Immobilization of marine fungi on silica gel, silica xerogel and chitosan for biocatalytic reduction of ketones

Article abstract of DOI:10.1016/j.molcatb.2012.05.025

The scanning electron microscopy (SEM) analysis showed that whole living hyphal of marine fungi Aspergillus sclerotiorum CBMAI 849 and Penicillium citrinum CBMAI 1186 were immobilized on support matrices of silica gel, silica xerogel and/or chitosan. P. citrinum immobilized on chitosan catalyzed the quantitative reduction of 1-(4-methoxyphenyl)-ethanone (1) to the enantiomer (S)-1-(4-methoxyphenyl)-ethanol (3b), with excellent enantioselectivity (ee > 99%, yield = 95%). Interestingly, ketone 1 was reduced with moderate selectivity and conversion to alcohol 3b (ee = 69%, c 40%) by the free mycelium of P. citrinum. This free mycelium of P. citrinum catalyzed the production of the (R)-alcohol 3a, the antipode of the alcohol produced by the immobilized cells. P. citrinum immobilized on chitosan also catalyzed the bioreduction of 2-chloro-1-phenylethanone (2) to 2-chloro-1-phenylethanol (4a,b), but in this case without optical selectivity. These results showed that biocatalytic reduction of ketones by immobilization hyphal of marine fungi depends on the xenobiotic substrate and the support matrix used.

Full text of DOI:10.1016/j.molcatb.2012.05.025

Journal of Molecular Catalysis B: Enzymatic 84 (2012) 160–165
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Immobilization of marine fungi on silica gel, silica xerogel and chitosan for
biocatalytic reduction of ketones
a
a
a
Lenilson Coutinho Rocha , Adriano Lopes de Souza , Ubirajara Pereira Rodrigues Filho ,
a
b
a,∗
Sergio Paulo Campana Filho , Lara Durães Sette , André Luiz Meleiro Porto
Instituto de Química de São Carlos, Universidade de São Paulo, Av. Trabalhador São-carlense, 400, CEP 13560-970, São Carlos, SP, Brazil
a
b
Divisão de Recursos Microbianos, Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas, CPQBA, Universidade Estadual de Campinas, Rua Alexandre Casellato, 999,
CEP 13140-000, Paulínia, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 14 July 2012
The scanning electron microscopy (SEM) analysis showed that whole living hyphal of marine fungi
Aspergillus sclerotiorum CBMAI 849 and Penicillium citrinum CBMAI 1186 were immobilized on sup-
port matrices of silica gel, silica xerogel and/or chitosan. P. citrinum immobilized on chitosan
catalyzed the quantitative reduction of 1-(4-methoxyphenyl)-ethanone (1) to the enantiomer (S)-1-
Keywords:
Biocatalysis
Penicillium citrinum
Aspergillus sclerotiorum
Whole living cells
Scanning electron microscopy
(4-methoxyphenyl)-ethanol (3b), with excellent enantioselectivity (ee > 99%, yield = 95%). Interestingly,
ketone 1 was reduced with moderate selectivity and conversion to alcohol 3b (ee = 69%, c 40%) by the
free mycelium of P. citrinum. This free mycelium of P. citrinum catalyzed the production of the (R)-alcohol
3a, the antipode of the alcohol produced by the immobilized cells. P. citrinum immobilized on chitosan
also catalyzed the bioreduction of 2-chloro-1-phenylethanone (2) to 2-chloro-1-phenylethanol (4a,b),
but in this case without optical selectivity. These results showed that biocatalytic reduction of ketones
by immobilization hyphal of marine fungi depends on the xenobiotic substrate and the support matrix
used.
©
2012 Elsevier B.V. All rights reserved.
1
. Introduction
of other ways, by covalent linkage, ion-coagulation, crosslinking
and/or entrapment methods, on a variety of matrices [6,7]. Among
the biocatalysts, purified enzymes and whole living cells of bac-
teria and yeasts are commonly immobilized in various supports
and matrices, but the immobilization of filamentous fungi has been
rarely described [8]. However, whole mycelia of filamentous fungi
are often used in biocatalytic processes, especially in redox reac-
tions with various organic compounds [9]. The morphology of fungi
is very complex, with different cellular structures at each stage of
their life cycle [10]. One way to preserve the morphological struc-
ture of these microorganisms is the immobilization of whole living
cells, such that the desired catalytic activity is preserved, and the
immobilized cells can then be used in the laboratory and on the
industrial scale [11]. The immobilization of whole living cells of
microorganisms is interesting in view of the increasing number of
industrial applications of biocatalytic process in the pharmaceuti-
cal and food industries, especially those involving filamentous fungi
with high production of enzymes [12].
Biocatalytic processes can take advantage of the habitat-related
properties of marine enzymes, such as salt tolerance, hyper-
thermostability, barophilicity and cold adaptivity, as well as the
common enzyme characteristics of substrate specificity and affin-
ity [1]. For a biocatalyst to be effective in a given process, it must be
protected from interaction with the solvent in which the reaction is
carried out, as this could lead to its inactivation, halting the catalytic
reaction [2]. In this context, new immobilization techniques have
been developed to provide stability to enzymes and facilitate their
recovery and reutilization [3]. Immobilized enzymes are defined
as enzymes that are physically confined or localized in a certain
region of space with retention of catalytic activities that may be
used continuously or repeatedly [4]. Enzymatic processes in the
immobilization of biocatalysts offers advantages such as increased
stability and improved process control, yield and purity of the final
product, besides enabling the use of reactors of a variety of con-
figurations [5]. One very simple immobilization technique is the
adsorption of the enzyme on porous material, such as celite, silica or
polyurethane foam. Immobilization has been achieved in a number
Most of the fungi contain chitin in the cell wall, forming 22–40%
of the wall constituents [13]. The presence of chitin, together
with that of other polysaccharides, has been used as a crite-
rion in fungal taxonomy. This polysaccharide, which forms fibrils
of different lengths, depending on the species and the cellular
location [10,14], is a homopolymer and it has a broad spectrum
of distribution in the biosphere, being formed in the shells of
∗ Corresponding author. Tel.: +55 16 3373 8103; fax: +55 16 3373 9952.
E-mail address: almporto@iqsc.usp.br (A.L.M. Porto).
1
381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2012.05.025

L.C. Rocha et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 160–165
161
Table 1
Reduction of ketones 1 and 2 by free and immobilized whole living cells of marine fungi (9 days, 120 rpm, 32 C).a
◦
1
-(4-Methoxyphenyl)ethanone (1)
Entry
Fungi marine
Supports matrices
c 1
60
100
100
–
c 3a; 3b
ee 3a; 3b (ac)
Yield (%)
1
2
3
4
Penicillium citrinum
Not immobilized
silica gel
silica xerogel
chitosan
40
0
0
69 (R)
35
–
–
–
–
100
>99 (S)
95
5
6
7
Aspergillus sclerotiorum
Not immobilized
silica gel
silica xerogel
–
–
100
100
100
0
>99 (S)
>99 (S)
–
90
97
–
2
-Chloro-1-phenylethanone (2)
Entry
Fungi marine
Supports matrices
c 2
c 4a; 4b
ee 4a; 4b (ac)
Yield (%)
8
9
0
1
Penicillium citrinum
Not immobilized
silica gel
silica xerogel
chitosan
30
35
100
–
70
65
0
31 (R)
25 (S)
–
66
62
–
1
1
100
0
98
1
1
1
2
3
4
Aspergillus sclerotiorum
Not immobilized
silica gel
silica xerogel
11
–
100
89
100
0
68 (R)
65 (R)
–
85
97
–
a
See Section 2.
Yield, obtained after purification by column chromatography; c (%), concentration determined by GC analyses using chiral column; ee (%), enantiomeric excess; ac, absolute
configuration.
crustaceans, such as crab, shrimp and lobster, and the exoskele-
tons of marine zoo-planktons such as coral and jellyfish, and
insects such as butterflies and ladybirds [15,16]. Chitosan, the N-
deacetylated derivative of chitin, is a ˇ(1-4)-linked copolymer built
of 2-amino-2-deoxy-d-glucopyranose (GlcN) and 2-acetamido-2-
deoxy-ˇ-d-glucopyranose (GlcNAc) units which is characterized
by its average degree of acetylation (DA), expressing the average
content of GlcNAc units along its chains [17,18].
In the present study, we report the immobilization of whole
living mycelial cells of the marine fungi Penicillium citrinum
CBMAI 1186 and Aspergillus sclerotiorum CBMAI 849 on silica gel,
xerogel and chitosan to catalyze the asymmetric reduction of 1-
and 2-chloro-1-phenylethanol (4a,b) were determined by GC-FID
analysis. The retention times of the alcohols (S)-enantiomer 3b,
(R)-enantiomer 3a, (S)-enantiomer 4b and (R)-enantiomer 4a were
15.2 min, 15.8 min, 16.5 min, and 17.0 min, respectively. For car-
rying out the gas chromatography-mass spectrometry analysis,
a Shimadzu GC 2010 plus gas chromatography system with a
30 m × 0.25 mm × 0.25 DB5 fused silica column (J&W Scientific)
coupled to a mass selective detector (Shimadzu MS 2010 plus) in
electron ionization (EI, 70 eV) mode was used.
2.2. Absolute configuration
(
(
4-methoxyphenyl)-ethanone (1) and 2-chloro-1-phenylethanone
2).
The optical rotation of the purified products 1-(4-
methoxyphenyl)-ethanol (3) and 2-chloro-1-phenylethanol
(4) were measured in a Perkin-Elmer (Waltham, MA, USA) 241
2
. Experimental
polarimeter with a 1 dm cuvette, at the sodium D-line. The absolute
configurations of the alcohols 3a,b and 4a,b were determined by
comparing the measurements of its specific rotations with those
reported in the literature [19,20].
2.1. General methods
The substrates 1-(4-methoxyphenyl)-ethanone (1) and 2-
chloro-1-phenylethanone (2) were purchased from Sigma–Aldrich.
Sodium borohydride, sodium hydroxide and ethanol were pur-
chased from Vetec or Synth. All manipulations involving the fungi
A. sclerotiorum CBMAI 849 and P. citrinum CBMAI 1186 were car-
ried out under sterile conditions in a Veco laminar flow cabinet.
Technal TE-421 or Superohm G-25 orbital shakers were employed
in the biocatalysed conversion experiments. The products of the
reduction reactions were purified out by column chromatography
2.3. Chemical synthesis of 1-(4-methoxyphenyl)-ethanol (3a,b)
and 2-chloro-1-phenylethanol (4a,b)
The racemic 1-(4-methoxyphenyl)-ethanol (3a,b) and 2-chloro-
1-phenylethanol (4a,b) were synthesized by reduction of the
1-(4-methoxyphenyl)-ethanone (1) (100.0 mg, 0.70 mmol) and 2-
chloro-1-phenylethanone (2) (100.0 mg, 0.65 mmol) with sodium
borohydride (30.0 mg, 0.77 mmol) in 25 mL methanol [21]. The
1
13
(
CC) over silica gel (230–400 mesh). The column was eluted with
spectroscopic data ( H and C NMR, MS and IR) of alcohols 3a,b
and 4a,b were in agreement with those reported in the literature
[19,20].
mixtures of n-hexane and ethyl acetate (Hex:EtOAc – 9:1 and 8:2)
and monitored by TLC, using pre-coated silica gel 60 F254 lay-
ers (aluminum-backed: Sorbent). The alcohols were analyzed in a
Shimadzu GC2010 gas chromatograph, equipped with a Varian Chi-
ral column, CP-Chiralsil-DEX, ˇ-Cyclodextrin (25 m × 0.25 mm i.d.;
2.4. Marine fungi
0.39 ␮m), an AOC 20i auto injector and a flame ionization detector
The marine fungi P. citrinum CBMAI 1186 and A. sclerotiorum
CBMAI 849 were isolated from the marine alga Caulerpa sp. and
a Brazilian cnidarian species, namely Palythoa variabilis, respec-
tively, which were collected by Prof. R.G.S. Berlinck in the town
of São Sebastião, on the coast of the State of São Paulo, Brazil.
The fungi used in this work were identified by both conventional
and molecular methods at the Chemical, Biological and Agricultural
(
FID). The chromatography analysis were carried out employing the
◦
following conditions: oven temperature initially 120 C (2 min) at
1
injector temperature 200 C; detector temperature 200 C; injector
split ratio 1:20; nitrogen carrier gas at a pressure of 69.2 kPa. The
enantiomeric excess (ee) of 1-(4-methoxyphenyl)-ethanol (3a,b)
◦
◦
65 C (8 min) and increased at 2 C/min; total run time 32.5 min;
◦
◦

1
62
L.C. Rocha et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 160–165
O
OH
OH
X
X
X
immobilized cells
marine fungi
and/or
R
R
R
(
S)-3b
(R)-3a
(S)-4b
R = OMe, X = H (1)
R = H, X = Cl (2)
(
R)-4a
Scheme 1. Reduction of ketones 1 and 2 by immobilized marine fungi.
Pluridisciplinary Research Center (CPQBA) at the State University
of Campinas (UNICAMP), SP, Brazil [20,22,23].
2.8. Scanning electron microscopy (SEM)
For SEM analysis, the surfaces of samples of immobilized
mycelia of fungi A. sclerotiorum and P. citrinum were washed with
water to remove the non-adhering support matrix. Samples were
dehydrated in a graded series of water–ethanol solutions (10%,
25%, 40%, 50%, 70%, 80%, 90% and 100%), 10 mL in 50 mL Erlen-
meyer flasks, for 15 min at each step. Samples were air dried at
room temperature, and coated with 8–10 nm of gold by argon ion
sputtering using a Baltec MCS 010 model sputter. Images were
obtained at a magnification of 200–5.00 K in a scanning electron
microscope (Leica-Zeiss LEO 440) operating at an accelerating volt-
age of 20 kV (Figs. 1 and 2) using a Secondary Electron Detector
positioned among 13–25 away from the sample.
2
.5. Biocatalytic reduction of ketones 1 and 2 by marine fungi
The marine fungi were grown in culture media and artificial
sea water [20]. Small slices of solid medium (0.5 × 0.5 cm) bearing
mycelia of A. sclerotiorum and P. citrinum were cut from the stock
solid culture and used to inoculate 1 L of liquid culture medium
contained in Erlenmeyer flasks (2 L). The mycelia were incubated
◦
at 32 C for 9 days in a rotatory shaker (120 rpm). Following, the
mycelia were harvested by Buchner filtration and suspended in a
buffer solution contained in Erlenmeyer flasks (250 mL). The bio-
catalytic reductions were carried out with 5.0 g (wet weight) of
mycelium and 0.50 mmol of ketones 1 or 2, previously dissolved in
3
00 ␮L of dimethyl sulfoxide, and mixed into 100 mL phosphate
2.9. Reduction of ketones 1 and 2 by immobilized mycelia of A.
buffer solution (Na HPO /KH PO , pH = 7, 0.1 M). The mixtures
sclerotiorum and P. citrinum
2
4
2
4
◦
were incubated for 9 days in an orbital shaker at 32 C and 120 rpm.
After 9 days the samples were extracted with 2.0 mL ethyl acetate
by mixing on a vortex and centrifuging at 6000 rpm for 6.0 min in a
HERMLE Z-200 A, and analyzed by GC-FID and GC–MS. The products
were purified by column chromatography over silica gel to yield the
alcohols 3a,b or 4a,b (Table 1 and Scheme 1). All the reactions were
performed in duplicates.
Biocatalytic reduction reactions were carried out with fungal
mycelia immobilized on (wet weight) 12.0 g silica gel, 12.0 g silica
xerogel or 7.0 g chitosan. The ketones 1 and 2, previously dissolved
in 300 ␮L of dimethyl sulphoxide, were added to 100 mL of 0.1 M
phosphate buffer solution (Na HPO /KH PO , pH = 7). The mix-
2
4
2
4
tures containing the substrates and the immobilized mycelia were
incubated for 9 days on an orbital shaker at 32 C and 120 rpm. The
◦
2
.6. Support matrix preparation
cultures were extracted with ethyl acetate (1.0 mL) by vortexing
followed by centrifugation (6000 rpm for 6.0 min in a HERMLE Z-
200 A centrifuge) and analyzed by GC-FID and GC–MS. The products
were purified by CC over silica gel, to yield the alcohols 3a,b and
4a,b (Table 1 and Scheme 1).
The support matrices of silica gel 60 F254 layer (aluminum-
backed: Sorbent) and chitosan [24] of high molecular weight
159,000 g/mol, average degree of acetylation 25%, lot code:
07490/123500), on the reagent tetraethylorthosilicate (TEOS),
(
4
were purchased from a commercial source (Sigma–Aldrich). All
reagents were utilized without washing or purification. The xero-
gel support was prepared by mixing the following chemicals in
3
. Results and discussion
Whole living cells of marine fungi are sometimes preferred for
®
a Teflon reactor TEOS (26.8 mL, 0.12 mol), ethanol (100.0 mL),
biocatalytic applications and the selection of an appropriate fungus
is an important part of this process. Recently we have investigated
the use of filamentous fungi from marine environments for the
reduction of prochiral ketones [20,26]. Due to the excellent results
obtained in the biocatalytic reduction of ketones with free cells, in
this study we investigated the immobilization of living mycelia of
marine fungi on various support matrices.
−
1
deionized water (1.5 mL, resistivity: 18.2 Mꢀ cm ) and 1.0 mL
−1
◦
of solution NaOH 0.1 mol L . This mixture was heated to 60 C
and kept at this temperature for 20 min under constant magnetic
stirring. Finally, the container with the mixture was left at room
temperature until complete drying and the xerogel was collected.
Hereafter, this silica is going to be named xerogel [25].
The biocatalytic reductions of 1-(4-methoxyphenyl) ethanone
1) and 2-chloro-1-phenylethanone (2) were attempted with
2
.7. Immobilization of whole living cells of A. sclerotiorum and P.
(
citrinum on the support matrices
immobilized whole cells of the marine fungi P. citrinum and A. scle-
rotiorum (Scheme 1). Control reactions were performed with free
fungal mycelia without the support matrices. The results are shown
in Table 1. The immobilization of marine fungi was effected with
three support matrices, namely silica gel, silica xerogel and chi-
tosan. The choice of these three supports was based on previous
reports and data about the immobilization of the fungi Aspergillus
sp. and Monascus kaoliang [3,6,18,27].
The whole living cells of A. sclerotiorum and P. citrinum were
grown as described in Section 2.5. The mycelia were harvested by
filtration and 5.0 g were suspended in 100 mL of phosphate buffer
solution (Na HPO /KH PO , pH = 7, 0.1 M) in a 250 mL Erlenmeyer
2
4
2
4
flask. The supports matrices of commercial silica gel (10.0 g), syn-
thesized silica xerogel (10.0 g) or chitosan (3.0 g) were added to
each Erlenmeyer flask. The mixtures were incubated for 24 h in
The whole living cells of P. citrinum and A. sclerotiorum were
successfully immobilized as shown on the SEM micrographs
◦
an orbital shaker maintained at 32 C and 120 rpm. After filtration,
the immobilized mycelia were immediately used in the biocatalytic
reductions. The supported fungi were analyzed by scanning elec-
tron microscopy as described in the following section (Figs. 1 and 2).
(
Figs. 1 and 2). Both silica matrices present irregular dense parti-
cles with average particle size for silica larger than for the xerogels,
respectively 77 ± 20 ␮m and 41 ± 24 ␮m. It can be observed that

L.C. Rocha et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 160–165
163
Fig. 1. Scanning electron microscopy micrographs. (A) Free cells of A. sclerotiorum. (B) Free cells of P. citrinum. (C and D) Whole cells of P. citrinum immobilized on silica gel.
E and F) Whole cells of P. citrinum immobilized on silica xerogel.
(
the support matrices of silica gel, silica xerogel and chitosan are
closely intertwined with hyphae (Figs. 1 and 2). The hypha diame-
ter in both silicas are statically the same, 3.7 ± 2.1 ␮m for silica gel
and 2.6 ± 2.2 ␮m for the xerogels.
hindered the access of the substrate to the reductases, preventing
the reduction of the ketone 1 under these conditions.
However, the fungus P. citrinum immobilized on chitosan
catalyzed quantitatively the reduction of 1-(4-methoxyphenyl)-
ethanone (1) to alcohol (S)-3b (c 100%). Interestingly, in this case,
the reduction was greatly enhanced by this type of organic sup-
port matrix. The reaction with the fungus immobilized on chitosan
led to a total inversion of the configuration produced by the free
mycelium, yielding the (S)-alcohol 3b (ee > 99%) instead of the (R)-
alcohol 3a (Entry 1, Table 1). The inversion of configuration of the
alcohol (R)-3a (not immobilized) to (S)-3b (immobilized) showed
strong influence of immobilization in according to type of sub-
strates used (supports and/or ketones). However, is not possible to
confirm in this study the inversion of configuration, because during
immobilization maybe favored the action of another dehydroge-
nase, and not necessarily is the same enzyme that catalyzed the
The free (not immobilized) live mycelia of P. citrinum catalyzed
the anti-Prelog stereospecific reduction of 1-(4-methoxyphenyl)-
ethanone (1), and after 9 days the (R)-1-(4-methoxyphenyl)-
ethanol (3a) was produced with moderate optical purity and
conversion (ee = 69%, c 40%, entry 1, Table 1). The (R)-alcohol 3a was
isolated with 35% yield after purification by column chromatogra-
phy over silica gel. When the mycelia of the fungus P. citrinum were
immobilized on silica gel and silica xerogel, the reduction of ketone
1
failed to occur (entries 2 and 3, Table 1).
As can be seen in the micrographs of silica gel and silica xerogel,
the fungal cells were covered by the support materials (Fig. 1C–F).
It is possible that the total coverage of the mycelia must have

1
64
L.C. Rocha et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 160–165
Fig. 2. Scanning electron microscopy micrographs. (A) Whole cells of P. citrinum immobilized on chitosan. (B) Whole cells of P. citrinum immobilized on chitosan. (C and D)
Whole cells of A. sclerotiorum immobilized on silica gel. (E and F) Whole cells of A. sclerotiorum immobilized on silica xerogel.
reduction in the absence of support. Free and immobilized whole
living hyphal of P. citrinum were efficient yielding both enantiomers
by using a single microorganism. As can be seen in the SEM micro-
graphs, mycelium of P. citrinum was completely intertwined with
the chitosan matrix (Fig. 2A–B). SEM images of the P. citrium immo-
bilized on chitosan show a higher dispersion than over inorganic
matrices. Such entanglement among hypha and the chitosan is
probably related to the surface charges on the surface of the matri-
ces. The interactions between the organic matrix and mycelium
probably enhanced the affinity with ketone 1 and preserved the
activity of the enzyme that catalyzed the reduction of ketone 1 to
enantiomerically pure (S)-alcohol-3b. It was therefore concluded
that chitosan was an excellent support matrix for the immobi-
lization of filamentous fungi, such as P. citrinum, with a beneficial
influence on enzyme activity.
The whole cells of A. sclerotiorum immobilized on silica gel and
the free mycelium catalyzed the reduction of ketone 1 to alco-
hol (S)-3b with excellent activity and selectivity (c 100%, ee > 99%,
entries 5 and 6, Table 1). However, when the mycelia of P. cit-
rinum were immobilized on silica xerogel there was no reduction
of ketone 1. Possibly, this was due to the silica xerogel adhering
strongly to the surface of the hyphae and hindering access of the
substrate to the enzymes (entry 7, Table 1).
The whole free cells of P. citrinum catalyzed the bioreduction
2-chloro-1-phenylethanone (2) to (R)-alcohol 4a in accordance to
Prelog’s rule after 9 days and with low optical purity (ee = 31%,
c 70%, Entry 8, Table 1). When the live mycelium of P. citrinum
was immobilized on silica gel, it gave similar results to those
obtained with free cells in the reduction of ketone 2 (ee = 25%, c 65%,
entry 9, Table 1), except for a stereopreference for (S)-alcohol 4b

L.C. Rocha et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 160–165
165
following anti-Prelog’s rule. In this case the support matrix used
Acknowledgements
did not impair enzyme activity, but altered the chiral selectivity of
the reduction reaction.
The authors wish to thank Prof. R.G.S. Berlinck (Instituto de
Química de São Carlos – USP) for providing the marine fungal strains
and wish to thank Prof. Timothy John Brockson (Universidade Fed-
eral de São Carlos – UFSCar) for optical rotation measurements
in a Perkin-Elmer 241 polarimeter (Waltham, MA, USA). We also
acknowledge to Conselho Nacional de Desenvolvimento Cientí-
fico e Tecnológico (CNPq) and Funda c¸ ão de Amparo à Pesquisa do
Estado de São Paulo (FAPESP) for financial support. We also thanks
to Coordena c¸ ão de Aperfei c¸ oamento de Pessoal de Nível Superior
(CAPES) for the scholarship grant to LCR.
In addition, when the P. citrinum was immobilized on the silica
xerogel matrix ketone 2 was not reduced. This result, as expected,
confirmed that the silica xerogel matrix formed a strong barrier on
the surface of the mycelium hindering the access of substrate to
one or more enzymes involved in the reduction (entry 10, Table 1
and Fig. 1E–F).
Again, the whole cells of P. citrinum immobilized on chitosan
catalyzed the reduction of ketone 2 to alcohol 4a,b in excellent
yield (98% after purification by CC), but without selectivity (entry
1
1, Table 1). However, when cells of P. citrinum were immobilized
on chitosan the conversion to alcohol (RS)-3a,b was dramatically
increased (entry 11, Table 1).
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4
a in accordance to Prelog’s rule, with good optical purity (entries
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1
2 and 13, Table 1). Previously, the fungus A. sclerotiorum was
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[
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(
(
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Silica gel proved to be an interesting support matrix to immobilize
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1
559–1563.
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[
[
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