Biotransformation of Nitriles by Marine Fungi 28.2 Experimental Methods 691
Part E | 28.2
28.2 Experimental Methods
28.2.1 Isolation, Identification,
and Preparation of Stock Cultures
of Marine Fungi
The fungal strains Aspergillus sydowii CBMAI 933 and
Penicillium miczynskii CBMAI 930 were isolated from
the marine sponge Geodia corticostylifera. A. sydowii
CBMAI 934, A. sydowii CBMAI 935, Bionectria sp.
CBMAI 936, and P. raistrickii CBMAI 931 were isolated from marine sponge Chelonaplysilla erecta. A.
sydowii CBMAI 1241, P. raistrickii CBMAI 1235,
P. decaturense CBMAI 1234, and Cladosporium sp.
CBMAI 1237 were isolated from marine sponge Dragmacidon reticulate. P. oxalicum CBMAI 1185 and P.
citrinum CBMAI 1186 were isolated from the marine alga Caulerpa sp. The sponges and algae were
collected in the South Atlantic Ocean off the northern coast of the state of São Paulo at São Sebastião,
Brazil. The fungi were identified by both conventional and molecular methods at the Chemical, Biological and Agricultural Pluridisciplinary Research Center
at UNICAMP, São Paulo, Brazil (http://www.cpqba.
unicamp.br/). Stock cultures of the fungi were stored on
a solid culture medium [K 2 HPO 4 (1:0 g L
1 ), MgSO 4
7 H 2 O (0:5 g L
1 ), KCl (0:5 g L
1 ), FeSO 4 7 H 2 O
(0:01 g L
1 ), CoCl 2 6H 2 O (0:001 g L
1 ), ZnSO 4 7
H 2 O (0:0067 g L
1 )], glucose (15:0 g L
1 ), phenylacetonitrile 1 (0:2, 0:4 or 0:6 g L
1 ) and agar (15:0 g L
1 )
in Petri dishes maintained at 4
ı C in the refrigerator.
28.2.2 Cultivation of Marine Fungi
on a Solid Medium in the Presence
of Phenylacetonitrile 1
Marine fungi were cultured on Petri dishes (10 1 cm)
containing solid mineral culture medium (glucose) supplemented with phenylacetonitrile 1
5:0 L (0:04 mmol), 10:0 L (0:08 mmol), 15:0 L
(0:12 mmol), and 20:0 L (0:17 mmol) per plate.
Phenylacetonitrile 1 was added to the agar at 4045
ı C
and the homogenous mixture was poured into Petri
dishes. Fungal mycelia were inoculated to the surfaces of the agar plates with an inoculating loop.
Next, the fungi were incubated at 32
ı C, and the
tolerance to phenylacetonitrile 1 was estimated by
the size of the colony formed on the surface of the
plates after 8 days. Control plates of mineral medium
and glucose were prepared, without the addition of
phenylacetonitrile 1.
28.2.3 Biotransformation
of Phenylacetonitrile 1
by Marine Fungi in a Liquid Medium
Small slices of solid medium (0:5 0:5 cm) bearing the
mycelia of eight marine fungal strains were cut from
the solid stock cultures and used to inoculate 100:0 mL
of liquid culture mineral medium containing 15:0 g
glucose in 250 mL Erlenmeyer flasks. The cultures
were incubated at 32
ı C on a rotary shaker (124 rpm),
with various volumes of phenylacetonitrile 1 added to
the medium (20:0 L, 0:17 mmol; 40:0 L, 0:34 mmol;
60:0 L, 0:52 mmol; Table 28.1). After the growth of
the strains in liquid medium for 8 days, the mycelia
were filtered on a Buchner funnel and the mycelial
dry weight was determined after drying at 70
ı C for
24 h. The results are summarized in Table 28.2. The
biotransformation of phenylacetonitrile 1 was monitored every 2 days by collecting 2:0 mL samples, which
were extracted by stirring with ethyl acetate (2:0 mL),
centrifuged (6000 rpm, 6 min), and analyzed by analyzed by GC-FID (Gas Chromatography-Flame Ionization Detector) and GC-MS (Gas Chromatography-Mass
Spectrometry) (Table 28.3).
28.2.4 Isolation
of 2-Hydroxyphenylacetic Acid 1a
from Culture Media Produced
by A. sydowii CBMAI 934
An additional experiment was performed with the
fungus A. sydowii CBMAI 934 for isolation and purification of the biotransformed product. Biocatalytic
reactions were carried out for 96 h in quintuplicate,
with 60:0 L (0:52 mmol) of phenylacetonitrile 1 in
100:0 mL medium per Erlenmeyer flask. Next, another
60:0 L of phenylacetonitrile 1 was added and the reactions were maintained on the orbital shaker (124 rpm,
32
ı C) for a further 96 h. The reaction mixtures were
filtered, the combined filtrates were extracted with
EtOAc (3 50:0 mL), and the organic phase was dried
over anhydrous Na 2 SO 4 , filtered and evaporated under
vacuum. The mycelia (16:0 g) were transferred to an
Erlenmeyer and stirred for 30 min with 50:0 mL of distilled water. Next, 50:0 mL of EtOAc was added, and
the mixture was stirred for 30 min. The mycelia were
filtered off, and the filtrate was extracted with ethyl acetate (3 50:0 mL). The organic phase was dried over
anhydrous Na 2 SO 4 , filtered, and the solvent evaporated
Part E | 28.2
28.2 Experimental Methods
28.2.1 Isolation, Identification,
and Preparation of Stock Cultures
of Marine Fungi
The fungal strains Aspergillus sydowii CBMAI 933 and
Penicillium miczynskii CBMAI 930 were isolated from
the marine sponge Geodia corticostylifera. A. sydowii
CBMAI 934, A. sydowii CBMAI 935, Bionectria sp.
CBMAI 936, and P. raistrickii CBMAI 931 were isolated from marine sponge Chelonaplysilla erecta. A.
sydowii CBMAI 1241, P. raistrickii CBMAI 1235,
P. decaturense CBMAI 1234, and Cladosporium sp.
CBMAI 1237 were isolated from marine sponge Dragmacidon reticulate. P. oxalicum CBMAI 1185 and P.
citrinum CBMAI 1186 were isolated from the marine alga Caulerpa sp. The sponges and algae were
collected in the South Atlantic Ocean off the northern coast of the state of São Paulo at São Sebastião,
Brazil. The fungi were identified by both conventional and molecular methods at the Chemical, Biological and Agricultural Pluridisciplinary Research Center
at UNICAMP, São Paulo, Brazil (http://www.cpqba.
unicamp.br/). Stock cultures of the fungi were stored on
a solid culture medium [K 2 HPO 4 (1:0 g L
1 ), MgSO 4
7 H 2 O (0:5 g L
1 ), KCl (0:5 g L
1 ), FeSO 4 7 H 2 O
(0:01 g L
1 ), CoCl 2 6H 2 O (0:001 g L
1 ), ZnSO 4 7
H 2 O (0:0067 g L
1 )], glucose (15:0 g L
1 ), phenylacetonitrile 1 (0:2, 0:4 or 0:6 g L
1 ) and agar (15:0 g L
1 )
in Petri dishes maintained at 4
ı C in the refrigerator.
28.2.2 Cultivation of Marine Fungi
on a Solid Medium in the Presence
of Phenylacetonitrile 1
Marine fungi were cultured on Petri dishes (10 1 cm)
containing solid mineral culture medium (glucose) supplemented with phenylacetonitrile 1
5:0 L (0:04 mmol), 10:0 L (0:08 mmol), 15:0 L
(0:12 mmol), and 20:0 L (0:17 mmol) per plate.
Phenylacetonitrile 1 was added to the agar at 4045
ı C
and the homogenous mixture was poured into Petri
dishes. Fungal mycelia were inoculated to the surfaces of the agar plates with an inoculating loop.
Next, the fungi were incubated at 32
ı C, and the
tolerance to phenylacetonitrile 1 was estimated by
the size of the colony formed on the surface of the
plates after 8 days. Control plates of mineral medium
and glucose were prepared, without the addition of
phenylacetonitrile 1.
28.2.3 Biotransformation
of Phenylacetonitrile 1
by Marine Fungi in a Liquid Medium
Small slices of solid medium (0:5 0:5 cm) bearing the
mycelia of eight marine fungal strains were cut from
the solid stock cultures and used to inoculate 100:0 mL
of liquid culture mineral medium containing 15:0 g
glucose in 250 mL Erlenmeyer flasks. The cultures
were incubated at 32
ı C on a rotary shaker (124 rpm),
with various volumes of phenylacetonitrile 1 added to
the medium (20:0 L, 0:17 mmol; 40:0 L, 0:34 mmol;
60:0 L, 0:52 mmol; Table 28.1). After the growth of
the strains in liquid medium for 8 days, the mycelia
were filtered on a Buchner funnel and the mycelial
dry weight was determined after drying at 70
ı C for
24 h. The results are summarized in Table 28.2. The
biotransformation of phenylacetonitrile 1 was monitored every 2 days by collecting 2:0 mL samples, which
were extracted by stirring with ethyl acetate (2:0 mL),
centrifuged (6000 rpm, 6 min), and analyzed by analyzed by GC-FID (Gas Chromatography-Flame Ionization Detector) and GC-MS (Gas Chromatography-Mass
Spectrometry) (Table 28.3).
28.2.4 Isolation
of 2-Hydroxyphenylacetic Acid 1a
from Culture Media Produced
by A. sydowii CBMAI 934
An additional experiment was performed with the
fungus A. sydowii CBMAI 934 for isolation and purification of the biotransformed product. Biocatalytic
reactions were carried out for 96 h in quintuplicate,
with 60:0 L (0:52 mmol) of phenylacetonitrile 1 in
100:0 mL medium per Erlenmeyer flask. Next, another
60:0 L of phenylacetonitrile 1 was added and the reactions were maintained on the orbital shaker (124 rpm,
32
ı C) for a further 96 h. The reaction mixtures were
filtered, the combined filtrates were extracted with
EtOAc (3 50:0 mL), and the organic phase was dried
over anhydrous Na 2 SO 4 , filtered and evaporated under
vacuum. The mycelia (16:0 g) were transferred to an
Erlenmeyer and stirred for 30 min with 50:0 mL of distilled water. Next, 50:0 mL of EtOAc was added, and
the mixture was stirred for 30 min. The mycelia were
filtered off, and the filtrate was extracted with ethyl acetate (3 50:0 mL). The organic phase was dried over
anhydrous Na 2 SO 4 , filtered, and the solvent evaporated