Immobilised derivatives of Williopsis californica, Williopsis saturnus,

Pachysolen tannophilus: New biocatalysts useful in the

stereoselective oxidation of 1-tetralol
a,1 a,1 a,1 b c,∗
J.D. Carballeira , C.A. Garc´ıa-Burgos , M.A. Quezada , E. Alvarez , J.v. Sinisterra-Gago
a b
Biotransformations Group, Department of Organic and Pharmaceutical Chemistry, Faculty of Pharmacy, Universidad Complutense, 28040 Madrid, Spain Centro
de Investigacion Basica, GlaxoSmithKline, 28760 Tres Cantos, Madrid, Spain
c
Scientific Park of Madrid, B.P. Solar Complex, 28760 Tres Cantos, Madrid, Spain
Received 19 January 2006; accepted 26 January 2006

Abstract
Four new whole cell biocatalysts have been selected after a high throughput screening of microorganisms from public collections: Williopsis
californica CBS 2158, Williopsis saturnus NCYC 2313, Pachysolen tannophilus NCYC 1597 and Coniochaeta velutina CBS 981.68, in basis to the
high yield and stereoselectivity in the oxidation of 1-tetralol. These microorganisms were immobilised in different supports by entrapment techniques,
being especially remarkable the results obtained with different tailor made agar matrix from Hispanagar (Spain). The selection of the immobilization
conditions for each strain was performed following a statistical design based on the response surface methodology. The immobilised biocatalysts –
Williopsis californica and Williopsis saturnus – immobilised in tailor-made agar (2.5%) from Pterocladia and Gracilaria (Hispanagar S.A.) carry to
100% yield in the oxidation of cyclohexanol. The immobilised biocatalysts displayed a high S-stereoselectivity (ee > 98% and 50% yield) in the
oxidation of (R,S)-1-tetralol and a high yield in the oxidation of bulky substrates as 2-adamantanol (100%). The kinetic studies showed that the diffusion
of reagents/products is the rate controlling step. The addition of glucose (0.5%) increases the oxidation yields.

Keywords: Whole-cell immobilization; Stereoselective alcohol oxidation; Biocatalysis

1. Introduction widely used and many natural and synthetic polymers have been
described [2], presenting advantages and disadvantages. Thus,
Immobilised microbial cells are used in organic synthesis to different matrix has been described such as polyvinyl alcohol,
fully exploit the technical and economical advantages that they polyacrylamide, alginate, agar, etc. [5,8–10]. The conditions for
display in comparison with free cell suspensions. They are able to the entrapment are very important because it is necessary that both
remain operationally active for more reaction cycles than actively substrate and product molecules can cross the barrier that
growing cell cultures [1,2]. In addition to this, the easy separation represents the matrix [8]. In addition, the shape of the immobilized
of the biocatalyst from the reaction mixture simplifies the catalyst (fibres, beads, foils or cylinders) is an important parameter
downstream process. Also, the immobilised derivatives can often to control the diffusion process.
be re-used repeatedly [3,4]. An ideal immobilised derivative
provides a well-balanced overall performance, with reasonable In the oxidation of alcohols, there are some cases where the
conversion yields, high operational stability and low mass transfer enzyme technology presents advantages over conventional
limitations [5]. chemical oxidants, for example, the oxidation of prochiral polyols,
the stereospecific oxidation of a racemic secondary alcohol [11]
Some of the most successful industrial biotransformations use and the oxidation of primary alcohols to aldehydes [12,13].
immobilised microbial cells [3,6,7]. The lyophilization of bacterial In the present paper we describe the results obtained in the
cells is also described as an alternative to the immobilisation immobilisation of three new yeasts: W. californicaCBS 2158, W.
methodology in order to obtain active biocatalysts, but the saturnus NCYC 2313 and Pachysolen tannophilus NCYC 1597
lyophilised derivatives were not as reusable as in the immobilized and one filamentous fungus, Coniochaeta velutina CBS 981.68,
whole cells, as reported in the case of Acetobacter acetii [4]. isolated after a microbial screening of 416 microorganisms from
Whole cells can be immobilised in a matrix by covalent binding, public collections. These studies allowed us to obtain and
physical adsorption or by gel entrapment. Gel entrapment is characterize different immobilised derivatives that displayed high
catalytic activity in the oxidation of alcohols.

2. Materials and methods
2.1. Chemicals
Cyclohexanol, cyclohexanone, sodium alginates with low,
medium and high viscosity values were obtained from Sigma–
Aldrich; acrylamide-bis acrylamide, and ammonium persulphate
were purchased to Bio-Rad. The chemical reagents were obtained
from Sigma–Aldrich. The culture media components were from
Difco Laboratories and Merck. Agarose D5 (lot F-5915)—gelling
point (g.p.) (1.5%) T = 36 ± 2 ◦C, sulphate < 0.012%. Several tailor
made agar types: Ref A27/03 (low methoxylation degree agar
from Pterocladia—g.p. 34 ± 2 ◦C), Ref A28/03 (high
methoxylation degree agar from Gracilaria—g.p. 42 ± 2 ◦C) and
Ref A90 (medium methoxylation degree from Gelidium—g.p. 36
±± 2 ◦C) were provided by Hispanagar S.A. (Spain). The amount
of sulphate groups was between 1.5% and 2.5%.
2.2. Microorganisms
The yeasts W. californica CBS 2158, W. saturnus NCYC 2313 and
P. tannophilus NCYC 1597 were conserved in Nunc® cryotubes at –80
◦
C in a 30% glycerol solution. The culture medium used for these
yeasts was YM: 3 g/L yeast extract (Difco); 3 g/L malt extract (Difco);
5 g/L bactopeptone (Difco); 10 g/L bactodextrose (Merck).
The filamentous fungus C. velutina CBS 981.68 was conserved as
a spore suspension in the same conditions. The culture medium used
for this fungus was HAGGS [14] (adjust to pH 6.6): 2 g/L glycine; 6
g/L soy broth; 20 g/L starch; mineral solution 10 mL/L. Mineral
solution: 1 g/L FeSO4·7H2O; 1 g/L MnSO4·4H2O; 0.025 g/L CuCl2;
0.10 g/L CaCl2; 0.056 g/L H3BO3; 0.2 g/L ZnSO4·7H2O; 0.019 g/L
(NH4)6Mo7O24·4H2O.
2.3. Factorial experimental design
In order to select the best immobilisation conditions for each
matrix using as less assays as possible, we performed a factorial
experimental design [15–18] using the software package Statgraphics
v.4.1 from Statistical Graph Corp (US). The number of experiments
were 2n–1, where n = number of independent variables. Two centre
points were performed to obtain standard deviation and confidence
limits.
2.4. Oxidation of cyclohexanol
2.4.1. Oxidation using growing cells
Yeasts: conical 100 mL flasks containing 20 mL of the selected culture m edium
were inoculated with 50 L of the glycerol suspension. The culture was
incubated in an orbital shaker at 250 rpm and 28 ◦C [19,20]. After 48 h
culture time the ketone used as substrate was added to the flask at a 10
mM final concentration [21,22]. 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 ethyl acetate (containing 1 mg/mL of
hexadecane as internal standard) were added [19,20]. After vortexing
for 10 s, the organic phase was transferred into a 2 mL Hewlett-
Packard vial. All the reactions were repeated three times and the
reported results are the average of the three experiments.
The experimental procedure followed with the fungus C. velutina
CBS 981.68 was the same as previously reported but the culture and
reaction times were 72 h.
2.4.2. Oxidation using resting cells
The culture conditions were the same as described above for
growing cells, but after the culture time defined for each taxonomic
group of microorganisms, the content of the conical flask was
transferred to a falcon tube, and centrifuged during 15 min at 4000
rpm. Then the biomass was removed and washed three times using 20
mL of 50 mM phosphate buffer (pH 6.5). When the cells were
apparently free of culture medium, they were resuspended using 20
mL of the same buffer in a 100 mL conical flask and the substrate was
added to the reaction media at a 5 mM final concentration. The flask
was shaken at 250 rpm and 28 ◦C in an orbital shaker.
2.4.3. Oxidation using lyophilised cells
Cells were obtained following the same experimental protocol used
for resting cells conditions. After washing cells, they were quickly
frozen at −80 ◦C and lyophilised for 72 h in a Lab-Conco
lyophilisation device. For the reactions, 100 mg of lyophilised cells
were added to 5 mL of 50 mM phosphate buffer (pH 6.5), and the
substrate was added to the reaction media at a 5 mM final
concentration. The flasks were shaken at 100 rpm and 28 ◦C in an
orbital shaker.
2.5. Immobilisation
2.5.1. Preparation of cells
The cells were cultured in 100 mL Erlenmeyer flasks containing 20
mL of YM culture medium. Incubations were performed at 28 ◦C and
250 rpm for 48 h, using a Khuner shaker. Afterwards, cells were
centrifuged for 15 min at 4500 rpm ¨ using 50 mL Falcon tubes. The
supernatant was discarded and the pellet was washed using 50 mM
phosphate buffer (pH 6.5) and centrifuged again. This process was
repeated twice.
The amount of cells in a culture flask was used as a unit for the
reactions with cyclohexanol. The biomass of 1, 2 and 3 Erlenmeyers
was used during the factorial experimental design.
2.5.2. Immobilisation in polyacrylamide
The immobilisation using polyacrylamide as matrix was performed
using different concentrations of the mixture acrylamide/bis-
acrylamide (2.5%, 7.5%, and 12.5%) [22] in water (Solution A). The
buffer used was Tris–HCl buffer 1.5 M, pH 8.8; these solutions were
mixed with N,N,N ,N -tetramethyl-ethylen diamine (TEMED) and
ammonium persulphate ((NH4)2S2O8) solution 10% (w/v) water. All
the components must be stored at 4 ◦C for optimum conservation.
Ammonium persulphate solution must be daily prepared [23]. After
addition of the corresponding volumes of TEMED and ammonium
persulphate the solution was stirred, and then the corresponding
amount of cells was added directly into the mixture. The acrylamide-
cell solution was mixed and rapidly transferred to a flat plastic
recipient. After approximately 30 min, a layer of solid polymer is
formed. The solid polymer was cut in small cubes of 0.5 cm side.
2.5.3. Immobilisation in barium alginate
For the alginate immobilisation, barium chloride was used as a
reticulating agent, according to Sinisterra and Dalton [24]. Sodium
alginates from Sigma with low (250 cP), medium (3500 cP) and high
viscosity (5500 cP) values were tested. The immobilisation was
carried out using a sterilised sodium alginate solution in 50 mM
phosphate buffer, pH 6.5, containing 0.5% glucose. This solution was
previously mixed with the cell pellets. The suspension was dropped
over a 0.05% BaCl2 solution in 50 mM phosphate buffer, pH 6.5,
containing 5% glucose from 15 cm height. This parameter determines
the size of the spheres produced.
 2.5.4. Immobilisation in agar
Three tailor-made agars from Hispanagar S.A. (Spain) were used
for immobilisation: Ref A27/03; Ref A28/03 and Ref A90. The agar
was emulsified at the desired concentration in 50 mM phosphate
buffer (pH 6.5) with 5% glucose and sterilised for 15 min at 121 ◦C
and 1 atm. Afterwards, the agar solution was cooled in a bath at 45 ◦C
and the temperature of the solution was monitored using a contact
thermometer. The cells were added when the agar suspension cooled
down till 42–45 ◦C. The cell-agar suspension was slowly stirred and
put into a plastic cast and allowed to stand for solidification. The solid
layer obtained was cut in small 0.5 cm cubes using a scalpel.
2.5.5. Immobilization in agarose
The immobilization of yeasts and of the fungus C. velutina in
agarose was performed in the same conditions than in agar (see
above). Agarose from His panagar S.A. (D5 lot F-5912) was used as
a matrix. The cells-agarose suspension was put in a plastic cast and
allowed to stand for solidification. The solid layer was cut in small
cubes (0.5 cm)
2.6. Oxidation of alcohols with immobilised biocatalysts
The oxidation of the alcohols was performed in the same way. The
reactions were carried out in 100 mL Erlenmeyer flasks containing
20 mL of 50 mM phosphate buffer, pH 6.5, with 5% glucose. The
cubes containing the immobilised biocatalyst were weighted and
introduced into the flask. The concentration of cyclohexanol in the
reaction mixture was 5 mM and the concentrations of 1- tetralol
enantiomers were set to 2.5 mM. Biotransformations were
performed at 28 ◦C and 200 rpm in a Khuner orbital shaker. The
reaction products were ¨ analysed by GC (Section 2.8.)
Table 1
Cyclohexanone (%)production by oxidation of cyclohexanol, using growing, resting or lyophilised cells
[cyclohexanol] 5 mM (10 mM for growing cells), reaction time was 72 h. Reactions were incubated at 28 ◦C and 250 rpm (lyophilised cells, 100 rpm).
3. Results and discussion
A hierarchical taxonomic screening of 416 strains was
performed [25] Three yeasts belonging to the Saccharomycetales
order were finally selected: W. californica CBS 2158, W. sat
urnus NCYC 2313 P. tannophilus NCYC 1597, as well as the
fungus C. velutina CBS 981.68.
3.1. Results obtained using growing, resting and
lyophilised cells in the oxidation of cyclohexanol
The oxidation of cyclohexanol was performed using the cells in
2.7. Determination of the cell concentration in cultures
For determination of productivity, the cell concentration was
measured in the different cultures. Cell densities were determined
using 250 mL Erlenmeyer flasks (50 mL medium) inoculated with 2
mL of a fresh culture (24 h incubation). Samples were picked up at
different culture times. The cell concentration was determined by
measuring the absorbance at 660 nm. The calculations were
performed according to the following expression:
Abs(D.O.) = 1.27 × 10−2c(106 cells/mL) + 1.26 × 10−2,
N = 9, R2 = 0.9692
The yeast culture medium was described above in Section 2.4.
2.8. Analysis of the reaction samples by gas chromatography
The oxidation of cyclohexanol was followed in a Hewlett Packard
5890 Series II gas chromatograph with an Agilent Technologies
automated sampler of 100 vials. Hydrogen was produced with an
electrolytic hydrogen generator (Domnick Hunter UHP-601).
Capillary glass column: Sugelabor SGL-1000, carbowax, 60 m, 0.25
mm, 0.25 m. Ti: 155 ◦C; ti: 1 min; Tf: 175 ◦C, tf: 10 min; rate: 4
◦
C/min; flow: 40 psi; split ratio: 100.
The analysis of the oxidation of (R) or (S)-1-tetralol and of 2-
adamantanol was performed using a Varian 3400 cx gas
chromatograph equipped with an automated sampler. A CP 7502
carbowax capillary column (25 m, 0.39 mm) from Sugelabor (Spain)
was employed in the following working conditions: Ti = 90 ◦C; ti = 5
min; heating rate = 5 ◦C/min; Tf = 175 ◦C; tf = 7 min. Carrier flow
(He) = 25 psi and split ratio = 100 mL/min. Injector and detector
temperatures =250 °C
three different physiological conditions (Table 1). The reaction
yields (%) were mean of two replications.
As can be observed the physiological conditions dramatically
affect the yields obtained. This effect has been described by
Molinari et al. [7] and associated to oxygen diffusion and the
alteration of biochemical mechanisms. Indeed, the yields achieved
using growing and resting cells are satisfactory but the lyophilised
cells carry to low yields. We postulate that the restrictions to the
oxygen transfer in the lyophilised cells could explain this poor
result. Both Williopsis strains were the most active
microorganisms. The filamentous fungus (C. velutina) showed a
very poor activity.
3.2. Immobilisation process
 Whole cell entrapment was selected as immobilisation method
and six different polymers were tested: polyacrylamide, barium
alginate, agar, -carrageenan, polyvinyl alcohol and chitosan. In
this first step, two main properties were considered: operational
stability and mass transfer. After these test assays, polyvinyl
alcohol, chitosan and -carrageenan immobilised derivatives, were
considered unsuitable as biocatalysts in the oxidation of
cyclohexanol, due to their low stability (chitosan and
-carrageenan) or the diffusion problems (polyvinyl alcohol).
Contrarily, Pterocladia (Ref A27/03) and Gracilaria (Ref A28/03)
agars, barium alginate and polyacrylamide carried to immobilised
derivatives with convenient physical and chemical properties.
3.2.1. Polyacrylamide immobilisation
Table 2
Cyclohexanol oxidation using immobilised derivatives of W. californica, W. saturnus, P. tannophilus and C. velutina using polyacrylamide as matrix
[cyclohexanol] 5 mM, reactions were incubated for 72 h at 28 ◦C and 100 rpm. Xa: percentage of polyacrylamide (%); Xb: biomass amount (x is the amount of biomass that
can be harvested from a 100 mL Erlenmeyer containing 20 mL of culture medium); Xc: gel volume (mL).
3.2.2. Immobilisation in agar
Different tailor made agars from Hispanagar S.A. (Spain)
have been tested during our research. The agar Ref A27/03 was
the best matrix for the immobilisation of these microorganisms.
This agar is strongly methoxylated and so, it shows the lowest
◦
gelling temperature 42 C. This special feature allows working in
a low temperature range avoiding cell damage. The factorial
design for the immobilisation of the strains using agar as the
matrix was done with a reduced matrix of four variables and
(4−1)
2 experiments. The maximum (+) and minimum (−) values
and the centre points are shown in Table 3. With this agar type
we achieved active biocatalysts for the oxidation of cyclo
hexanol. The best condition was I-2 for both Williopsis strains
but the centre points (I-1 and I-10) in the case of P. tannophilus
(Table 3). The immobilised biocatalyst obtained shows the best
yields and optimum physical properties that ensure a large
operational stability.
As in the case of polyacrylamide derivatives, C. velutina did not
present activity as biocatalyst. Only in the case of I-9 experiment,
an acceptable yield could be achieved with Williopsis strains.
Contrarily, the yeast P. tannophilus gave better yields in centre
point conditions. These results indicate to us that differ ent genera
Table 3
Cyclohexanol oxidation using immobilised derivatives of W. californica, W. saturnus, P. tannophilus and C. velutina using agar Ref A27/03 as matrix
The factorial design for the immobilisation of the
microorganisms in polyacrylamide as matrix was done with a
(3−1)
reduced matrix of three variables, and so with 2 experiments.
The maximum (+) and minimum (−) values and the centre points
(0) are shown in Table 2. We used as unit of biomass the amount
of cells that grow per Erlenmeyer in the described culture
conditions, Xb. The biomass from 1 ((−) minimum), 2 ((0) centre
point) or 3 ((+) maximum) Erlenmeyers was used during the
factorial design. The other variables were the percentage of poly
acrylamide in the matrix (Xa) and the total volume of gelified
solution (Xc).
Only the gels made up using low concentrations of poly
acrylamide (2.5%) and low gel volume (I-4) showed moderated
results, in the case of Williopsis genus yeasts. Nevertheless poly
acrylamide was not a suitable immobilisation matrix for the
biocatalysts involved in oxidation reactions because low yields
are obtained.
carry to optimum yields in different immobilization conditions.
These results again indicate that this fungus is not as interesting as
yeasts for the oxidation reaction.
3.2.3. Barium alginate
The factorial experiment design for this immobilization was
performed using a reduced matrix of four variables and so, 2
(4−1)
experiments. In this case, we have considered the viscosity as
another variable due to our experience in the control of beads
formation with this ionic gel. In addition only barium alginate was
used for immobilization of the cells due to our experience [24] in
the gelling of Ca(II) and of Ba(II) alginates. The maxi mum (+)
and minimum (−) values and the centre points (0) are shown in
Table 4.
The results obtained with this matrix were less satisfactory than
using agar (Ref A27/03), in the case of W. saturnus and W.
californica. Especially negative was this immobilisation in the
case of P. tannophilus compared to immobilisation in agar.
Contrarily, this methodology was very useful for the fungus C.
velutina, because this was the only matrix where this fungus
displayed activity. The yields achieved were greater than those
described for this microorganism in the case of resting cells (Table
1). The experiment conditions I-4 and I-9 could be considered the
most interesting for yeasts and fungus.
[cyclohexanol] 5 mM, reactions were incubated for 72 h at 28 ◦C and 100 rpm. Xa: % agar; Xb: biomass amount (x is the amount of biomass that can be harvested
from a 100 mL Erlenmeyer containing 20 mL of culture medium); Xc: agar volume (mL); Xd: stirring speed (rpm).

Table 4 4
Cyclohexanol oxidation using immobilised derivatives of W. californica, W. saturnus, P. tannophilus and C. velutina using barium alginate as matrix

[cyclohexanol] = 5 mM, reactions were incubated for 72 h at 28 ◦C and 100 rpm. Xa: alginate volume (mL); Xb: (%) alginate; Xc: viscosity; Xd: biomass amount— x is the
amount of biomass that can be harvested from a 100 mL Erlenmeyer containing 20 mL of culture medium.

immobilisation conditions (Table 3). Indeed when maximum

3.3. Immobilisation conditions selected for the oxidation of
alcohols
From the results indicated till now, we could resume the best
experimental immobilization conditions for each strain as:
Agar from Hispanagar is the best matrix for the immobilisation
of yeasts, according to the yields achieved, the physical properties
and the operational stability. Barium alginate is the most general
matrix to be used but lower yields in cyclohexanone were obtained
compared to those achieved with the immobilisation in agar.
From the factorial experimental design, the effect (bi) of each
variable (Xi) was obtained from the surface response. In our case a
polynomial model was used (Eq. (1)):
2
yield = b0 + biXi + biX i + bibjXiXj (1)
From this analysis we can deduce that in the case of agar, both
Williopsisstrains show a similar behaviour in the same
percentage of agar (Xa) and biomass volume (Xb) and minimum
the values of agar volume (Xc) and stirring speed (Xd) the yields
are the best. The significant effects (bi) were (for agar):
W. californica : ba = 3.0; bb = 4.7; bc = −1.8; bd = −8.5,
W. saturnus : ba = 5.2; bb = 8.7; bc = −3.3; bd = −9.0
The strong negative effect of stirring speed must be related to
the soft structure of the immobilised biocatalyst that is destroyed
under mechanical stirring conditions. Therefore, a maximum
percentage of agar – that makes the beads stronger – favours the
process (ba > 0). A high value of the volume of biomass (bb)
increases the yield because increase the catalyst if Xa and Xc are
constant.
In the case of P. tannophilus clearly the best conditions for the
immobilisation are those of the centre point. In these conditions
we obtained the best results in the oxidation of cyclohexanol.
Therefore, each genus carries to different optimum immobilization
conditions.
When the response surface model (Eq. (1)) was applied to the
results obtained with barium alginate (Table 4) we obtain the
following effect values (barium alginate):
W. californica : ba = −4.0; bb = −14.5; bc = −8.0;
bd = −10.0,
 W. saturnus : ba = 15.5; bb = −17.2;
bc = −17.0; bd = −5.0,
P. tannophilus : ba = 12.75; bb = −16.3; bc = −42;
bd = −30.7
We observe that in the case of barium alginate all the effects
are negative and the values of bi are greater than those obtained in
the case of agar. These values indicate that barium alginate
formation is more sensitive to the variation of the experimental
conditions (ionic gel) than agar (thermogel). In addition, P.
tannophilus is the most sensible microorganism to the variation in
the immobilisation conditions (highest bi value). The most
significant effect is the percentage of alginate that should be
minimum (Xa = 0.5%) in all cases and the second is the viscosity
value of the sodium alginate (Xc) used in the immobilisation that
should be minimum, too. These effects indicate that the restriction
in the diffusion of reagents and/or products within the cells is very
high. Finally, the added biomass exerts (Xd) a negative effect,
especially in the case of P. tannophilus. This effect was described
by Sinisterra and Dalton [24] in this immobilisation.

Table 5
Maximum yield achieved in the oxidation of cyclohexanol using cells immobilised cells in 2.5% agar in the presence of 0.5% glucose Microorganism (%) Glucose

Reactions were performed in 50 mL of 50 mM phosphate buffer (pH 6.6) at 28 ◦C and 200 rpm; 25 g of wet cubes of biocatalyst (agar).

3.4. Oxidation of cyclohexanol using immobilised whole results obtained with W. californica as an example. The
cells immobilization conditions were I-1 or I-2 (Table 3).
From Fig. 1, we can deduce the existence of an “induction like”
The addition of sugars to the reaction medium is a well period (Tind) in the case of immobilised cells in agar. This period
established methodology to increase the yield in the reduction of is smaller in the case of agar with 3.75% (w/v) than in the case of
ketones by means of alcohol dehydrogenases of yeasts [26,27]. To agar 2.5% (w/v) and it is not observed both in the case of
explore this topic in the oxidation of cyclohexanol, parallel fermenter and in resting cells conditions. Therefore, we can
experiments were performed using the same culture deduce that agar matrix is the responsible of this delay. It is related
microorganism and the same experimental conditions. Glucose, to diffusion restrictions of cyclohexanone and/or cyclohexanol by
fructose, glycerol and sucrose – in different proportions – were the water included in the small agar cubes.
used. All the reactions were performed with similar amounts of Indeed, the greater the percentage of agar, the lower the amount of
cells (45,000 ± 2000) (10 cells) and 25 g of biocatalyst. The best water inside the cubes and the smaller is the delay. It has been
6

results are obtained when adding 0.5% glucose (Table 5). confirmed by diffusion experiments of cyclohexanol and
P. tannophilus displayed lower activity than Williopsis strains, cyclohexanone trough this matrix (data not shown). Nevertheless,
as described in Table 1. Comparing the results shown in Tables 2 strong cubes of I-2 sample (3.75% agar) carry to lower yields than I-
and 5 we can see how the statistical design of experiments and the 1 biocatalyst, probably because of cyclohexanone (hydrophobic)
addition of glucose contributed to increase the yields in the remains inside the cubes—as observed in different experiments and
oxidation of alcohols. The toxicity of cyclohexanol for the cells previously described [29]. Very good yields are achieved in all cases
can be deduced from the reduction in the yield and the increase in with I-1 biocatalyst, similar to those obtained in fermenter conditions
the reaction time to achieve the maximum yield. This effect is (Table 6).
more important for P. tannophilusthan in Williopsis strains. The reaction progress curves can be fitted to a pseudo-first
Nevertheless the immobilization in agar A27/03 reduces the order kinetic expression (2) but not to a Michaelis–Menten kinetic
toxicity effect compared to free cells in fermenter conditions [28], profile.
where concentrations of alcohol greater than 5 mM dramatically
reduce the yield. V = kap[S] = ksp × [cat][S] (2)

The apparent kinetic constants (kap) and the apparent specific

3.5. Kinetic runs
The oxidation of cyclohexanol (10 mM) was performed using
whole cells in different situations: fermenter conditions, resting
cells and immobilised in agar A27/03. In Fig. 1, we show the
constants (kesp) are shown in Table 7. As expected, immobilised
derivatives gave lower apparent (kap) and specific (ksp) kinetic
constant values than those observed for resting or fermenting
conditions. This finding is related to well known diffusion
restrictions produced by the immobilisation process. Besides, we
can see that specific kinetic constants for immobilised Williopsis
 strains are greater than that of P. tannophilus.

Table 6
Oxidation of cyclohexanol to cyclohexanone using different biocatalysts

Fig. 1. Oxidation of cyclohexanol using whole cells of W. californica. Reaction

conditions: T = 28 ◦C; 200 rpm; [cyclohexanol] = 10 mM; V = 50 mL phosphate
buffer (pH 6.5); glucose = 0.5%. Immobilised conditions: I-1, agar A27/03 2.5%
T = 28 ◦C; 200 rpm; V = 50 mL phosphate buffer (pH 6.5); glucose 0.5%;
(w/v) and I-2: agar A27/02 3.75% (w/v) (Table 3). Resting cells = 1.2 g wet pellet;
cyclohexanol 10 mM. I-1 = 2.5% agar A27/03; I-2 = 3.75% agar A27/02
immobilised biocatalysts = 25 g wet cubes (1 cm × 1cm × 0.2 cm).

3.6. Oxidation of bulky alcohols

The oxidation of 2-adamantanol, as an example of bulky alcohol, was performed using the immobilised derivatives in agar. In Fig. 2
we show – as an example – the oxidation of the alcohol by immobilised whole cells of P. tannophilus in different tailor made agars and
in a commercial agarose D5 (Hispanagar S.A.) using I-9 conditions (Table 3). Due to the low water solubility of 2-adamantanol (solid)
only 2.5 mM can be achieved.
Immobilised biocatalyst in agar Ref A28/03 obtained from Gracilaria algae gave similar yields than free whole cells. The
immobilisation in agars from Gelidium (Ref A-90) or Pterocladia A27/03 gave lower yields than in tailor made agar Ref A28/03. These
results can be related to a lower diffusion resistance of the hydrophobic 2-adamantanol molecule in the case of highly methoxylated agar
(Ref A28/03). This methoxylation takes place in the CH 2OH group (C-6) of the galactose units [30] making the agar from Gracilaria
(A28/03), slightly more hydrophobic than the others.

Fig. 2. Oxidation of 2-adamantanol by immobilised whole cells ofP. tannophilus immobilised in tailored made agar A28/03, A27/03, A-90 and agarose D5 Lot F-5912. [2-
adamantanol] = 2.5 mM; T = 28 ◦C, 175 rpm, 25 g wet cubes biocatalyst; [2-adamantanol] = 2.5 mM, cubes of 2 cm × 2 cm × 0.2 cm.

3.7. Stereospecific oxidation of 1-tetralol enantiomers with immobilised whole cells

These rigid structures were selected as substrate models. The oxidation of chiral alcohols was performed with the most active strains: W.
saturnus and W. californica. It is remarkable that the immobilised whole cells showed a high stereospecificity in the oxidation of 1-
tetralol as it has been previously described in fermenter conditions [28]. Indeed, only S-1-tetralol was oxidised and no one of our
biocatalysts displayed activity in the oxidation of the R-enantiomer (Fig. 3).
In accordance with data of Section 3.4, highly methoxylated agar (Ref A 28/03) was selected as immobilisation matrix using the
conditions I-2 (Table 3) (3.75% agar). In Fig. 3 we show the results obtained using free and immobilised cells of W. californica and W.
saturnus that were the most active strains using free and immobilised cells. P. tannophilus (55% yield using free cells) was immobilised
in agar using I-1 conditions (Table 3) showing yields below 25%. The catalyst loading were selected in order to obtain similar
concentration of cells (free or immobilised) in the reaction flasks. None of the immobilised derivatives showed activity against 1R-
tetralol. Williopsis saturnus could be considered as the best strain – both as free and as immobilised cells – for the stereospe cific
oxidation of 1S-tetralol. The immobilised biocatalyst could be reused giving better yields in the recycles than immobilised W.
californica that seems to be sensitive to the immobilisation methodology.

Table 7
Kinetic values obtained in the oxidation of cyclohexanol using different biocatalysts

T = 28 ◦C; 200 rpm; 50 mL phosphate buffer (pH 6.5); glucose 0.5%. I-1 = 2.5% (w/v) agar Ref 27/03; I-2 = 3.75% (w/v); fermented cells for 48 h; 25 g of wet cubes of
biocatalyst (agar); 1.2 g of wet pellet in the case of resting cells.
After dilution of the pellet (1.2 g) in 50 mL reaction volume.
Fig. 3. Kinetic profiles for the oxidation of 1S-tetrahydronaphtol using free and immobilised cells of W. californica and W. saturnus in agar.
Cell concentrations—W. saturnus free cells assays: 7419 millions of cells/mL; immobilised cells assays: 25 g biocatalyst, 6882 millions of
cells/mL. W. californica—free cell assays: 6882 millions of cells/mL; immobilised assays: 25 g biocatalyst, 6348 millions of cells/mL.