCC 4740, Cunninghamella echinulata var. elegans ATCC 8688a, Aspergillus niger ATCC 9142, Phanerochaete chrysosporium ATCC 24725 and Whetzelinia sclerotiorum ATCC 18687. The results show, for the first time, that encapsulated mycelial fragments essentially carry out the same bioconversions as those observed with growing cells. As the immobilized cells were "resting", the products formed were free of contamination by natural products, and this greatly aided the purification of the metabolites. Conditions for bead preparation were optimized. Furthermore, it was noted that the beads could be reused, once they had been subjected to a rejuvenation process.

Full text of DOI:10.1016/j.steroids.2011.10.008

Steroids 77 (2012) 85–90
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Entrapment of mycelial fragments in calcium alginate: A general technique
for the use of immobilized filamentous fungi in biocatalysis
Patrice C. Peart a, Avril R.M. Chen a, William F. Reynolds b, Paul B. Reese a,
⇑
a Department of Chemistry, University of the West Indies, Mona, Kingston 7, Jamaica
b Department of Chemistry, University of Toronto, Toronto, Ontario, Canada M5S 3H6
a r t i c l e i n f o
a b s t r a c t
Article history:
Transformation reactions on 3b,17b-dihydroxyandrost-5-ene using free fungal cells were compared with
those carried out by macerated mycelia, immobilized in calcium alginate beads. Six fungi were utilized in
this study, namely Rhizopus oryzae ATCC 11145, Mucor plumbeus ATCC 4740, Cunninghamella echinulata
var. elegans ATCC 8688a, Aspergillus niger ATCC 9142, Phanerochaete chrysosporium ATCC 24725 and
Whetzelinia sclerotiorum ATCC 18687. The results show, for the first time, that encapsulated mycelial frag-
ments essentially carry out the same bioconversions as those observed with growing cells. As the immo-
bilized cells were ‘‘resting’’, the products formed were free of contamination by natural products, and this
greatly aided the purification of the metabolites. Conditions for bead preparation were optimized. Fur-
thermore, it was noted that the beads could be reused, once they had been subjected to a rejuvenation
process.
Received 22 August 2011
Received in revised form 24 September
2011
Accepted 19 October 2011
Available online 28 October 2011
Keywords:
Filamentous fungus
Calcium alginate
Immobilization
Hydroxylation
Biotransformation
Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction
polysaccharide, resulting in spontaneous polymerization to form
spherical pellets. These mild conditions allow for higher cell viabil-
There is increasing interest in the immobilization of whole cells
of microorganisms. This thrust is partly fuelled by the desire to
replace the usage of isolated enzymes in industrial and laboratory
processes. Unfortunately, most of the work on cell entrapment has
been performed using bacteria and yeasts. These microorganisms
are unicellular which makes immobilization of their whole cells
relatively simple. However, the majority of fungi form filamentous
strands and, therefore, are not so easily immobilized using conven-
tional methods. Nevertheless, there have been many modifications
of the different methods to make them applicable to the filamen-
tous fungi [1].
Entrapment is one such technique that is widely used. Polyacryl-
amide and calcium alginate gels have been employed for this tech-
nique. In the first method cells are entrapped by the polymerization
of an aqueous solution of acrylamide monomers in which the fungal
cells were suspended, followed by subdivision into pellets. The
process can result in loss of cell activity. Studies have shown that
calcium alginate gives a higher retention of cell activity when com-
pared with polyacrylamide [1]. Fungi are trapped by dripping a sus-
pension of fungal spores in aqueous sodium alginate into a stirred
solution of chilled calcium chloride. The monovalent sodium ion
is replaced by the divalent calcium ion, which cross-links the
ity. Furthermore, these pellets are well suited for usage in commer-
cial reactors [2].
Yeast cells and the spores of several fungi have been entrapped in
alginate. Using this method Baker’s yeast, Saccharomyces cerevisiae,
has been employed in the asymmetric reduction of ketones [3–9]. In
some cases the fermentation was carried out in the presence of or-
ganic solvents, for example, hexane [5,10]. More recently immobi-
lized cells of S. cerevisiae have been used in the production of
ethanol from sucrose [11]. Spores of the filamentous fungi Rhizopus
stolonifer [12] and Curvularia lunata [13], trapped in a calcium algi-
nate matrix, were used in the bioconversion of progesterone. Freshly
germinated spores of Penicillium digitatum, another filamentous fun-
gus, enmeshed in alginate, have found utility in the transformation
of limonene to a-terpineol [14]. In a rare case Mortierella isabellina
mycelium has also been immobilized in calcium alginate and used
to transform dehydroabietic acid [15]. Normally the fungus is grown
for few days and the spores are harvested and trapped in the alginate
gel (and possibly allowed to germinate in situ) [16,17]. In some cases
the gel network affected the growth of the mycelia.
There is only one previous report where homogenization was
used in the preparation of entrapped cells in alginate [18]. It was
unclear whether this was done using spores or mycelia. Addition-
ally, the immobilized cells were dried and used for soil treatment.
This is the first report where macerated fungal cells are immobi-
lized and used for fermentation purposes.
⇑
Corresponding author. Tel.: +1 876 927 1910; fax: +1 876 977 1835.
E-mail address: paul.reese@uwimona.edu.jm (P.B. Reese).
0039-128X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2011.10.008

86
P.C. Peart et al. / Steroids 77 (2012) 85–90
extracted with ethyl acetate (2 Â 500 mL). The mycelia were placed
2. Experimental
2.1. General procedure
in ethyl acetate (500 mL) and homogenized, warmed, and then fil-
tered. The organic extracts were dried using sodium sulfate. The sol-
vent was removed in vacuo, and the residues were analyzed by thin
layer chromatography. The extracts were combined and absorbed
on silica gel and purified by flash column chromatography or by pre-
parative thin layer chromatography. After isolation of the untrans-
formed substrate (2), the mixture of products of biotransformation
was acetylated (acetic anhydride-pyridine) as an aid to purification.
Melting points were obtained using a Reichert Hot Stage melting
point apparatus and are uncorrected. Infrared data was acquired
using a Perkin Elmer Fourier transform infrared spectrophotometer
1000 using sodium chloride disks. NMR spectra were obtained on
Bruker Avance 200, Bruker Avance 500 and Varian Unity 500 spec-
trometers. Compounds were analyzed using CDCl3, unless other-
wise stated, with tetramethylsilane as the internal standard.
Optical rotations were performed using a Perkin Elmer 241 MC
polarimeter and solutions were prepared in CH2Cl2, unless other-
wise stated. Purifications were done by column chromatography
using silica gel (230–400 mesh) as the stationary phase. Addition-
ally, preparative thin layer chromatography (PLC) glass backed
2.3. Preparation of immobilized fungal cells
One slant was used to inoculate four Erlenmeyer flasks each
containing 125 mL liquid culture medium. The fungus was allowed
to grow for 3 d. At the end of the incubation period the cells were
harvested by filtration. The cells were suspended in water (40 mL)
and 3% aqueous sodium alginate solution (140 mL) was added. The
cells were macerated for 1 min at 8000 rpm using an IKA Ultra-
Turrax T25 homogenizer. The cell-alginate suspension was then
added dropwise to stirred chilled aqueous 0.1 M calcium chloride
(200 mL). The alginate beads formed were allowed to harden for
30 min in the calcium chloride solution. The calcium chloride solu-
tion was decanted, the beads were rinsed with water and were
stored under water at 4° until used.
plates with silica gel (60 Å,250 lm and 1000 lm thicknesses) were
used. Thin layer chromatographic (TLC) analyses were carried out
using polyester backed plates. Both the TLC and PLC plates were
visualized under ultraviolet light or by spraying with ammonium
molybdate-sulfuric acid or methanol-sulfuric acid reagents. After
spraying the plates were warmed for colour development using a
heat gun. Petrol refers to the petroleum fraction boiling between
60° and 80°. 3b-Hydroxyandrost-5-en-17-one (dehydroepiandros-
terone, DHEA) (1) was obtained from Productos Químicos
Naturales, S.A. de C.V. (Orizaba, Mexico). 3b,17b-Dihydroxyand-
rost-5-ene (2) was prepared from 1 in methanol using sodium
borohydride. The fungi used for these experiments were obtained
from the American Type Culture Collection, Rockville, MD, USA.
2.4. Immobilized cell fermentation conditions
The alginate beads derived above were divided into equal por-
tions and placed into four 500 mL Erlenmeyer flasks, each contain-
ing water (125 mL). The substrate (200 mg) in ethanol (5 mL) was
distributed among the flasks. The flasks were shaken at 180 rpm
for 5 d. After the fermentation was complete the aqueous solution
was decanted from the beads and the former was extracted using
ethyl acetate (2 Â 300 mL). The organic solution was dried using
sodium sulfate, filtered, and the solvent was removed in vacuo.
The residue was analyzed by TLC and purified by column chroma-
tography. After isolation of the untransformed substrate (2), the
mixture of products of biotransformation was acetylated (acetic
anhydride-pyridine) as an aid to purification. The used beads were
covered with distilled water and stored at 4°.
R3
R2
H
H
R1
H
H
H
H
R1
R2
O
7 R1=H2, R2=OH
7a R1=H2, R2=OAc
8 R1=O, R2=OH
8a R1=O, R2=OAc
1 R1=OH, R2=H2, R3=O
2 R1=OH, R2=H2, R3=βOH,αH
3 R1=OH, R2=αOH,βH, R3=βOH,αH
3a R1=OAc, R2=αOAc,βH, R3=βOAc,αH
4 R1=OH, R2=βOH,αH, R3=βOH,αH
4a R1=OAc, R2=βOAc,αH, R3=βOAc,αH
5 R1=OH, R2=βOH,αH, R3=O
2.5. Rhizopus oryzae ATCC 11145
R3
This fungus was maintained on malt agar slants at 28°. Five
slants were used to inoculate twenty 500 mL Erlenmeyer flasks,
each containing 125 mL liquid culture. The medium was prepared
from glucose (20 g/L), peptone (5 g/L), sodium chloride (5 g/L) and
yeast extract (5 g/L) [19]. The flasks were shaken at 250 rpm.
H
5a R1=OAc, R2=βOAc,αH, R3=O
6 R1=OH, R2=αOH,βH, R3=O
H
H
R1
OH
6a R1=OAc, R2=αOAc,βH, R3=O
R2
2.5.1. Free cell fermentation
9 R1=R2=R3=OH
The combined extract (2.08 g) was purified using column chro-
matography. Elution with ethyl acetate/petrol (1:19 v/v) yielded
fed substrate 2 (28 mg). Further elution using ethyl acetate/petrol
9a R1=R2=R3=OAc
(1:9 v/v) afforded 3b,7a,17b-trihydroxyandrost-5-ene (3). This
compound was characterized as the triacetate (3a) (77 mg),
2.2. Free cell fermentation conditions
Rf = 0.89, acetone/dichloromethane (1:19 v/v), which crystallized
from acetone/methanol as plates, m.p. 142–144°, [
(c 0.22), lit. [20] 156–158°, [ À152°; IR: mmax 1747, 1723,
1246 cmÀ1 1H NMR: d 0.80 (3H,s,H-18), 1.03 (3H,s,H-19), 2.04
a
]
À176.8°
D
One slant was used to inoculate four 500 mL Erlenmeyer flasks,
each containing 125 mL of liquid culture medium. The substrate
(2) (1 g) in ethanol (20 mL) was distributed among the flasks, in
the following way. A solution of 10% of the total mass of the sub-
strate was fed 24 h after inoculation. Then 20%, 30% and 40% of the
substrate in ethanol were fed at 36, 48 and 60 h after inoculation
respectively. The fermentation was allowed to proceed for an addi-
tional 5 d after the final feed. The mycelia were filtered from the
broth. The broth was divided into two portions. Each portion was
a]
D
;
(9H,s,3 Â CH3CO2),
(1H,dd,J = 9.8 Hz, H-3
(1H,d,J = 5 Hz,H-6).
4.68
),
(1H,m,w/2 = 12 Hz,H-17
4.98 (1H,t,J = 4.5 Hz,H-7b),
a
),
4.69
5.59
a
Further elution with ethyl acetate/petrol (1:9 v/v) gave
3b,7b,17b-trihydroxyandrost-5-ene (4). This compound was charac
terized as the triacetate 4a (38 mg), Rf = 0.88, acetone/dichloro-
methane (1:19 v/v), which crystallized as needles from

P.C. Peart et al. / Steroids 77 (2012) 85–90
87
acetone-methanol, m.p. 178–179°, [
a
]
D +42.9° (c 0.15), lit. [21] m.p.
prepared using glucose (20 g/L), yeast extract (5 g/L), soya meal
(5 g/L), sodium chloride (5 g/L) and di-potassium hydrogen phos-
phate (5 g/L) [24]. The flasks were shaken at 180 rpm.
210–211°, [ ]D +52°; IR:
a
m
max 2947, 1739, 1733, 1370, 1238 cmÀ1; 1H
NMR: d 0.79 (3H,s,H-18), 1.05 (3H,s,H-19), 2.10 (9H,s,3 Â CH3CO2),
4.60 (2H,d,J = 8.1 Hz,H-3
5.37 (1H,s,H-6).
a,H-17a), 5.08 (1H,d,J = 8.2 Hz, H-7b),
2.7.1. Free cell fermentation
The mycelia and broth extracts were combined (2.04 g) and par-
tially purified by column chromatography to give the fed com-
pound 2 (469 mg). Acetylation and further purification gave two
products, 3a (18 mg) and 4a (12 mg).
2.5.2. Immobilized cell fermentation
The extract (147.5 mg) was partially purified by column
chromatography to give the fed substrate (2) (12.3 mg). Further
purification using ethyl acetate/petrol (1:9 v/v) afforded
3b,7a,17b-trihydroxyandrost-5-ene (3), which was characterized
as the triacetate (3a) (36.9 mg).
2.7.2. Immobilized cell fermentation
The broth extract (170 mg) was partially purified by column
chromatography to give the fed compound 2 (24.5 mg). Acetylation
and further purifications yielded two metabolites, 3a (23.6 mg)
and 4a (16.9 mg).
Further elution with ethyl acetate/petrol (1:9 v/v) gave
3b,7b,17b-trihydroxyandrost-5-ene (4) which was characterized
as the triacetate 4a (65.7 mg).
Elution with ethyl acetate/petrol (3:17 v/v) afforded a third
metabolite, 3b,7b-dihydroxyandrost-5-en-17-one (5) (10.3 mg),
which was characterized as the diacetate 5a, and which did not
2.8. Aspergillus niger ATCC 9142
crystallize, Rf = 0.76, acetone/dichloromethane (1:19 v/v), [
+200° (c 0.09), lit. [22] m.p. 164–165°, [ +105°; IR: mmax 2947,
1736, 1373, 1241, 1032 cmÀ1 1H NMR: d 0.91 (3H,s,H-18), 1.12
(3H,s,H-19), 2.04 (3H,s,CH3CO2-3), 2.06 (3H,s,CH3CO2-7), 4.61
a]
D
This fungus was maintained on potato dextrose agar slants at
28°. Five slants were used to inoculate twenty 500 mL Erlenmeyer
flasks, each containing 125 mL of liquid culture. The medium was
prepared using glucose (20 g/L), yeast extract (5 g/L), soya meal
(5 g/L), sodium chloride (5 g/L) and di-potassium hydrogen phos-
phate (5 g/L) [24]. The flasks were shaken at 180 rpm.
a]
D
;
(1H,m,w/2 = 17.5 Hz,H-3
(1H,t,J = 2 Hz,H-6).
a), 5.17 (1H,dt,J = 8.7,2.1 Hz,H-7a), 5.29
2.6. Mucor plumbeus ATCC 4740
2.8.1. Free cell fermentation
The combined extracts (1.22 g) were purified using column
chromatography. Elution with ethyl acetate/petrol (1:19 v/v)
afforded 254 mg of the fed compound (2). After acetylation, further
purification gave three transformed products. Compound 4a
(10 mg) was identified by comparison of its spectral data with that
of an authentic sample.
Elution with ethyl acetate/petrol (1:4 v/v) afforded a second
metabolite, 17b-hydroxyandrost-4-en-3-one (7), which was char-
acterized as the acetate 7a (17 mg), Rf = 0.14, acetone/dichloro-
methane (1:19 v/v), which crystallized from acetone as needles,
This fungus was maintained on potato dextrose agar slants at
28°. Five slants were used to inoculate twenty 500 mL Erlenmeyer
flasks, each containing 125 mL liquid culture medium. The medium
was prepared using glucose (30 g/L), potassium chloride (0.5 g/L),
corn steep solids (5 g/L), sodium nitrate (2 g/L), magnesium sulfate
heptahydrate (0.5 g/L) and iron(II) sulfate heptahydrate (0.02 g/L)
[23]. The flasks were shaken at 250 rpm.
2.6.1. Free cell fermentation
The extracts were purified separately using column chromatog-
raphy. Purification of the mycelial extract (746 mg) gave only the
fed steroid (2) (109.6 mg), which was collected in the fractions
eluted with ethyl acetate/petrol (1:19 v/v). The broth extract
(626 mg) afforded two transformed products. Elution with ethyl
m.p. 130–132°, [
+96.2°; IR: mmax 2932, 1737, 1675, 1247 cmÀ1
(3H,s,H-18), 1.20 (3H,s,H-19), 2.05 (3H,s,CH3CO2-17), 4.60
(1H,dd,J = 7.7,8.8 Hz,H-17 ), 5.74 (1H,s,H-4).
a]
+92° (c 0.01), lit. [25] m.p. 141–142°, [a]
D
D
;
1H NMR: d 0.84
a
Further elution with ethyl acetate/petrol (3:7 v/v) gave a third
compound, 17b-hydroxyandrost-4-ene-3,16-dione (8), which was
characterized as the monoacetate 8a (104 mg), Rf = 0.62, acetone/
dichloromethane (1:19 v/v), which crystallized from acetone-
acetate/petrol (1:9 v/v) gave 3b,7a,17b-trihydroxyandrost-5-ene
(3), which was characterized as the triacetate 3a (148 mg).
Further elution with ethyl acetate/petrol (3:17 v/v) yielded a
second metabolite, 3b,7
was characterized as the diacetate 6a (52 mg), Rf = 0.81, acetone/
dichloromethane (1:19 v/v), which resisted crystallization, [
À228° (c 0.21), lit. [22] m.p. 168–170°, [ À178°; IR: mmax 1722,
1622, 1228 cmÀ1 1H NMR: d 0.88 (3H,s,H-18), 1.05 (3H,s,H-19),
), 5.12
a-dihydroxyandrost-5-en-17-one (6). This
ethanol as plates, m.p. 202–204°, [
a
]
D À43.4° (c 0.15), lit. [26] m.p.
200–201.5°, [
a
]
D
À48°; IR: mmax 2939, 1742, 1672, 1234 cmÀ1
;
1H
a]
D
NMR: d 0.88 (3H,s,H-18), 1.22 (3H,s,H-19), 2.18 (3H,s,CH3CO2-
a]
D
17), 5.02 (1H,s,H-17a), 5.76 (1H,bs,H-4).
;
2.05 (6H,s,2 Â CH3CO2), 4.68 (1H,m,w/2 = 16.5 Hz,H-3
a
2.8.2. Immobilized cell fermentation
(1H,dt,J = 8.7,2.1 Hz,H-7b), 5.62 (1H,dd,J = 5.3,1.5 Hz,H-6).
The broth extract (161.8 mg) was purified using column chro-
matography. Elution with ethyl acetate/petrol (1:19 v/v) afforded
133 mg of the fed compound (2). Acetylation and further purifica-
tion gave three transformed products, namely, 4a (5 mg), 7a
(3 mg), and 8a (4 mg).
2.6.2. Immobilized cell fermentation
Partial purification of the broth extract (172 mg) allowed for the
recovery of the fed substrate (2) (30.5 mg). Further percolation using
ethyl acetate/petrol (1:9 v/v) gave 3b,7
ene (3) (37.4 mg), which was characterized as the triacetate 3a.
Further elution with ethyl acetate/petrol (3:17 v/v) yielded 3b,7
a,17b-trihydroxyandrost-5-
a
-
2.9. Phanerochaete chrysosporium ATCC 24725
dihydroxyandrost-5-en-17-one (6), characterized as the diacetate
6a (53.8 mg).
This fungus was maintained on potato dextrose agar slants at
28°. Five slants were used to inoculate twenty 500 mL Erlenmeyer
flasks each containing 125 mL of liquid culture. The medium was
made using potassium nitrate (10 g/L), magnesium sulfate hepta-
hydrate (1.5 g/L), potassium di-hydrogen phosphate (2.5 g/L), glu-
cose (40 g/L) and yeast extract (2 g/L) [27]. The flasks were
shaken at 180 rpm.
2.7. Cunninghamella echinulata var. elegans ATCC 8688a
This fungus was maintained on maltose-peptone slants at 28°.
Five slants were used to inoculate twenty 500 mL Erlenmeyer
flasks, each containing 125 mL of liquid culture. The medium was

88
P.C. Peart et al. / Steroids 77 (2012) 85–90
The fermentation of 3b,17b-dihydroxyandrost-5-ene (2) by
indicated that the corresponding proton showed correlations to
protons at H-6 and H-8, and as such the position of hydroxylation
was determined to be C-7. The proton at C-7 showed T-ROESY cor-
relations to protons at H-8 and H-16b. The transformed product
P. chrysosporium produced several metabolites, however, the
quantities were insufficient to allow for their isolation and
characterization.
was, therefore, determined to be 3b,7a,17b-trihydroxyandrost-5-
ene (3), which was characterized as the triacetate (3a) [19]. The
NMR data of the triacetate of the second metabolite (4a) was quite
similar to that of 3a. Loss of the methylene at d 31.4, C-7, was
accompanied by the appearance of a new methine at d 75.3. The
metabolite was determined to be 3b,7b,17b-trihydroxyandrost-5-
ene (4), which was characterized as the triacetate (4a) [21].
2.10. Whetzelinia sclerotiorum ATCC 18687
This fungus was maintained on potato dextrose agar slants at
28°. Five slants were used to inoculate twenty 500 mL Erlenmeyer
flasks, each containing 125 mL of liquid culture. The medium was
made using potassium nitrate (10 g/L), magnesium sulfate hepta-
hydrate (1.5 g/L), potassium dihydrogen phosphate (2.5 g/L), glu-
cose (0.5 g/L), yeast extract (0.5 g/L) and cellulose (10 g/L) [28].
The flasks were shaken at 180 rpm.
3.1.2. Immobilized cell fermentation
This fermentation afforded three metabolites. 3b,7a,17b-Tri-
hydroxyandrost-5-ene (3) and 3b,7b,17b-trihydroxyandrost-5-ene
(4) were characterized as their respective acetates (3a and 4a),
both found in the free cell fermentation. The 13C NMR spectrum
of the diacetate (5a) of the third metabolite showed the appear-
ance of a new peak at d 220.3. The spectrum also revealed the loss
of the methine at d 83.1, C-17. There was a new methine signal at d
74.9 which was coupled to a proton resonance at d 5.17. This was
accompanied by the disappearance of the methylene at d 31.8,
indicating that hydroxylation had occurred at C-7. The COSY data
showed correlations between the H-6 and H-8 with the proton at
d 5.17. The stereochemistry of the hydroxyl group was determined
2.10.1. Free cell fermentation
The both extracts were combined (782 mg) and partially purified
by column chromatography to give the fed substrate (2) (468 mg).
Acetylation and further purification gave 3a (133 mg), 4a (134 mg)
and 5a (4.9 mg). Elution with acetone/petrol (1:9 v/v) afforded
3b,6b,17b-triacetoxy-5a-hydroxyandrostane
(9a)
(36.2 mg),
Rf = 0.43, acetone in dichloromethane (1:19 v/v), which resisted
crystallization, [
a
]
À97.1° (c 0.21); IR: mmax 3420, 2943, 1735,
D
1373, 1247 cmÀ1; HREIMS: m/z: 473.2533, MNa+ (C25H38O7Na
requires 473.2509); 1H NMR: d 0.81 (3H,s,H-18), 1.16 (3H,s,H-19),
2.02 (3H,s,CH3CO2-3), 2.04 (3H,s,CH3CO2-17), 2.08 (3H,s,CH3CO2-
as b, as there were correlations between H-7 and H-9, H-14, H-15a.
Compound 5 was determined to be 3b,7b-dihydroxyandrost-5-en-
17-one, characterized as the diacetate 5a [22].
6), 4.59 (1H,dd,J = 7.5,8.8 Hz,H-17
a), 4.69 (1H,t,J = 2.5 Hz,H-6a),
5.14 (1H,tt,J = 12,6 Hz,H-3
a
). 13C NMR: d 12.3 (CH3-18), 16.4
(CH3-19), 20.5 (CH2-11), 21.2 (CH3CO2-17), 21.4 (CH3CO2-3), 21.5
(CH3CO2-6), 23.4 (CH2-15), 26.6 (CH2-2), 27.5 (CH2-16), 30.6 (CH-
8), 31.0 (CH2-7), 31.8 (CH2-1), 36.8 (CH2-12), 36.9 (CH2-4), 38.6 (C-
10), 42.8 (C-13), 45.0 (CH-9), 50.1 (CH-14), 70.5 (CH-3), 74.9 (C-5),
76.0 (CH-6), 82.7 (CH-17), 170.1 (CH3CO2-6), 170.6 (CH3CO2-3),
171.2 (CH3CO2-17).
3.2. Biotransformations using M. plumbeus
3.2.1. Free cell fermentation
The NMR data confirmed that the first metabolite, after acetyla-
tion, was 3a. The 13C NMR spectrum of the second metabolite
showed the loss of the methine at d 83.1, C-17, along with the loss
of the signal at d 4.56 in the 1H NMR. There was a new peak in the
13C NMR spectrum at d 220.3, indicating the presence of an uncon-
jugated ketone moiety. The HMBC data showed that H-12, 16, and
18 were coupled to the carbon at d 220.3. A new methine was ob-
served at d 67.0, which was attached to a proton resonating at d
5.12. The COSY data showed that there was correlation of this
new proton with H-6 and H-8. This confirmed that hydroxylation
had occurred at C-7. T-ROESY data showed that H-7 was coupled
to H-8. The acetylated compound 6a was determined to be
2.10.2. Immobilized cell fermentation
The broth extract (182.6 mg) was partially purified by column
chromatography to give the fed substrate (2) (57.9 mg). Acetyla-
tion and further purification gave 3a (28.9 mg), 4a (26.7 mg) and
9a (8 mg).
3. Results
A summary of the products of transformation obtained is found
in Table 1.
3b,7
metabolite 3b,7
a
-diacetoxyandrost-5-en-17-one (6a) [22], derived from the
-dihydroxyandrost-5-en-17-one (6).
a
3.2.2. Immobilized cell fermentation
3.1. Biotransformations using R. oryzae
The two products of transformation obtained from this incuba-
tion were the same as those that were found in the free cell fer-
3.1.1. Free cell fermentation
The 13C NMR spectrum for the first acetylated metabolite (3a)
showed the occurrence of a new methine at d 67.6. COSY data
mentation, namely, 3b,7
3b,7 -dihydroxyandrost-5-en-17-one (6), which were character-
ized as 3a and 6a.
a,17b-trihydroxyandrost-5-ene (3) and
a
Table 1
3.3. Biotransformations using C. echinulata var. elegans
Products of transformation under different fermentation conditions.
Fungi
Fermentation method
Products
3.3.1. Free cell fermentation
Two metabolites were isolated from this incubation, com-
pounds 3 and 4. Both were characterized as their respective acetate
derivatives, 3a and 4a.
R. oryzae
R. oryzae
M. plumbeus
M. plumbeus
C. echinulata var. elegans
C. echinulata var. elegans
A. niger
A. niger
W. sclerotiorum
W. sclerotiorum
Free cell
Immobilized cell
Free cell
Immobilized cell
Free cell
Immobilized cell
Free cell
Immobilized cell
Free cell
Immobilized cell
3, 4
3, 4, 5
3, 6
3, 6
3, 4
3.3.2. Immobilized cell fermentation
The results of this biotransformation paralleled those of the free
cell fermentation. Two metabolites were obtained, 3b,7a,17b-tri-
hydroxyandrost-5-ene (3) and 3b,7b,17b-trihydroxyandrost-5-ene
(4), characterized as 3a and 4a respectively.
3, 4
4, 7, 8
4, 7, 8
3, 4, 5, 9
3, 4, 9

P.C. Peart et al. / Steroids 77 (2012) 85–90
89
3.4. Biotransformations using A. niger
3.6.2. Immobilized cell fermentation
This incubation afforded three transformed products: 3b,7
a,17b-
3.4.1. Free cell fermentation
trihydroxyandrost-5-ene (3), 3b,7b,17b-trihydroxyandrost-5-ene
The first metabolite 4 was characterized as the acetate 4a. The
13C NMR spectrum of the acetate (7a) of second metabolite showed
that the methine at d 74.2 had disappeared, and this was accompa-
nied by the appearance of a new peak at d 199.8. The position of this
resonance indicated that the ketone was conjugated. This was cor-
roborated by the changes in the signals for the unsaturated carbons.
Therefore, there had been oxidation of the 3-hydroxyl, followed by
migration of the carbon-carbon double bond from C-5,6 to C-4,5.
Compound 7a was determined to be 17b-acetoxyandrost-4-en-3-
one [25], which was derived from 17b-hydroxyandrost-4-en-3-
one (7). The final transformed product (8) was acetylated to yield
8a. The 13C NMR spectrum showed the disappearance of the peak
at d 74.2, along with the appearance of a new nonprotonated carbon
at d 199.2. HMBC data revealed that the protons at C-1 were cou-
pled to this conjugated ketone, establishing that this moiety was lo-
cated in ring A. The hydroxyl group at C-3 had been oxidized to a
ketone, and this was followed by a migration of the double bond
from C-5,6 to C-4,5. Additionally, there was the loss of a methylene
at d 27.9, C-16, accompanied by the appearance of a new signal at d
210.3. The HMBC spectrum showed that there were correlations
between H-15 and H-17 to the carbon at d 210.3. This confirmed
that the group was present in ring D. The final metabolite was
17b-hydroxyandrost-4-ene-3,16-dione (8), which was character-
ized as the acetate 8a [26].
(4) and 3b,5 ,6b,17b-tetrahydroxyandrostane (9). These were the
a
same as those obtained from the free cell incubation with the excep-
tion that the free cell fermentation also produced 3b,7b-dihydroxy-
androst-5-en-17-one (5).
3.7. Optimization of immobilized cell fermentations
3.7.1. Mycelial fragmentation time
It was envisaged that the size of the mycelial fragments would
affect the viability of the fungal cells within the alginate beads.
This is because the smaller the hyphae fragments, the lower the
likelihood that intact cells remained. It was observed that myce-
lium macerated for periods longer than 3 min retained very little
or no enzymatic activity (Table 2). Yields were determined from
the masses of transformed metabolites separated from the organic
extract of the fermentation.
3.7.2. Alginate bead diameter
In any fermentation the rate at which xenobiotes in the culture
medium diffuse into the mycelia will affect the yield of trans-
formed products. This same is true for incubations using immobi-
lized cells. The substrate has to diffuse through not one, but two
semi-permeable barriers (gel matrix and cell membrane). The
surface area:volume ratio is a function of the diameter of the algi-
nate beads. With this in mind the search for an alginate bead size
that would give the highest transformation yields was investi-
gated. This was executed by varying the diameter of the bore of
the dropping tube. These results indicated that with larger alginate
beads the transformation yield was reduced. The optimal diameter
was found to be 3 mm (Table 3). Yields were determined from the
masses of transformed metabolites separated from the organic
extract of the fermentation.
3.4.2. Immobilized cell fermentation
The biotransformed compounds isolated from this fermentation
were the same as those from the free cell fermentation: 3b,7b,17b-
trihydroxyandrost-5-ene (4), 17b-hydroxyandrost-4-en-3-one (7)
and 17b-hydroxyandrost-4-ene-3,16-dione (8). These metabolites
were converted to their acetates (4a, 7a and 8a) for characterization.
3.5. Biotransformations using P. chrysosporium
3.7.3. Rejuvenation of used immobilized cells
Some enzymes must be associated with other compounds in
order to carry out their reactions. Exhausted cofactors have to be
replenished before further reactions can occur. Sonomoto showed
that incubation of immobilized cells in a nutrient medium could
rejuvenate these systems [12]. Replenishment of the cofactor
NADPH is crucial for the success of cytochrome P450 hydroxyl-
ations. In this regard four possible media were investigated: water,
1% glucose solution, potato broth (PB), and potato dextrose broth
(PDB). The exhausted immobilized cells were incubated in each
medium with shaking for 12 h. The beads were then washed with
water and reincubated with the steroid. Rejuvenation with PDB
3.5.1. Free cell fermentation
The fermentation produced several metabolites, however, their
quantities were not significant enough to allow for their isolation
and characterization.
3.5.2. Immobilized cell fermentation
As with the free cell fermentation, the immobilized cells of
P. chrysosporium produced several metabolites in poor yields, and
as such it was not deemed practical to isolate and characterize
these products.
Table 2
Effect of maceration time on transformation yields (%) using different fungi.
3.6. Biotransformations using W. sclerotiorum
Fungi
Maceration time @ 8000 rpm
3.6.1. Free cell fermentation
1 min
3 min
5 min
7 min
The first three metabolites were identified as compounds 3, 4
and 5 by examination of the NMR data of their respective acetates
3a, 4a and 5a. The fourth product of biotransformation, compound
9, was converted to the triacetate 9a. The 13C NMR spectrum re-
vealed the loss of signals representing the carbon-carbon double
bond. There was a new methine resonating at d 75.9 as well as a
new nonprotonated carbon at d 74.9. This showed that there was
a hydroxyl group at C-5. For these reasons it was deduced that
epoxidation of the carbon-carbon double bond had taken place,
and this was followed by hydrolysis. This compound was deter-
M. plumbeus (% transformation)
A. niger (% transformation)
R. oryzae (% transformation)
10
4
9
31
7
12
8
1
8
2
1
1
Table 3
Effect of bead diameter on transformation yields (%) using different fungi.
Fungi
Alginate bead diameter/mm
1
3
5
M. plumbeus (% transformation)
A. niger (% transformation)
R. oryzae (% transformation)
10
3
31
5
12
7
1
8
mined to be 3b,6b,17b-triacetoxy-5a-hydroxyandrostane (9a),
which was derived from metabolite 9. The latter is completely
characterized here for the first time.
6

90
P.C. Peart et al. / Steroids 77 (2012) 85–90
[3] Nakamura K, Higaki M, Ushio K, Oka S, Ohno A. Stereochemical control of
Table 4
microbial reduction. 2. Reduction of b-keto esters by immobilized bakers’
yeast. Tetrahedron Lett 1985;26:4213–6.
Effect of different media (for rejuvenation) on transformation yields (%) using
different fungi
[4] Sakai T, Nakamura T, Fukuda K, Amano E, Utaka M, Takeda A. Enantioselective
reduction of ethyl 2-acyloxy-3-oxobutanoate with immobilized baker’s yeast.
Bull Chem Soc Jpn 1986;59:3185–8.
[5] Griffin DR, Yang F, Carta G, Gainer J. Asymmetric reduction of acetophenone
with calcium-alginate-entrapped baker’s yeast in organic solvent. Biotechnol
Prog 1998;14:588–93.
[6] Naoshima Y, Maeda J, Munakata Y, Nishiyama T, Kamezawa M, Tachibana H.
Bioreduction with immobilized bakers’ yeast in hexane using alcohols as an
energy source. J Chem Soc Chem Commun 1990:964–5.
Fungi
Media for rejuvenation
H2O
Glucose
PB
PDB
M. plumbeus (% transformation)
A. niger (% transformation)
R. oryzae (% transformation)
19
1
23
3
20
1
7
49
5
11
3
7
[7] Naoshima Y, Nakamura A, Munakata Y. Synthesis of (5Z, 13S)-5-tetradecen-13-
olide, aggregation pheromone of the Cryptolestes grain beetle, employing
asymmetric bioreduction with immobilized bakers’ yeast entrapped in calcium
alginate beads. Bull Chem Soc Jpn 1990;63:1263–5.
[8] Fadnavis NW, Deshpande A, Chauhan S, Bhalerao UT. Peptide synthesis
mediated by immobilized and viable baker’s yeast in reverse micelles:
gave the best results overall (Table 4). Yields were determined
from the masses of transformed metabolites separated from the
organic extract of the fermentation. Immobilized cells rejuvenated
in the glucose solution exhibited outgrowth of mycelia from the
alginate matrix. This led to the collapse of the beads. It was con-
cluded from the data that the cells needed glucose and inorganic
salts for cofactor regeneration.
synthesis of leucine enkephalin analogues.
1990:1548–50.
J Chem Soc Chem Commun
[9] Bhalerao T, Chanderaprakash Y, Babu RL, Fadnavis NW. b-Ketoester reduction
by baker’s yeast immobilized in calcium alginate: an examination of pH effects
on enantiospecificity. Synth Commun 1993;23:1201–8.
[10] Griffin DR, Gainer JL, Carta G. Asymmetric ketone reduction with immobilized
yeast in hexane: biocatalyst deactivation and regeneration. Biotechnol Prog
2001;17:304–10.
4. Discussion
[11] Najafpour G, Younesi H, Tsmail KSK. Ethanol fermentation in an immobilized
cell reactor using Saccharomyces cerevisiae. Bioresource Technol 2004;92:
250–1.
Our investigations have led to the development of a general
technique for the immobilization of filamentous fungi. This was
accomplished by growing the microorganism in a suitable med-
ium, after which the mycelium was harvested. The latter was sub-
jected to a fragmentation process and this was followed by
entrapment in the alginate matrix. Incubation of the alginate beads
in water with 2 led to the preparation of transformed analogues.
This study showed that, for the first time, transformation reactions
by immobilized cells paralleled those from the growing cells, in
that, to a large extent, they produced the same metabolites with
most of the fungi that were used. The reason that they are not
exactly the same is probably due to the fact that, in the free versus
immobilized cell fermentations, there must be some small differ-
ences in fungal metabolism. Additionally, in a majority of the en-
trapped cell incubations there was an improvement in yields
over those from the free cells. Purification of the transformed prod-
ucts was significantly facilitated (and reduced solvent volumes
were utilized in chromatography), since cells in the matrix were
not producing secondary metabolites. The procedure is straightfor-
ward, and the fact that the immobilized cells can be reused makes
the technique effective and efficient. An additional advantage of
this new method over previous ones is its applicability in cases
where the mould does not produce spores, for example, as is the
situation with many endophytic fungi.
[12] Sonomoto K, Nomura K, Tanaka A, Fukui S. 11a-Hydroxylation of progesterone
by gel-entrapped living Rhizopus stolonifer mycelia. Eur
J Appl Microbiol
Biotechnol 1982;16:57–62.
[13] Tan Q, Day DF. Bioconversion of limonene to
a-terpineol by immobilized
Penicillium digitatum. Appl Microbiol Biotechnol 1998;49:96–101.
[14] Mazumder TK, Sonomoto K, Tanaka A, Fukui S. Sequential conversion of
cortexolone to prednisolone by immobilized mycelia of Curvularia lunata and
immobilized cells of Arthrobacter simplex. Appl Microbiol Biotechnol 1985;21:
154–61.
[15] Kutney JP, Choi LSL, Hewitt GM, Salisbury PJ, Singh M. Biotransformation of
dehydroabietic acid with resting cell suspensions and calcium alginate-
immobilized cells of Mortierella isabellina. Appl Environ Microbiol
1985;49:96–100.
[16] Ohlson S, Flygare S, Larsson PO, Mosbach K. Steroid hydroxylation using
immobilized spores of Curvularia lunata germinated in situ. Eur
J Appl
Microbiol Biotechnol 1980;10:1–9.
[17] Sonomoto K, Hoq MM, Tanaka A, Fukui S. 11b-Hydroxylation of cortexolone
(Reichstein compound S) to hydrocortisone by Curvularia lunata entrapped in
photo-cross-linked resin gels. Appl Environ Microbiol 1983;45:436–43.
[18] Walker HL, Connick WJ, Quimby PC (inventors). Method for mycoherbicide-
containing pellets. The United States of America (assignee). US Patent
4,718,935. January 12, 1988.
[19] Hufford CD, Badria FA, Abou-Karram M, Shier WT, Rogers RD. Preparation,
characterization, and antiviral activity of microbial metabolites of stemodin. J
Nat Prod 1991;54:1543–52.
[20] Wilson MR, Gallimore WA, Reese PB. Steroid transformations with Fusarium
oxysporum var. Cubense and Colletotrichum musae. Steroids 1999;64:834–43.
[21] Defaye G, Luche MJ, Chambaz EM. Microbiological 7- and 15-hydroxylations of
C-19 steroids. J Steroid Biochem 1978;9:331–6.
[22] Dodson RM, Nicholson RT, Muir RD. Microbiological transformations. IV.
The oxidation of dehydroisoandrosterone at C-7. J Am Chem Soc 1959;83:
6295–7.
Acknowledgments
[23] Lamm AS, Chen ARM, Reese PB. Steroid hydroxylation by Mucor plumbeus,
Whetzelinia sclerotiorum and Phanerochaete chrysosporium. Steroids 2007;72:
713–22.
[24] Belan A, Bolte J, Fauvre A, Gourcy JG, Veschambre H. Use of biological systems
for the preparation of chiral molecules. 3. Application in pheromone synthesis:
preparation of sulcatol enantiomers. J Org Chem 1987;52:256–60.
[25] Tada H, Sawa YK. Preparation of A-ring conjugated enones and the
corresponding.alpha.,.beta.-epoxy ketones of 17.beta.-acetoxy-5.alpha.-
androstane. J Org Chem 1968;33:3347–9.
The authors wish to thank the University of the West Indies,
Mona/Inter-American Development Bank (UWI/IDB) Programme
for partial funding. PCP and ARMC are grateful to Tanaud
International B.V. (Jamaica) and the University of the West Indies,
respectively, for the granting of postgraduate scholarships. PCP
and ARMC also acknowledge the University of the West Indies for
providing Tutorial Assistantships.
[26] Dodson RM, Mizuba S. Microbiological transformations. VIII. The oxidation of
androstenedione at C-16. J Org Chem 1962;27:698–700.
[27] Bhutani KK, Thakur RN. The microbiological transformation of parthenin by
Beauveria bassiana and Sporotrichum pulverulentum. Phytochemistry 1991;30:
3599–600.
References
[28] Chen ARM, Ruddock PLD, Lamm AS, Reynolds WF, Reese PB. Stemodane and
stemarane diterpenoid hydroxylation by Mucor plumbeus and Whetzelinia
sclerotiorum. Phytochemistry 2005;66:1898–902.
[1] Anderson JG. Immobilized cell and film reactor systems for filamentous fungi.
In: The Filamentous Fungi. In: Smith JE, Berry DR, Kristiansen B, editors. Fungal
Technology, IV. London, UK: Arnold; 1983. p. 145–9.
[2] Smidsrød O, Skjåk-Bræk G. Alginate as immobilization matrix for cells. Trends
Biotechnol 1990;8:71–8.