Multicomponent thermosensitive systems for biocatalysts

Article abstract of DOI:10.1007/s11172-005-0273-9

Composite matrices based on macroporous silica modified by N-vinylcaprolactam copolymers with diallyldimethylammonium chloride and with 2-hydroxyethyl methacrylate were obtained. Lipase from Pseudomonas fluorescens was immobilized on the obtained material

Full text of DOI:10.1007/s11172-005-0273-9

452
Russian Chemical Bulletin, International Edition, Vol. 54, No. 2, pp. 452—457, February, 2005
Multicomponent thermosensitive systems for biocatalysts
a
a
a
D. V. Kapustin,a A. A. Vikhrov, I. V. Gorokhova, A. N. Generalova,
ꢀ
O. V. Kalyazina,b T. G. Murzabekova,b and V. P. Zubova
аM. M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
16/10 ul. MiklukhoꢀMaklaya, 117997 Moscow, Russian Federation.
Fax: +7 (095) 335 1011. Eꢀmail: kapustin@mail.ibch.ru
bState Research Institute of the Genetics and Selection of Industrial Microorganisms,
1, 1st Dorozhnyi proezd, 113545 Moscow, Russian Federation.
Fax: +7 (095) 315 0510. Eꢀmail: tamos@mail.ru
Composite matrices based on macroporous silica modified by Nꢀvinylcaprolactam copolyꢀ
mers with diallyldimethylammonium chloride and with 2ꢀhydroxyethyl methacrylate were
obtained. Lipase from Pseudomonas fluorescens was immobilized on the obtained materials.
The temperature dependence of the hydrolytic activity of the immobilized lipase preparations
in the triacetin hydrolysis was investigated. The hydrolytic activity of lipase immobilized on the
matrix modified by the Nꢀvinylcaprolactam copolymer with 2ꢀhydroxyethyl methacrylate can
be regulated by varying the temperature of the reaction medium. The temperature dependence
of the hydrolytic activity of the immobilized enzyme has a maximum at 40 °C, the activity of
the immobilized lipase being ∼3.5 times higher compared to that at 20 °C. After immobilization
on these composite materials, lipase retained the activity in the acetylation of 1ꢀ(RS)ꢀphenylꢀ
ethanol with vinyl acetate in ButOMe.
Key words: composite materials, lipase, immobilization, copolymers, Nꢀvinylcaprolactam,
diallyldimethylammonium chloride, 2ꢀhydroxyethyl methacrylate, regulation of enzyme acꢀ
tivity.
In recent decades, rapt attention of researchers is atꢀ
polymers incorporated in gels. In some cases, the enzyꢀ
matic activity can also be regulated in these systems by
varying the temperature. However, the accessibility of the
active sites of the enzymes incorporated in the gel bulk
usually decreases due to the diffusion restrictions for the
transport of the substrate and the enzymatic reaction prodꢀ
ucts. Apparently, immobilization of the enzymes on solid
surfaces modified by thin layers of thermally sensitive
polymers would increase the efficiency of the biocatalysts
obtained, in particular, by increasing their mechanical
strength, high sorption capacity and the lack of diffusion
restrictions for substrate transport toward the enzyme.
Thus it has been shown13 that the adsorption of subtilisin
on a silica surface results in a thousandꢀfold increase in
the enzymatic activity. However, the use of such systems
modified by smart polymers implies a more efficient reguꢀ
lation of the functional activity of immobilized bioꢀ
molecules, in particular, by changing their hydrophobicꢀ
hydrophilic environment as the temperature changes. In
this study, we prepared biocatalysts by using macroporous
silica modified by thermosensitive cоpolymers of Nꢀvinylꢀ
caprolactam with diallyldimethylammonium chloride
(cоpolymer 1) and with 2ꢀhydroxyethyl methacrylate
(cоpolymer 2). These polymeric modifying agents were
chosen due to the possibility of performing efficient modiꢀ
tracted by synthetic polymers able to change their conforꢀ
mation and properties in response to minor changes in
the environment, in particular, temperature.1 These smart
polymers are used for enzyme and cell immobilization,2
controlled delivery of drugs,3 preconcentration of metal
ions,4 affinity precipitation,5 and so on.
By using thermoꢀ and/or pHꢀsensitive polymeric maꢀ
terials, one can prepare systems ensuring the retention of
functional properties of the biomolecules incorporated
into it and control the preparation activity by measuring
the temperature and/or pH. A series of biocatalysts obꢀ
tained by enzyme immobilization into gels based on
thermosensitive polymers are currently known. These are,
in particular, urease and alkaline phosphatase incorpoꢀ
rated into poly(Nꢀisopropylacrylamide),6,7 trypsin in the
poly(Nꢀisopropylacrylamide)—methacrylic acid cоpolyꢀ
mer gel,8 asparaginase and βꢀgalactosidase incorporated
into gels of poly(Nꢀisopropylacrylamide) copolymer with
acrylic acid,9,10 and trypsin, В carboxypeptidase,11 αꢀchyꢀ
motrypsin12 incorporated in the poly(Nꢀvinylcaproꢀ
lactam)ꢀCaꢀalginate gel. It was shown in the aboveꢀmenꢀ
tioned studies that enzymes immobilized in composite
gels retain the catalytic activity in both aqueous and mixed
water—organic media, as they are stabilized by the smart
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 2, pp. 443—448, February, 2005.
1066ꢀ5285/05/5402ꢀ0452 © 2005 Springer Science+Business Media, Inc.

Thermosensitive systems for biocatalysts
Russ.Chem.Bull., Int.Ed., Vol. 54, No. 2, February, 2005
453
inorganic salts, and sodium hydroxide (Reachim, Russia). The
commercial preparation of lipase from Pseudomonas fluorescens
was provided by the RÖHM Pharma Polymers company (Gerꢀ
many); the specific activity of the initial sample with respect to
triacetin was 120 (mmol of the product) h–1 (g of the prepaꢀ
fication of the silica surface and some structural features
that determine differences between the protein affinities
of these compounds.
The methods for modification of porous silica matriꢀ
ces by these polymers were chosen from the following
considerations. Previously,14 it has been shown that
poly(diallyldimethylammonium chloride) (polymer 3) can
be sorbed from solutions in aqueous alcohols on a bulkꢀ
porous silica to give a stable coating due to the charge
difference between the support surface and the nitrogenꢀ
containing groups of the polymer. Procedures for the
preparation of polymerꢀcontaining composite sorbents
based on bulkꢀporous silicas by immobilization of polyꢀ
mers from solutions on the silica surface with solvent
evaporation have been proposed.15 We immobilized
cоpolymer 1 on the silica surface by adsorption from a
solution, while coating by cоpolymer 2 was performed by
distilling off the solvent. The introduction of monomer
units differing in the protein affinity into Nꢀvinylcaproꢀ
lactamꢀbased cоpolymers may induce structural differꢀ
ences in the microenvironment of enzymes immobilized
on polymer surfaces to produce biocatalysts. Previously,
it has been shown16 that silica sorbents modified by polyꢀ
mer 3 are suitable for the isolation of the total protein
fraction from complex mixtures containing polysacchaꢀ
rides, nucleic acids, and monomeric compounds, together
with proteins, because this polymer has an enhanced proꢀ
tein and peptide affinities. Conversely, the 2ꢀhydroxyethyl
methacrylate polymer is known to have low protein and
peptide affinities; hence, it can be used, in particular, as a
capillary modifier in the capillary electrophoresis, and
nonspecific sorption in both organic17 and aqueous18 meꢀ
dia is reduced.
ration)–1
.
Radical cоpolymerization of Nꢀvinylcaprolactam with diꢀ
allyldimethylammonium chloride and 2ꢀhydroxyethyl methacryꢀ
late. The reaction was carried out in anhydrous MeOH in the
presence of AIBN as an initiator under oxygenꢀfree conditions
for 16 h at 60 °C with gentle stirring. The Nꢀvinylcaprolactamꢀ
enriched fractions of the cоpolymers were precipitated with acꢀ
etone. The unreacted Nꢀvinylcaprolactam and acetoneꢀsoluble
fractions were removed by the thermal precipitation of cоꢀ
polymers at 75 °C. The total yields of the acetoneꢀinsoluble
fractions of cоpolymers 1 and 2 determined by gravimetry were
76 and 83% of the initial content of comonomers in the mixture,
respectively.
To estimate the weightꢀaverage molecular masses of copolyꢀ
mers 1 and 2, their diffusion coefficients (D) were determined in
solutions (in water for cоpolymer 1 and in methanol for
cоpolymer 2) at 20 °C. The resulting D values were substituted
into the Polson equation
D = aM–1/3
,
where а = 2.74•10–5 20
,
and the weight average molecular masses
were calculated.
The structure of cоpolymer 1 was determined by 13C NMR
spectroscopy. 13C NMR (D2O), δ: 123 (C=O); 35.4 (NMe);
34.4 (αꢀCH2); 32.5 (δꢀCH2); 29.7 (γꢀCH2); 27.4 (βꢀCH2); 23.1
(εꢀCH2); 16.2 (Me); 15.9 (CH2); 6.8 (CH). The structure of the
waterꢀinsoluble cоpolymer 2 was determined by IR spectroꢀ
scopy (film from MeOH). IR, ν/cm–1: 1400 (CH2OH); 1650
(CONR2); 1700 (CO); 2900 (CH2). The composition of cоꢀ
polymers was confirmed by elemental analysis. Cоpolymer 1.
Found (%): C, 64.86; H, 9.59; Cl, 9.59; N, 10.00; O, 6.49.
Cоpolymer 2. Found (%): C, 67.42; H, 9.18; N, 8.89; O, 14.39.
The lower critical solubility temperatures (LCST) of the
cоpolymers were determined based on light scattering using a
Coulter N4 submicron particle analyzer (France).
As the model protein for the preparation of the
thermosensitive biocatalyst, we chose the thermally stable
lipase from the microorganisms Pseudomonas fluorescens
(CF 3.1.1.3), whose activity does not change much over a
broad temperature range.19 In addition, lipases occupy a
special place in the series of hydrolytic enzymes used as
catalysts of organochemical transformations, as they can
be used both in the hydrolysis of lipids and esters and in
the enantioselective esterification and transesterification
in organic solvents or in waterꢀorganic media.
The temperature dependence of hydrophobicꢀhydrophilic
properties of the cоpolymers was studied by the capillary method
using an improved procedure21 in which the capillaries with an
inner diameter of 2 mm were filled with solutions of cоpolymers
(0.5 g ml–1) in MeOH and then the solvent was evaporated
in vacuo. Then the capillaries were immersed into a dish with
water at different temperatures and the height of the liquid colꢀ
umn was rapidly measured.
Preparation of the composite sorbents. The specified weight
portions of the Trisoporꢀ500 macroporous glass were placed into
an evacuated reactor. The samples (10 g) were evacuated to
remove air and the vapor that condensed during the storage of
silica. The layer of the support particles was wetted with 60 mL
of a solution of a thermosensitive cоpolymer with a concentraꢀ
tion of 0.033 g mL–1 (in the case of cоpolymer 1, an aqueous
solution was used and for cоpolymer 2, a solution in MeOH).
The resulting suspension was treated in an ultrasonic bath for
15 min to attain a more uniform distribution of macromolecules
over the sorbent pore bulk. When immobilizing cоpolymer 1
containing diallyldimethylammonium chloride residues on the
Experimental
The study was performed using the Trisoporꢀ500 macroꢀ
porous silica with an average pore diameter of 50 nm, a specific
pore volume of 1.2 cm3 g–1, and a specific surface area of
108 m2 g–1 (Shuller GmbH, Germany); Nꢀvinylcaprolactam,
diallyldimethylammonium chloride, 2ꢀhydroxyethyl methacryꢀ
late (Sigma—Aldrich GmbH, Germany); 2,2´ꢀazobis(isobutyroꢀ
nitrile) (AIBN), ButOMe, vinyl acetate, 1ꢀ(RS)ꢀphenylethanol
(Fluka Chemie AG, Switzerland); triacetylglycerol (triacetin)
(Merck, Germany); chemically pure grade methanol, acetone,

454
Russ.Chem.Bull., Int.Ed., Vol. 54, No. 2, February, 2005
Kapustin et al.
support surface, the suspension was kept for 3 h with gentle
stirring on a magnetic stirrer and the sorbent thus produced was
washed with water (a 10ꢀfold volume ) and acetone (a 10ꢀfold
volume) on a Büchner funnel. For immobilization of cоpolyꢀ
mer 2 containing 2ꢀhydroxyethyl methacrylate residues, the solꢀ
vent was evaporated using a rotary evaporator. The resulting
sorbent was also washed with water (a 10ꢀfold volume ) and
acetone (a 10ꢀfold volume). The products were dried in vacuo at
30 °C to a constant weight.
To estimate the strength of retention of the polymer modifiꢀ
ers by the support surface, the weight portions of the sorbents
(300 mg) were incubated in water for 1 h at 20, 30, and 50 °C,
the sorbent particles were precipitated by centrifuging for 10 min
at 3000 rpm, supernatants were withdrawn, and UV spectra
were recorded on a Beckman DUꢀ70 spectrophotometer (USA).
The morphology of the resulting material was studied
by mercury porosimetry on a PoreSizer 9300 instrument
(Micromeritics, USA).
taining 5 mmol of the racemic substrate, 1ꢀ(RS)ꢀphenylethanol,
and 15 mmol of vinyl acetate. The suspension was shaken with a
velocity of 200 vibrations per min at 20—40 °C. Aliquots (0.5 mL
each) were taken from the reaction mixture. The solid catalyst
particles were separated on a centrifuge and the supernatant was
analyzed by HPLC on a Beckman liquid chromatograph (Sysꢀ
tem Gold, USA) with a LiChroCART RP 18 column (5 µm)
(240×4 mm), Merck (Germany). The absorbance was reꢀ
corded at λ = 260 nm at a flow rate of 0.8 mL min–1 using a
MeCN—water mixture (1 : 1) for elution. The retention times of
1ꢀ(RS)ꢀphenylethanol and 1ꢀ(R)ꢀphenylethyl acetate were
5.9 and 13.5 min, respectively. The results were used to calꢀ
culate the esterification activity of the enzyme in (µmol
1ꢀ(R)ꢀphenylethyl acetate) h–1. The scatter did not exceed 2%.
Results and Discussion
We synthesized polymeric modifiers 1 and 2 by radical
cоpolymerization. The model composite biocatalysts were
prepared using random cоpolymers of Nꢀvinylcaprolactam
containing, according to elemental analysis, 40% diallylꢀ
dimethylammonium chloride or 12% 2ꢀhydroxyethyl
methacrylate. Cоpolymer 2 swells in water and dissolves
upon the addition of 40% (v/v) MeOH.
Determination of the hydrolytic stability of the sorbents. The
hydrolytic stability of the obtained materials was estimated by
incubating samples of the initial silica and the composite sorꢀ
bents (1 g) in 10 mL of a buffer solution (pH 9.5) containing
sodium borate (0.025 mol L–1) and NaOH (0.1 mol L–1) for 8 h
at ∼20 °C. The aliquot portions (0.25 mL) taken every hour were
mixed with equal volumes of a 0.05 М solution of sodium moꢀ
lybdate acidified with conc. H2SO4 (1/200 v/v with respect to
the sodium molybdate solution), and then the absorbance was
measured on a Beckman DUꢀ70 spectrophotometer (USA) at
λ = 320 nm.
Lipase immobilization on a composite sorbent. To prepare the
biocatalyst, a sample (1 g) of the obtained composite material
was reꢀsuspended in a 3 mL of a lipase solution (50 mg in a 0.2 M
solution of KH2PO4, pH 7.5), and the mixture was incubated for
24 h at 4 °C with gentle stirring. The resulting suspension was
centrifuged for 10 min at 3000 rpm and the supernatant was
withdrawn. The biocatalyst was washed on a Schott filter with a
20ꢀfold volume of the phosphate buffer and subjected to freezeꢀ
drying. The enzyme content incorporated in the composite maꢀ
terial was calculated from the residual hydrolytic activity of the
enzyme in the supernatant and in the filtrates.
The weightꢀaverage molecular masses calculated from
the Polson equation20 using the diffusion coefficient found
experimentally by correlation laser spectroscopy were
20000 and 25000 for cоpolymers 1 and 2, respectively.
The structures of cоpolymers 1 and 2 were established
by 13C NMR and IR spectroscopy, respectively. The data
thus obtained were compared with the spectra of the
poly(Nꢀvinylcaprolactam) homopolymer (homopolyꢀ
mer 4). As the analytical 13C NMR signals of cоpolymer 1,
we used the signal at δ ∼29 from the methylene bridge,
which links the ring units of the macromolecule of polyꢀ
mer 3, and the multiplet signal at δ 47—50, corresponding
to the αꢀC atom in macromolecule 4. The presence of
diallyldimethylammonium chloride residues in the cоꢀ
polymer results in a lower intensity of the signal at δ 47. In
addition, the broad line at δ 180.3 corresponding to the
lactam carbonyl in homopolymer 4 decreases and a weak
signal at δ 181—182 simultaneously increases. The specꢀ
tra of the obtained cоpolymers exhibit additional signals
at δ 27 and 32.5; the group of signals at δ 30—31.5 beꢀ
comes more intense, which, in our opinion, is also inꢀ
dicative of the appearance of the methylene group adjaꢀ
cent to the βꢀC atom of the Nꢀvinylcaprolactam unit and
the methylene group near the αꢀC atom of this unit. Analyꢀ
sis of the IR spectra of cоpolymer 2 showed wellꢀresolved
peaks at 1400 cm–1 corresponding to the absorption of
hydroxyethyl groups of 2ꢀhydroxyethyl methacrylate, in
addition to the absorption peaks at 1650, 1700, and
2900 cm–1, typical of homopolymer 4. Thus, the IRꢀspecꢀ
troscopy data confirmed the formation of cоpolymers unꢀ
der these conditions. The structural formulas of the cоꢀ
polymers are presented below.
Determination of the hydrolytic activity of the enzyme. Triꢀ
acetin (2 mL) was dissolved in 100 mL of a solution of NaCl
(0.05 mol L–1) and CaCl2 (0.05 mol L–1) in water (pH 7.0). The
resulting solution of the substrate (5 mL) was placed in the cell
of a Radiometer Copenhagen TTT80 titrator and 50 µL of a
solution containing 5 µL of the enzyme or 120 mg of the immoꢀ
bilized lipase (containing 5.88 µg of the enzyme in the biocataꢀ
lyst modified by cоpolymer 1, or 4.44 µg of the enzyme in the
biocatalyst modified by cоpolymer 2). The acetic acid formed in
the enzymatic reaction was titrated with a 0.01 М solution of
NаOH at pH 7.0 for 10—15 min. The results of titration were
used to calculate the hydrolytic activity of the enzyme in (µmol
AcOH) h–1 22
The scatter of the values did not exceed 2.6%.
.
Determination of the esterification activity of the enzyme.
The lipase activity in esterification was determined from the
initial rate of 1ꢀ(RS)ꢀphenylethanol acetylation with vinyl acꢀ
etate to give 1ꢀ(R)ꢀphenylethyl acetate. The initial lipase prepaꢀ
ration (14 mg; protein content, 35 µg) or the solid support
(100 mg; protein contents of 4.9 and 3.7 mg for the biocatalysts
based on silicas modified by cоpolymers 1 or 2, respectively) was
placed into a conical flask containing 10 mL of ButOMe conꢀ

Thermosensitive systems for biocatalysts
Russ.Chem.Bull., Int.Ed., Vol. 54, No. 2, February, 2005
455
Specific pore volume/cm3 g–1
1.2
2
a
1.0
0.8
0.6
0.4
0.2
1
0.01
Specific pore volume/cm3 g–1
0.9
0.1
1
Pore radius/µm
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
2
b
The LCST for a solution of cоpolymer 1 was 38—39 °C.
For a solution of cоpolymer 2, a sharp change in the
turbidity with an increase in the temperature was obꢀ
served at 32—33 °C. For comparison, the LCST for a
solution of homopolymer 4 (M = 20000) was determined
by this method; this was equal to 37 °C. The influence of
the temperature around the LCST on the hyrophobicꢀ
hydrophilic properties of cоpolymers 1 and 2 was also
studied. This was done using an improved procedure21
based on the measurement of the height of the liquid
meniscus in capillaries with inner walls modified by the
layer of the thermosensitive polymer. The height of the
liquid column in the capillaries treated with cоpolymers 1
and 2 proved to be lower than the value found for the
untreated capillary (32 mm), being equal to 31 and 28 mm
in cold water and 25 and 20 mm in hot water (70 °C),
respectively. These data indicate the ability of the thermoꢀ
sensitive cоpolymers to change the hydrophilicꢀhydroꢀ
phobic balance on passing the LCST. A more pronounced
decrease in the height of the liquid column in the capilꢀ
lary treated with cоpolymer 2 attests to its higher hydroꢀ
phobicity with respect to cоpolymer 1. Thus, on phase
transition due to temperature rise above the LCST,
cоpolymer 2 loses the hydration shell to a greater extent.
Cоpolymer 1 was immobilized by incubating the supꢀ
port in an aqueous solution of the cоpolymer. This was
accompanied by spontaneous sorption of the cоpolymer
on the support surface due to the charge difference beꢀ
tween the silica surface and the diallyldimethylammonium
chloride units incorporated in the cоpolymer. Waterꢀinꢀ
soluble cоpolymer 2 was deposited onto the support surꢀ
face from a methanol solution followed by solvent evapoꢀ
ration. The contents of the polymer modifiers 1 and 2 in
the obtained composite materials were 12 and 15%, reꢀ
spectively.
1
0.01
0.1
1
Pore radius/µm
Fig. 1. Differential (1) and integral (2) pore distribution over
effective radii (mercury porosigrams) for nonmodified silica (а)
and silica modified by cоpolymer 1 (b).
tained (Fig. 1). It follows from their analysis that not only
the external surface of particles but also the internal surꢀ
face of the pores is modified by the polymer, which is
accompanied by a decrease in the average pore diameter
from 48 to 43 nm. Thus, the average effective thickness of
the polymer coating was ∼25 Å. In addition to the deꢀ
crease in the average diameter, their specific volume also
decreases (from 1.2 to 0.87 and 0.78 cm3 g–1 for cоꢀ
polymers 1 and 2, respectively), which is also indicative
of immobilization of the polymer layer on the inner surꢀ
face of the pores. The porosity of the support is largely
retained. The presence of a continuous polymer film on
the pore surface is also confirmed by testing the stability
of the resulting materials against alkaline hydrolysis. The
incubation of the nonmodified support samples and the
materials obtained in an alkaline buffer (pH 9.5) for 4 h
resulted in 7 and 9 times greater amounts of silicic acid in
the case of nonmodified silica than in the case of samples
containing cоpolymers 1 and 2, respectively.
The composite sorbents obtained are able to efficiently
bind lipase with retention of its enzymatic activity. The
degree of inclusion of the enzyme into silicaꢀbased sorꢀ
bents modified by cоpolymers 1 and 2, determined from
the residual activity of the supernatants and the filtrates
obtained by washing of the biocatalysts after lipase immoꢀ
The morphology of the polymerꢀcontaining composꢀ
ite sorbents was studied by mercury porosimetry. For
samples of both sorbents, similar porosigrams were obꢀ

456
Russ.Chem.Bull., Int.Ed., Vol. 54, No. 2, February, 2005
Kapustin et al.
Table 1. Change in the lipase enzymatic activity in the obtained
biocatalysts depending on temperature
taining mainly Nꢀvinylcaprolactam residues. Therefore,
the activity of the lipase within the biocatalyst does not
increase substantially on temperature rise.
T/°С
Activitya
The possibility of regulating the enzymatic activity by
changing the temperature is demonstrated in relation to
the biocatalyst based on silica modified by cоpolymer 2. It
follows from the data presented in Table 1 that the depenꢀ
dence of the enzymatic activity of immobilized lipase
toward hydrolysis passes through a maximum at 40 °C
(the activity increases more than 3.5ꢀfold with respect to
the activity at 20 °C), then the activity decreases someꢀ
what still remaining ∼3 times higher at 45 °C than at 20 °C
(Fig. 3). This pattern of dependence of the lipase activity
on the temperature is apparently due to enzyme immobiꢀ
lization on the fragments of cоpolymer macromolecules
containing poly(Nꢀvinylcaprolactam), because the secꢀ
tions containing 2ꢀhydroxyethyl methacrylate residues
have a low protein affinity (see Fig. 2, b). The lack of a
narrow temperature range in which the catalyst activity
would sharply change is probably related to the random
nature of cоpolymer 2. Apparently, a sharp activity change
can be expected upon modification of the supports by
block or graft thermosensitive cоpolymers.
The phase transition that takes place as the temperaꢀ
ture increases above the LCST of the thermosensitive
cоpolymer and determines the change in the activity of
the biocatalyst is caused by the presence of Nꢀvinylꢀ
caprolactam residue in the cоpolymers and is accompaꢀ
nied by changes in the structure of the hydration shell of
the cоpolymer macromolecule. One should expect that in
nonpolar media without a hydration shell, no activation
of immobilized lipase will be observed. Indeed, the esteriꢀ
fication activity of lipase incorporated in the biocatalyst
based on cоpolymer 2 in an organic solvent (ButOMe) in
the 20—45 °C range changes insignificantly (from 89.3
to 93.9%) with respect to the activity of native lipase (see
Table 1).
hydrolyticb
cоpolymer 1 cоpolymer 2
esterificationc
cоpolymer 2
20
25
30
35
40
45
37000
37100
36600
36200
36600
35500
7300
11300
17800
20600
27000
21400
1340
1350
1380
1400
1400
1400
a Expressed in (µmol of the product) h–1 (mg of the enzyme)–1
.
b The activity of the initial lipase was 52000 µmol h–1 (mg of
enzyme)–1 at 20 °C.
c The activity of the initial lipase was 1500 µmol h–1 (mg of
enzyme)–1 at 20 °C.
bilization, was 98 and 74%, respectively. The greater inꢀ
clusion of lipase into the sorbent based on silica modified
by cоpolymer 1 is, apparently, due to the electrostatic
interaction of the lipase macromolecule, which is negaꢀ
tively charged at neutral pH (pI 4.46),23 with the posiꢀ
tively charged diallyldimethylammonium chloride on the
matrix surface.
The variation of the hydrolytic activity of lipase in the
biocatalysts obtained as a function of temperature is deꢀ
picted in Table 1. These data indicate, in particular, that
the hydrolytic activity of lipase incorporated in the sorꢀ
bent modified by cоpolymer 1 varies insignificantly (the
scatter does not exceed 2.6%) over the temperature range
under study (20—45 °C). The use of the silicaꢀbased sorꢀ
bent modified by cоpolymer 1 does not seem to allow
regulation of the enzymatic activity by varying the temꢀ
perature under these conditions. This can be due to the
fact that the lipase molecules are accumulated in the surꢀ
face sections composed of poly(diallyldimethylammonium
chloride) (Fig. 2, а), which are not involved in the phase
transition on temperature change, unlike the sections conꢀ
Thus, no temperature sensitivity of the systems studꢀ
ied is observed in an anhydrous medium.
Lipase activity (rel.u.)
4
a
Enz
Enz
T > LCST
2
3
2
1
1
b
T > LCST
Enz
Enz
25
30
35
40
T/°С
Fig. 3. Variation of the enzymatic activity of lipase in the triaceꢀ
tin hydrolysis: initial lipase (1) and lipase immobilized on silica
modified by copolymer 2 (2) (lipase activity at 20 °C is taken to
be unity).
Fig. 2. Schematic diagram illustrating the degree of accessibility
of the lipase immobilized on the surface of sorbents modified by
cоpolymers 1 (a) and 2 (b) depending on temperature.

Thermosensitive systems for biocatalysts
Russ.Chem.Bull., Int.Ed., Vol. 54, No. 2, February, 2005
457
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15, 476.
11. E. A. Markvicheva, A. S. Bronin, N. E. Kudryavtseva, I. F.
Kuz´kina, I. I. Pashkin, Yu. E. Kirsh, L. D. Rumsh, and
V. P. Zubov, Bioorgan. Khim., 1994, 20, 257 [Russ. J. Bioorg.
Chem., 1994, 20, 242 (Engl. Transl.)].
The relatively low activity of lipase incorporated in the
sorbent modified by cоpolymer 2 attests to the need to opꢀ
timize the ratio of the Nꢀvinylcaprolactam to 2ꢀhydroxyꢀ
ethyl methacrylate residues in the macromolecules of the
modifying agent.
Thus, these studies resulted in the preparation of
biocatalysts based on polymerꢀmodified silicas with immoꢀ
bilized lipase from the microbes Pseudomonas fluorescens.
The possibility of regulating the catalytic activity of the
resulting material in aqueous media on changing the temꢀ
perature was demonstrated.
12. N. L. Eremeev, L. V. Sigolaeva, A. O. Kost, and N. F.
Kazanskaya, Biotekhnologiya, 1994, 8, 25 [Biotechnol. Rusꢀ
sia, 1994, 8, 19 (Engl. Transl.)].
13. J. Partidge, P. J. Halling, and B. D. Moore, Chem. Commun.,
1998, 7, 841.
14. L. L. Zavada, L. Yu. Fillipova, I. V. Bobyleva, T. V.
Ovchinnikova, D. V. Kapustin, V. V. Saburov, V. P. Zubov,
and V. A. Bykov, Mezhdunar. Konf. "Farmatsevticheskaya
Bioetika" [Int. Conf. "Pharmaceutical Bioethics"] (Moscow,
November 10, 1997), Abstrs, Moscow, 1997, 25 (in Russian).
15. D. V. Kapustin, V. V. Saburov, L. L. Zavada, A. V. Evstratov,
G. B. Barsamyan, and V. P. Zubov, Bioorgan. Khim., 1998,
24, 868 [Russ. J. Bioorg. Chem., 1998, 24, 770 (Engl. Transl.)].
16. D. V. Kapustin, V. P. Zubov, L. L. Zavada, V. A. Bykov,
N. E. Gruzinova, A. V. Evstratov, and O. A. Trifonova,
Biomeditsinskie Tekhnol. [Biomedical Engineering], 1998, 8,
53 (in Russian).
This work was financially supported by the Russian
Foundation for Basic Research (Project No. 01ꢀ03ꢀ
33115).
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Received July 22, 2004;
in revised form November 18, 2004