Williopsis californica, Williopsis saturnus,

and Pachysolen tannophilus: Novel

Microorganisms for Stereoselective
Oxidation of Secondary Alcohols

J. D. Carballeira Rodrı´guez,1 C. Garcı´a-Burgos,1 M. A. Quezada Alvarez,1

E. Alvarez Ruiz,2 J. V. Sinisterra Gago1
1
Biotransformations Group, Department of Organic and Pharmaceutical
Chemistry, Faculty of Pharmacy, Universidad Complutense, 28040 Madrid,
Spain; telephone:+34-91-394-18-20; fax:+34-91-394-18-22;
e-mail: jvsgago@farm.ucm.es
2
Centro de Investigacio´n Ba´sica, GlaxoSmithKline, Tres Cantos, Madrid, Spain
Received 30 January 2004; accepted 29 March 2004

Published online 6 August 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20150

Abstract: A screening of 416 microorganisms from different
taxonomical groups (bacteria, actinomycetes, yeasts, and
filamentous fungi) has been performed looking for active strains
in the stereoselective oxidation of secondary alcohols. The
working collection was composed of 71 bacterial strains, 45
actinomycetes, 59 yeasts, 60 basidiomycetes, 33 marine fungi,
and 148 filamentous fungi. All microorganisms selected were
mesophilic. Yeasts were the most active microbial group in the
whole-cell-catalyzed oxidation. Williopsis californica, Williopsis
saturnus, and Pachysolen tannophilus were the strains of
greatest interest, both as growing cells and as resting cells. The
oxidation of the alcohols takes place when cells are in the
stationary growth phase (after 48 h of culture). These three
strains are S-stereoselective for the oxidation of racemic
secondary alkanols and show stereospecificity in the oxidation of
menthol or neo-menthol, whereas iso-menthol is not oxidized. In
the case of the 1-tetrahydronaphtol enantiomers, only the S-
enantiomer is oxidized. The three strains were immobilized by
entrapment using agarose and agar from algae of the Gracilaria
genus. The agarose derivatives displayed significant
improvement in the stereospecificity of the reactions..
Keywords: alcohol oxidation; stereospecific; stereoselective;
whole cells; biocatalysts
INTRODUCTION
The oxidation of alcohols rarely exhibits insurmountable
problems to the synthetic organic chemist, but there are
some situations where the use of biocatalysts for the
oxidation of alcohols could be of particular interest: (i)
Enzymatic oxidation of primary alcohols for the production
of aldehydes (Gandolfi et al. 2001; Molinari et al., 1995;
Villa et al. 2002). (ii) Stereospecific oxidation of one
enantiomer of a racemic secondary alcohol (Faber, 2000).
This reaction has a drawback in that 50% of the reagent is
destroyed. There are two methodologies for overcoming
this problem: (a) coupled redox reactions catalyzed by
alcohol dehydrogenases (one of the stereomers is
oxidized to a ketone that is stereoselectively reduced to
the secondary alcohol), and (b) Consecutive reactions
catalyzed by alcohol dehydrogenases. Both processes
are used in the stereoinversion of a secondary alcohol
and 100% of the opposite stereomer can be achieved. (iii)
Regioselective oxidation of polyols. In this case, the use
of biocatalysts for oxidation is of much interest because
the oxidation of these compounds requires a number of
protection and deprotection steps if performed by
chemical means. Sugars and related polyhydroxy
compounds have been oxidized selectively to produce the
corresponding keto-ols and keto acids using strains such
as Acetobacter suboxydans or Pseudomonas sp. (Faber,
2000).
The oxidation of alcohols could be carried out by alcohol
dehydrogenases and other oxidative enzymes (Holland,
1992). The alcohol dehydrogenases (ADHs) are
ubiquitous intracellular enzymes that are present in
microorganisms, plants, and animals. ADHs are
enzymes that use either NAD + or NADP+ as cofactor
(Duester et al., 1999; Jornwall, 1977). Because a
continuous enzymatic reaction needs regeneration of the
cofactor, the oxidation of alcohols is usually performed
using whole cells. Cofactor recycling can be efficiently
achieved when whole microbial cells are used as
biocatalysts for redox reactions. In this case, cheap
carbon sources, such as carbohydrates, can be used
because the microorganism possesses all enzymes and
cofactors required for the recycling (Faber, 2000).
In this work, a hierarchical screening process has been
performed in looking for new whole-cell biocatalysts
useful in the oxidation of alcohols. As a result from the
screening campaign, three active strains for the
oxidation of secondary alcohols are reported for the first
time: Williopsis californica, Williopsis saturnus, and
Pachysolen tannophilus. The catalytic activity of the
three strains has been studied extensively and
 characterized both as growing and as resting cells. The
synthetic activity of their immobilized derivatives has
been determined.
METHODS
Chemicals
All substrates for the oxidation reactions were obtained
from Sigma-Aldrich. All culture media components were
from Difco Laboratories or Merck. Agarose LM3 and agar
from Gracilaria algae genus were generously provided by
Hispanagar.
Microbial Collection
The microorganisms used in this study were obtained
from different public collections: the American Type
Culture Collection (ATCC); the Fungal Biodiversity Center,
Utrecht, The Netherlands (CBS); the International
Mycological Institute, UK (IMI); the German Collection of
Cell Cultures, Germany (DSMZ); and the National
Collection of Yeast Cultures, UK (NCYC).
Conservation of Microorganisms
Bacteria, actinomycetes, yeasts, filamentous, and marine
fungi were conserved as suspensions in a 30% glycerol
solution. All these strains were stored at 80jC in Nunc
cryotubes. Basidiomycetes were also conserved as cell
suspensions in 30% glycerol and stored in a liquid
nitrogen tank.
Culture Media
A preliminary screening was performed to select the most
appropriate culture media for each type of microorganism.
The following culture media were then selected for the
different microbial groups: Bacteria: LB medium (Frenken
et al., 1992): Tryptone (Difco), 10 g/L; yeast extract
(Difco), 5 g/L; NaCl (Merck), 5 g/L; KH 2PO4 buffer (pH
6.5). Actinomycetes: ABME (Carballeira, 2003) medium:
CaCO3 (Merck), 10 g/L; FeSO 4 7H2O (Merck), 0.003 g/L;
KCl (Merck), 0.5 g/L; MgSO4 7H2O (Merck), 0.5 g/L; meat
extract (Oxoid), 5 g/L; malt extract (Difco), 40 g/L. Yeasts:
yeast extract (Difco), 3 g/L; malt extract (Difco), 3 g/L;
bactopeptone (Difco), 5 g/L; bactodextrose (Merck), 10
g/L. Basidiomycetes: lyophilized potato, 22 g/L; dextrose
(Merck), 20 g/L. Filamentous fungi: HAGGS medium
(Vázquez et al., 2003) (adjust to pH 6.6): glycine, 2 g/L;
tryptic soy broth, 6 g/L; starch, 20 g/L; mineral solution, 10
mL/L. Mineral solution: FeSO4 7H2O, 1 g/L; MnSO4 4H2O,
1 g/L; CuCl2, 0.025 g/L; CaCl2, 0.10 g/L; H3BO3, 0.056
g/L; ZnSO4 7H2O, 0.2 g/L; (NH4)6Mo7O24 4H2O, 0.019 g/L.
Marine fungi: lyophilized potatoes, 22 g/L; dextrose
(Merck), 20 g/L; NaCl (Merck), 5 g/L.
Screening Methods
Screening Method A: Growing Cells
Bacteria and yeasts. Conical 100-mL flasks containing 20
mL of the selected culture media were inoculated with 50
uL of the microbial cells suspension in glycerol (previously
thawed). Cultures were performed in an orbital shaker at
28°C and 250 rpm (Alphand et al., 1990; Sandey and
Willets, 1992). After 48-h incubation, the ketone used as
substrate was added to the flasks at a 10 mM final
concentration (Alphand and Furstoss, 1992; Sandey and
Willets, 1992). The reaction time was 72 h. When the
reaction was finished, the content of the conical flask was
transferred into a Falcon tube and 5 mL of ethylacetate
(containing 1 mg/mL of hexadecane as internal standard)
was added. After vortexing for 10 s, the organic phase
was transferred to a 2-mL Hewlett-Packard vial. All the
reactions were repeated three times and the results
displayed are the arithmetic mean values.
Other microorganisms. For the other microbial groups,
the screening process was essentially the same as just
described. Culture and reaction times were 72 h for
actinomycetes and filamentous fungi. For basidiomycetes
and marine fungi the culture time was set to 120 h and the
reaction time was 72 h.
Screening Method B: Resting Cells
The culture conditions were the same as in the case of
growing cells. Once the culture time defined for each
group of microorganisms was reached, the content of the
conical flask was transferred into a Falcon tube, and
centrifuged during 15 min at 4000 rpm. The cells were
then washed three times using 20 mL of 50 mM KH 2PO4
buffer (pH 6.5) and sedimented by centrifugation using the
conditions previously described. When the cells were free
of culture media they were resuspended in 20 mL of the
same KH2PO4 buffer, in a 100-mL conical flask. Then, the
substrate was added to the reaction media at a 5 mM final
concentration. The flasks were shaken at 28°C and 250
rpm in an orbital shaker (Khuner).
Screening Method C: Lyophilized Cells
The experimental protocol followed in this case was similar
to that used for the resting cells. After washing, the cells
were quickly frozen at 80°C and lyophilized for 72 h in a
Lab-Conco lyophilizer. The reaction was performed using
100 mg of lyophilized cells in 5 mL of 50 mM KH 2PO4
buffer (pH 6.5) and the substrate concentration was 5 mM.
Reactions were performed in nonreactive plastic flasks
(pyrolysis type) at 28°C with stirring at 100 rpm (Molinari et
al., 1999a).
Analysis: Gas Chromatography
Analysis of the samples from the cycloalkanol reactions
was performed with a Hewlett Packard 5890 Series II gas
chromatograph equipped with an automated sampler (Agi
lent Technologies) and an electrolytic hydrogen generator
UHP-601 (Dominick Hunter). The capillary column was a
Carbowax SGL-1000 (60 m, 0.25 mm, 0.25 Am) from
Sugelabor (Spain). Starting from the conditions proposed
by Carnell and Willets (1992), we defined the following
analytical parameters: Ti = 155°C; ti = 1 min; Tf = 175°C; Tf
= 10 min; heating rate = 4jC/min; carrier flow (He) = 40
psi; and split ratio = 100. Injector and detector
temperatures = 250°C.
Analysis of the oxidation of 1-furyl-ethanol, 1-phenyl
ethanol, 1-tetrahydronaphtol, and menthol stereoisomers
was performed using a Varian 3400cx gas chromatograph
equipped with an automated sampler. A CP-7502 Carbo
wax capillary column (25 m, 0.39 mm) from Sugelabor
(Spain) was employed in the following conditions: T i =
90°C; ti = 5 min; heating rate = 5jC/min; Tf = 175°C; and tf
= 7 min. Carrier flow (He) = 25 psi, and split ratio = 100
mL/min. Injector and detector temperatures = 250°C.
Calculation of the yield was performed by dividing the
peak area of product by that corresponding to the
standard extraction value of the substrate in the same
conditions. The standard extraction value was established
using data from 10 conical flasks with the same volume of
culture medium, stirring speed, temperature, and reaction
time. The substrate was extracted using ethylacetate and
analyzed by gas chromatography. No significant variation
in the value of the area was detected between flasks. The
arithmetic mean of these areas was used for yield
determination. The standard extraction value is a
reference that serves to avoid potential problems owing to
the physicochemical properties of the organic substrates
tested.
Determination of Cell Concentration in Cultures
For determination of productivity, cell concentration was
measured in the different cultures. Cell densities were
determined using 250-mL conical flasks (50 mL of culture
medium) inoculated with 20 mL of preincubated culture (24
h). Samples were picked up at different culture times. The
cell concentration was determined using absorbance at
660 nm (1). The calculations were performed according to
the following expression: Abs (DO) = 1.27 10 2 c (106 Immobilization in Agarose LM3 (Hispanagar)
cells/mL) + 1.26 10 2 (N = 9, R2 = 0.9692). The yeast
culture medium was as described earlier.
Influence of Culture Time in Oxidation
of Cyclohexanol
The reactions were performed at 10 mM cyclohexanol (pH 7.0). The substrate was then added at a 2.5 mM
concentration, using the experimental procedure concentration and the reaction was performed at 28jC and
described earlier. The alcohol was added to the culture 100 rpm. Samples were taken at different reaction times,
flask at different times (ti in Table IV). The addition time
was referred as t = 0. The samples were picked up and
analyzed as described earlier.
Oxidation of Chiral Substrates Using Whole Cells
The substrate concentration was set to 2.5 mM and the
reaction time was 48 h. The rest of the procedure was
described earlier in the subsections on screening methods
of A (growing cells) and B (resting cells).
Immobilized Derivatives
Whole cells can be immobilized in a matrix by covalent
binding, by physical adsorption, or by gel entrapment
(Bickerstaff, 1997; Mosbach, 1986; Tischer and
Wedekind, 1999; Wijffels et al., 1996). We used the
entrapment technique due to our extensive experience
with this technique in our laboratory. The conditions for
entrapment are very important because it is necessary
that substrate and product molecules are able to cross the
barrier that the matrix re presents (Faber, 2000). Thus, the
immobilization conditions could affect the activity and
selectivity of the biocatalysts (Sinisterra and Dalton,
1996).
Cell Preparation
The yeasts were cultured following the conditions
previously defined, and the cells were obtained by
centrifugation at 4000 rpm (15 min).
Immobilization in Agar From Gracilaria Algae
A solution of 2% agar was prepared and sterilized (0.5
atm, 15 min). The hot agar solution was allowed to stand
for cooling, and the temperature was monitored using a
contact thermometer. When the temperature of the
solution reached 45j to 50jC, the cells corresponding to 20
mL of culture were added (to have a comparative value
with free cells assays), maintaining vigorous stirring using
a glass bar. The mixture was poured into a plastic
container with 0.5-cm depth and, after a couple of hours
of hardening, the gel was cut using a scalpel into cubes of
approximately 0.5 cm per side.
Immobilization in agarose LM3 was performed following
the same method as described for Gracilaria agar, but the
concentration of agarose was set to 6%.
The immobilized derivatives were added to a 100-mL
conical flask containing 20 mL of 30 mM phosphate buffer
as shown in Figures 5 and 6.
 Table I. Microorganisms showing yields of >20% cyclohexanol oxidation [cyclohexanol] at 10 mM.
Reaction time 72 h; T = 28jC; stirring speed 250 rpm. B, bacteria; BS, basidiomycete; FF, filamentous fungi; MF, marine fungi; Y, yeast. Boldface: strains
selected for the second phase of screening.
RESULTS AND DISCUSSION
Primary Screening: Cyclohexanol Oxidation
In the experimental conditions of the screening, the ability to
oxidize cyclohexanol to cyclohexanone (yield >10%) was
more common in yeasts (24% of tested yeasts) and in
bacteria (19% of the tested bacteria) than in the other
taxonomic groups tested (marine fungi 12%, actinomycetes
11%, filamentous fungi 3%, and basidiomycetes 2%). Very
few filamentous fungi strains display activity in the oxidation
of cyclohexanol. This situation is opposite of that observed
in the case of the inverse reaction, in which this microbial
group was the most active (Carballeira, 2003). The
microorganisms showing the highest yields are shown in
Table I.
Among the strains displaying ketone yields of >40%, the
most active strains are also yeasts. Two strains from the
Williopsis genus are the most active strains. There are
only two active bacteria, Rhodococcus rhodochrous DSMZ
11097 and the well-reported Rhodococcus erythropolis
(Van der Werf et al., 1989; Van der Werf and Boot, 2000).
During this study, several microorganisms were found to
produce compounds other than cyclohexanone. These
products were detected using the coupled gas
chromatography/ mass spectrometry technique. They were
identified and studied when possible, but this topic is
beyond the scope of the present study. It is obvious that
the absence of secondary reactions is very important for
selection of new strains for biotransformations. Therefore,
only the microorganisms showing the best conversion
rates and not displaying side-reactions were selected. The
selected strains are also defined in Table I (bold type).
Coniochaeta velutina and Tetracladium setigerum were the
only non-yeast strains that displayed considerable activity
without side-reactions.
The microorganisms selected, capable of becoming new
useful biocatalysts, were assayed in different physiological
conditions—growing cells, resting cells, and lyophilized
cells—to determine whether the physiological state of the
microorganisms can affect the oxidation process. The
results obtained in each condition are shown in Table II.
From these results we can deduce that lyophilization of
the microbial cells is not a good methodology to carry out
the oxidation of cyclohexanol when compared with
conventional growing conditions. This result contrasts with
that described by Molinari (1999b), who described the
lyophilization of yeasts as a useful technique to obtain
active biocatalysts, and was previously described by our
group for reduction of biocatalysts (Carballeira, 2003).
Also, the product yields obtained using resting cells are
lower than those obtained with growing cells. The strains
that displayed better yields in the experimental conditions
tested—growing, resting, and immobilized cells—are W.
californica CBS 2158, W. saturnus NCYC 2313, and P.
tannophilus NCYC 1597. Therefore, these strains could be
used in resting cells conditions, and they are candidates to
be immobilized in a different matrix.

Table II. Cyclohexanone yield using growing, resting, and lyophilized cells
[cyclohexanol] at 5 mM (10 mM for growing cells).
Figure 1. Tolerance of the microorganisms to cyclohexanone. Profile of
cyclohexanol oxidation using different substrate concentrations. The
selected microorganisms were tested as growing cells. The reaction
was performed at 28jC and 250 rpm with growing yeast cells (48-h
culture time) or filamentous fungi (72-h culture time). The contact time
for strain– substrate was 72 h.

Kinetic Profile of Oxidation Reactions

The microorganisms selected for cyclohexanol oxidation
reached maximum activity near 48 h (Fig. 2). All strains
displayed an induction-like period in the production of
cyclohexanone, between 0 and 24 h, with the exception
of P. tannophilus. This fact could be related to problems
in the diffusion of substrate/products through the cell
membranes. Finally, we can deduce from Figure 2 that
W. californica, W. saturnus, and P. tannophilus reach
higher yields in the oxidation of cyclohexanol.

Reaction time 72 h; T = 28°C; shaking speed 250 rpm (100 rpm for
lyophilized cells).

Influence of Substrate Concentration on Microbial

Activity: Substrate Toxicity Assays
All microorganisms showed the same qualitative
behavior, showing low tolerance to cyclohexanol (Fig.
1). Concentrations of cyclohexanol at >20 mM produced
a large decrease in the maximum yield obtained.
Therefore, to scale-up the process, the cells should be
immobilized (Villa et al., 2002) and a two-phase system
should be used (Miro liaci and Nemat-Gorgani, 2002).
Both Williopsis strains and P. tannophilus were the most
resistant microorganisms.

Figure 2. Kinetic profile of the cyclohexanol oxidation reaction using whole

cells as biocatalysts. The reaction was performed at 28jC and 250 rpm
with growing yeast cells (48-h culture time) or filamentous fungi (72-h
culture time). The substrate concentration

Oxidation of Monocyclic Alcohols
The oxidation of cycloalkanols with a different ring size
was used to explore the selectivity of the strains against
the molecular size of the substrates. All microorganisms
from Table II were tested for activity against cyclobutanol
2, cyclopentanol 3, cyclohexanol 1, cycloheptanol 4, cyclo
octanol 5, and cyclododecanol 6, and used as substrates.
The results obtained are shown in Table III. In all cases,
the cycloalkanol concentration was 10 mM to avoid a
decrease in catalytic activity due to alcohol toxicity:
From these data we can conclude that all the strains
are active against cycloalkanols of four (2), five (3), and
six (1) carbon atoms. Dodecanol (6) is oxidized, with
moderated yield, by P. tannophilus, C. velutina, W.
saturnus Fel lomyces sp., and A. fermentans.
W. saturnus and K. Lactis are selective against cyclo
hexanol and small cycloalkanols, whereas W. californica,
P. tannophilus, and Fellomyces sp. can be considered
non selective strains. Finally, taking into the account the
results reported up to now, in addition to the scale-up of
the culture conditions and the absence of secondary
reaction products, three strains were selected for further
studies. Two strains were selected as nonselective strains
(W. californica and P. tannophilus), whereas W. saturnus
was found to be strain selective for small cycloalkanols.
Tabla III. Oxidation of different monocytic alcohols
Reaction time 72 h; T = 28°C; 250 rpm. Yeasts were cultured for 48 h
and filamentous fungi for 72 h. Substrate concentration 10 mM.
Enzyme Productivity
The next step was to determine the culture time for
maximum production of the enzymes involved in the
process. This point is very important if we intend to carry
out the reactions with immobilized whole cells, because
the immobilization must be performed using cells with the
maximum enzymatic activity.
Figure 3 shows the growth profiles of the three strains.
The biomass produced by W. saturnus was higher than
that obtained with W. californica or P. tannophilus.
Afterwards, the oxidation of cyclohexanol was performed
using whole cells grown at different culture times (t i). At
these ti times, the cell number was determined as
described in Methods. Cyclohexanol was added (see
Methods) to the culture medium and the
biotransformation was followed by GC until 48 h
(according to Fig. 2). At the end of the experiment, the
cell number was measured by absorption at 660 nm. The
values of the initial and final results of cell concentration
and the yield obtained, after 48 h, are shown in Table IV.
Some interesting results can be concluded from the
data in Table IV. After addition of cyclohexanol, the
microorganisms are able to grow up to the maximum
value achieved in normal growth conditions (Fig. 3).
Therefore, the concentrations of cyclohexanol used
are not toxic for the cells. For W. saturnus, the yield
achieved after 48 h is constant if the addition of
cyclohexanol is carried out at culture times of >10 h.
However, the longer the addition times the lower the
yield in the case of W. californica. This result seems to
indicate that the enzyme of the second yeast is less
stable than the enzyme from W. saturnus.
To get a better understanding of this topic, the reaction
profiles from each microorganism were recorded. The
results obtained with the microorganisms tested are
shown in Figure 4.
In all cases, we can see that the shorter the addition
times the longer the time needed to start the
biotransformation process. It seems that cyclohexanol
is biotransformed only after cell growth is finished. This
result is general for the three strains W. californica, P.
tannophilus, and W. sa turnus. Therefore, the next
biotransformations were performed after a 48-h culture
period.
Figure 3. Growth profiles for W. saturnus, W. californica, and
P. tannophilus.
Figure 4. Reaction profiles in the oxidation of cyclohexanol when the
addition of cyclohexanol was performed at different culture times (T i). (A)
W. saturnus t1 = 10 h; t2 = 20 h; t3 = 23 h; and t4 = 48 h; (B) W. californica
t1 = 7 h; t2 = 10 h; t3 = 13 h; and t4 = 48 h. (C) P. tannophilus t 1 = 10 h; t2 =
20 h; t3 = 24 h; and t4 = 48 h.
Table IV. Influence of the culture time in the oxidation of cyclohexanol
using W. saturnus, W. californica, and P. tannophilus.
Reaction conditions: [cyclohexanol] 10 mM; T = 28!C;
shaking speed
250 rpm.
a
Concentration of cells when 10 mM cyclohexanol was
added
b
Concentration of cells when the reaction was stopped at
48 h.
 Figure 5. Oxidation of (1R)-furyl-ethanol and (1S)-furyl-ethanol by
immobilized derivatives of W. californica and P. tannophilus using
Gracilaria agar (2%) as immobilization matrix. Reactions were
performed at a 2.5 mM substrate concentration and 100-rpm
shaking speed.

Figure 6. Oxidation of (1R)-furyl-ethanol and (1S )-furyl-ethanol by immobilized derivatives of W. californica and P. tannophilus using agar ose LM3 (6%)
as immobilization matrix. Reactions were performed at a 2.5 mM substrate concentration and 100-rpm shaking speed

Table V. Product yields obtained using W. californica CBS 2158, W. saturnus NCYC 2313, and P. tannophilus NCYC 1597 as whole-cell biocatalysts
in oxidation of 7, 8, 9, 10, 11, 13, and 14.
 Reaction time 48 h; T = 28°C; shaking speed 250 rpm. Substrate concentration 2.5 mM.
a
Yields are the arithmetic mean values of three assays. No activity was observed against iso-menthol 15.

Oxidation of Alcohols With Different Structures

To explore stereocontrol in the oxidation of alcohols catalyzed by the three selected strains, the oxidations of (1R)-(7)
or (1S)-(2-furyl)-ethanol (8), (1R)-(9) or (1S)-1- phenyl ethanol (10), (1R)-tetrahydronaphtol (11), and (1S)-
tetrahydronaphtol (12), and the oxidations of neo-menthol (13), menthol (14), and iso-menthol (15) were performed.
The addition of alcohol was performed after a 48-h culture time, according to the data indicated earlier. Oxidation was
carried out in batch culture or in resting cells conditions. Lyophilized whole cells were not used due to the low yields
obtained (Table II, see earlier). The three selected microorganisms were poorly active in the oxidation of primary
alcohols to aldehydes (yield < 10%). The detailed results obtained against these chiral compounds are displayed in
Table V.
According to the results in Table V we can observe that all the strains are stereoselective versus the S-stereomer. The
enantio preference is remarkable in the case of tetra hydronaphtol, where no conversion is detected against the R-
enantiomer, whereas the S-enantiomer is well oxidized by all the strains, especially by W. californica, showing the best
conversion rates and the highest enantiopreference. Interestingly, the biocatalysts show higher enantiopreference
against the most rigid of the structures tested. Depending on the type of substrate, resting cells lead to better yields
than growing cells in the cases of W. californica and P. tannophilus, while using W. saturnus the results are very
similar. These results are different from those in Table II, where the growing cells gave the best results, according to
data reported by Molinari et al. (1999b) in the case of (R,S)- 2-phenyl-1-propanol. Therefore, the structure of the
substrate and/or the product should be considered when selecting the best experimental conditions.
The oxidations of (-)-neo-menthol ((1R,2R,5S)-2-isopropyl-5-methyl-cyclohexanol), 13, (+)-menthol ((1S,2R,5S)- 2-
isopropyl-5-methyl-cyclohexanol), 14, and iso-menthol ((1S,2R,5R)-2-isopropyl-5-methyl-cyclohexanol), 15, were
performed to explore the stereocontrol in the oxidation of chiral secondary cycloalkanols. No activity was observed
against iso-menthol. P. tannophilus showed minor activity in the oxidation of menthol and neo-menthol, both with growing
cells and in resting cells conditions:

From the results obtained in the reactions with the menthol stereoisomers, it may be concluded that the equatorial
position of methyl group is determinant for the enzymatic activity, and the OH position—equatorial or axial—not so
important. Nevertheless, in all cases, the equatorial position of the OH (menthol) is preferred to the axial position (neo
menthol). As in the cases of (1R) or (1S)-(2-furyl)-ethanol, enantiomeric excess is low, indicating high tolerance of these
strains to different structures and geometries.

Immobilization

According to previous studies on the immobilization of whole-cell biocatalysts for Baeyer–Villiger monooxygenation
(Carballeira, 2003), we attempted to define specific immobilization conditions that could improve the enantiopreference
of the strains against the flexible structures where selectivity is low. Growing cells from W. californica and from P.
tannophilus were immobilized by entrapment techniques using Gracilaria agar (2%) and agarose LM3 (6%). The
immobilization conditions were fixed according to the best results obtained from a statistical experimental design of the
parameters (Carballeira, 2003). The substrates selected were the furyl-ethanol enantiomers due to the low
enantiopreference shown against these substrates. Thus, an improvement in the selectivity of the immobilized
derivatives could be easily detectable.
Figure 5 displays the behavior of the strains immobilized in Gracilaria agar as matrix. The immobilization derivatives of
W. californica display similar conversion values to the reactions performed using free growing cells. However, the
immobilization of P. tannophilus leads to a decrease in the activity against both enantiomers. Consequently, no
enhancement in the enantiopreference is achieved using Gracilaria agar as matrix.
The immobilization in 6% agarose LM3 maintains the activity of W. californica against the (1S)-furyl-ethanol in the
same values as free cells, but activity against (1R)-furyl ethanol is not detectable. This effect is also observed in the
case of P. tannophilus, but in this case the activity against the S-enantiomer is lower than when using free cells.
According to these results, the agarose LM3-immobilized derivatives may be a suitable tool for producing
deracemization of a racemic mixture of both alcohols.
From these results, the presence of two enzymes could be hypothesized, each one with an opposing enantiopre
ference, with different kinetic properties, and affected in a different way for the immobilization process. Another
explanation for this behavior is that the mechanism of the membrane transport could be different for each enantiomer
and may be influenced in a different manner by the immobilization matrix.This point is currently being investigated.