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Z. Boros et al. / Process Biochemistry 48 (2013) 1039–1047
resulted in improved immobilized CE biocatalysts with enhanced
recyclability and thermal stability [22].
2. Materials and methods
2.1. Materials
MPSs with amino functions on their surfaces enable either
adsorptive or covalent immobilization. For example, an
tive immobilization of lipase from porcine pancreas (PPL),
resulting in a higher hydrolytic activity and better reusabil-
ity than when the enzyme was adsorbed on non-grafted MPS
[23].
presence of a precipitant enable supportless immobilization by the
preparation of cross-linked enzyme aggregates (CLEAs) which are
efficient immobilized biocatalysts [24] even on an industrial scale
[25].
ther means of upgrading of the efficacy of biotransformations. In
attention and is becoming a promising alternative to batch pro-
cesses [26,27]. Most of the continuous-mode biocatalytic syntheses
[28]. Stainless steel continuous-flow packed-bed bioreactors can
be effectively used to study the effects of temperature, pressure
catalyzed kinetic resolutions [29–32].
Racemic 1-phenylethanol (rac-1a), racemic 1-phenylethanamine (rac-1b),
glutardialdehyde (GDA) solution (25%, w/v in H2O), vinyl acetate, sodium
chloride, mono- and dibasic sodium phosphate, Trizma® base (2-amino-2-
hydroxymethyl-1,3-propanediol; Tris base), hydrochloric acid and TritonTM X-100
(4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) were purchased from
Sigma–Aldrich.
Davisil® 150 [35–70 m] (Dv150), Davisil® 250 [40–63 m] (Dv250), Daraclar®
915 (Dc915) and Daraclar® 920 (Dc920) were the products of Grace (Deer-
field, USA). Geduran® Si 60 [63–200 m] (Ged60) was purchased from Merck
(Darmstadt, Germany). Silica supports functionalized with phenyltrimethoxysi-
lane (PTMOS), octyltrimethoxysilane (OTMOS), (3-aminopropyl)trimethoxysilane
(APTMOS), [3-(2-aminoethylamino)propyl]trimethoxysilane (AEAPTMOS), [3-(2-
aminoethylamino)propyl]methlydimethoxysilane (AEAP-MDMOS) and with vari-
ous mixtures of such organosilanes were produced by SynBiocat Ltd (Budapest,
Hungary). The labels for the functionalized silica supports indicate both the nature of
the grafted silica gel and the grafting reagents. For example, Geduran® Si 60 grafted
with OTMOS is labeled as Ged60O or Davisil® 250 grafted with a PTMOS:AEAP-
MDMOS 1:1 mixture is labeled as Dv250PAEAP11.
Novozym® 435 (CaLB N 435, recombinant lipase B from Candida antarctica
expressed in Aspergillus niger and adsorbed on acrylic resin) and Novozym® CaLB
L recombinant (lipase from Candida sp. expressed in Aspergillus niger with a pro-
tein content ∼4%, ≥5000 LU/g) were obtained from Sigma–Aldrich (Saint Louis, MO,
USA). CaLB T2-150 (Candida antarctica lipase B covalently attached to dry acrylic
beads with a 150–300 m particle size) was the product of ChiralVision BV (Leiden,
The Netherlands).
Solvents (toluene, ethyl acetate, acetone, n-hexane, methyl tert-butyl ether,
dichloromethane and 2-propanol) from Molar Chemicals (Budapest, Hungary) were
dried and/or freshly distilled prior to use.
Among readily available lipases lipase B from Candida antarctica
(CaLB) exhibits many attractive characteristics and has thus
become one of the most widely used biocatalysts in both indus-
the character of the temperature effect in the range of 0–70 ◦C
depended significantly both on the substrate and on the mode of
immobilization [36].
Immobilization of CaLB on butyl- [37] or octyl-silica [19,38]
has indicated the usefulness of surface-modified silica as lipase
carrier. Surface-modified silica supports, especially butyl silica,
have proven to be suitable carriers for CaLB-catalyzed reactions
in ionic liquid/supercritical carbon dioxide biphasic media [39].
A study with a series of grafted silica gels indicated phenyl-
silica as ideal support for CaLB securing satisfactory selectivity
in the kinetic resolution of racemic 1-phenylethanol rac-1a,
while the octyl-silica-adsorbed CaLB had poor activity with
adsorption resulted in CaLB biocatalysts of moderate performance
[39].
Covalent immobilization of CaLB on amino-silica supports indi-
cated that the thermal stability of such biocatalysts was better than
those prepared by physical adsorption only [41]. CaLB adsorbed
and cross-linked on a polypropylene carrier maintained its activity
when dispersed in ionic liquids [42].
Coating an MPS with a mixture of the grafting reagents
4-aminophenyltrimethoxysilane and phenyltrimethoxysilane at
different ratios demonstrated that the density of the amino groups
present on the silica surface can be successfully controlled while
keeping the overall number of grafts constant [43]. Thus, a simple
method can secure tunable and even dispersion of amino function-
ality on the surface.
2.2. Methods
Thin-layer chromatography was carried out using Kieselgel 60 F254 (Merck)
sheets. Spots were visualized under UV light (Vilber Lourmat VL-6.LC, 254 nm and
365 nm) or by treatment with 5% ethanolic phosphomolybdic acid solution and
Reactions yielding 2a from 1a were analyzed by gas chromatography (GC)
on Agilent 4890 equipment [FID: 250 ◦C, injector: 250 ◦C, carrier gas: H2
(12 psi), split ratio: 1:50] using a Hydrodex -6TBDM column (Machery-Nagel,
25 m × 0.25 mm × 0.25 m, heptakis-(2,3-di-O-methyl-6-O-t-butyldimethylsilyl)-
-cyclodextrin); GC data (oven program: 120 ◦C, 8 min−1; molar response factor
for 2a/1a: 1.23): tr (min): 4.0 [(S)-2a], 4.4 [(R)-2a], 5.8 [(R)-1a], 6.0 [(S)-1a. Reac-
tions yielding 2b from 1b were analyzed on Agilent 5890 equipment [FID: 250 ◦C,
injector: 250 ◦C, carrier gas: H2 (12 psi), split ratio: 1:50] using a Hydrodex -TBDAc
column (Machery-Nagel; 25 m × 0.25 mm × 0.25 m, heptakis-(2,3-di-O-acetyl-6-
8
◦C min−1, 5 min at 180 ◦C; molar response factor for 2b/1b: 1.17): tr (min): 2.9 [(S)-
1b], 3.1 [(R)-1b], 9.8 [(R)-2b], 10.0 [(S)-2b]. All data in Tables and Figures arose from
a precise integration of chromatograms in which both enantiomers of the substrates
1a, b and the products 2a, b were clearly visible. When peaks for the minor enan-
tiomers were indistinguishable from noise, solid curves were replaced by dashed
lines in Figs. 4 and 5.
Conversion (c), enantiomeric excess (ee) and enantiomeric ratio (E) were deter-
mined by GC. Enantiomeric ratio (E) was calculated from c and enantiomeric excess
of the product (eeP) using the equation E = ln[1 − c(1 + eeP)]/ln[1 − c(1 − eeP)] [44].
Due to its sensitivity to experimental error E values in the 100–200 range are given
as >100, in the range of 200–500 as >200 and above 500 as ꢀ200.
In batch reactions, the specific activity of the biocatalyst (UB) was determined
using the equation UB = (nrac × c)/(t × mB) [32]. To characterize the productivity of the
biocatalysts, the specific reaction rates in the batch reactions (rbatch) were calculated
using the equation rbatch = nP/(t × mB) (where nP [mol] is the amount of the product,
t [min] is the reaction time and mB [g] is the mass of the biocatalyst) [44]. Specific
reaction rates in continuous-flow systems (rflow) were calculated using the equation
rflow = [P] × v/mB (where [P] [mol mL−1] is the molar concentration of the product,
v [mL min−1] is the flow rate and mB [g] is the mass of the biocatalyst) [44]. Because
the rate of product formation is not a linear function of c, rigorous comparisons by
the r values between the productivity of a continuous-flow reaction and its batch
mode counterpart can only be made at similar degrees of conversions [44].
2.3. General procedure for lipase adsorption to silica supports
In the present work we set out to compare the usefulness of
biocatalysts prepared by simple adsorption of CaLB with adsorp-
tion combined with cross-linking and covalent binding onto
surface-functionalized silica supports with dispersed amino func-
tions as novel biocatalysts in both in batch and continuous-flow
mode.
CaLB
L
(1.25 mL, ≥6250 LU/g, ∼5 mg protein) was dissolved in Tris buffer
(11.25 mL, 100 mM, pH = 7.5, ionic strength controlled with NaCl to 175 mM), an
then the support (250 mg) was added to the solution. The enzyme-support suspen-
sion was incubated at 400 rpm and 4 ◦C for 18 h. The immobilized CaLB was filtered
off on glass filter (G4), washed with 2-propanol (2 × 5 mL) and hexane (5 mL), dried
for 2 h at room temperature and stored at 4 ◦C. The properties of CaLBs immobilized