Proteinases immobilized on poly(vinyl alcohol) cryogel: Novel biocatalysts for peptide synthesis in organic media

Article abstract of DOI:10.1023/A:1014350600760

Covalent immobilization of subtilisin and thermolysin on cryogel of poly(vinyl alcohol) was carried out. The biocatalysts obtained are characterized by high stability in water and in DMF-MeCN mixtures of various compositions The synthetic efficiency of im

Full text of DOI:10.1023/A:1014350600760

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a
b
b
a
1
896
Russian Chemical Bulletin, International Edition, Vol. 50, No. 10, pp. 1896—1901, October, 2001
Proteinases immobilized on poly(vinyl alcohol) cryogel: novel biocatalysts
for peptide synthesis in organic media
I. Yu. Filippovaa , A. V. Bacheva , O. V. Baibak , F. M. Plieva , E. N. Lysogorskaya ,
E. S. Oksenoit , and V. I. Lozinskyb
a
àDepartment of Chemistry, Lomonosov Moscow State University,
Leninskie Gory, 119899 Moscow, Russian Federation.
Fax: +7 (095) 932 8846. E-mail: irfilipp@genebee.msu.su
bA. N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences,
2
8 ul. Vavilova, 117813 Moscow, Russian Federation.
Fax: +7 (095) 135 5085
Covalent immobilization of subtilisin and thermolysin on cryogel of poly(vinyl alcohol)
was carried out. The biocatalysts obtained are characterized by high stability in water and in
DMF—MeCN mixtures of various compositions. The synthetic efficiency of immobilized
subtilisin in the multiple iterative synthesis of the peptide Z—Ala—Ala—Leu—Phe—pNA was
examined in organic mixtures of different solvent compositions. Immobilized subtilisin exhibits
high synthetic activity in organic media. A series of N-acylated p-nitroanilides of tetrapeptides
of the general formula Z—Ala—Ala—Xaa—Yaa—pNA (Z is benzyloxycarbonyl, Xaa = Leu,
Lys, or Glu; Yaa = Phe or Asp; pNA = 4-NO —C H NH-) were synthesized in 70–98%
2
6
4
yields using immobilized subtilisin as a biocatalyst without activation and protection of the
ionogenic groups of polyfunctional amino acids. Immobilized thermolysin in a DMF—MeCN
mixture catalyzed the formation of the peptide Z—Ala—Ala—Leu—pNA, which was obtained
in 90% yield (during 1 h). It was demonstrated that the biocatalyst can be used repeatedly and
that it retained activity after storage in an aqueous buffer during 6 months.
Key words: immobilized subtilisin and thermolysin, poly(vinyl alcohol) cryogel, peptide
synthesis, organic solvents, proteinases.
6
The biological role of proteinases is to hydrolyze
cryogel (PVAC), which was prepared by freezing-thaw-
ing of concentrated aqueous solutions of this polymer,
has great potential for these purposes. After introduction
of reactive groups, this support was used for covalent
immobilization of trypsin,5 α-chymotrypsin,7 and pan-
creatic lipase.8 The two last-mentioned types of
biocatalysts have been successfully tested in enzyme
catalysis in organic media with low water contents.
In the present study, we examined the properties of
serine proteinase subtilisin-72 (SL) and metalloendo-
proteinase thermolysin (TL), which were covalently at-
tached to PVAC granules. These immobilized biocatalysts
(ISL and ITL, respectively) were used for the chemical
enzymatic synthesis of peptides in media with high
contents of polar organic solvents (MeCN and DMF).
bonds between amino acid residues in polypeptides and
proteins. Under certain conditions, proteolytic enzymes
can also be used in the reverse reaction, viz., in the
synthesis of peptide bonds.1 Obvious progress has been
achieved in the field of the enzymatic peptide synthesis
in recent years, which provides evidence that protein-
ases are promising catalysts of the peptide formation.1,2
However, the scope of the enzymatic peptide synthesis
in aqueous media is limited by low solubility of pro-
tected peptides and the possibility of undesirable hy-
drolysis of the products. The use of organic solvents
instead of aqueous media promotes the shift of the
thermodynamic equilibrium toward the formation of the
target compounds and the enhancement of the solubility
of protected peptides, but it is unfavorable for enzymes
because organic solvents cause enzyme inactivation. In
this connection, a search for systems, which ensure
preservation of the functional properties of enzymes in
organic solvents and protect them from inactivation
over a long period, is an urgent problem. One of
efficient approaches, which provides favorable condi-
tions for both the functioning of the enzyme and solu-
bility of peptides, involves immobilization of proteinases
on appropriate insoluble supports.3,4 Thus, it has been
demonstrated5 that the so-called poly(vinyl alcohol)
Results and Discussion
The choice of the enzymes, which differ by the
nature and the catalysis mechanism, for immobilization
was dictated by their high synthetic potential as catalysts
of the peptide formation.1,2 The support and the proce-
dure for immobilization of these enzymes should satisfy
some specific requirements associated with the charac-
teristic features of functioning of the immobilized
biocatalysts in nonaqueous media. First, the support
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 10, pp. 1811—1816, October, 2001.
066-5285/01/5010-1896 $25.00 © 2001 Plenum Publishing Corporation
1

Immobilized proteinases in the peptide synthesis
Russ.Chem.Bull., Int.Ed., Vol. 50, No. 10, October, 2001
1897
and the procedure for the attachment of the enzyme
must provide reliable fixation of protein macromol-
ecules on a support in order to prevent enzyme leakage
from catalyst granules. It should be noted that protein
immobilization on insoluble supports through physical
adsorption is not sufficiently efficient in the functioning
of biocatalytic systems in polar organic media. This is
associated with irreversible losses of the enzyme through
its desorption into the reaction medium, high sensitivity
with respect to the change in the medium composition
and, as a consequence, with the need for the use of very
large amounts of enzymes. Although the applicability of
this approach to the enzymatic peptide synthesis in
nonaqueous media was exemplified by the preparation
of a number of peptides and peptide mimetics in or-
ganic media,9—13 the covalent attachment of the protein
to a support matrix seems to be preferable. Second, the
support should be compatible both with the aqueous (at
least at the stage of enzyme immobilization) and or-
ganic media. It is necessary to fulfill this condition for
prevention of undesirable phenomena, such as collapse
of polymeric materials or species agglutination on going
from one medium to another. In this connection, a
gentle procedure for immobilization based on the physi-
cal entrapment of the enzyme into a matrix of poly-
meric hydrogel can be used only with great caution due
to subsequent strong collapse of such a support in polar
dehydrating solvents. Finally, the support used should
possess good performance characteristics, i.e., mechani-
cal, chemical, and biological stabilities. Poly(vinyl alco-
hol) cryogel, which was successfully used for the prepa-
ration of immobilized enzymes intended for functioning
in nonaqueous media (see above), adequately satisfies
the above-mentioned criteria.
Preparation of immobilized biocatalysts. Subtilisin
was immobilized on two cryogel derivatives containing
reactive aldehyde (PVAC-A) or epoxide (PVAC-E)
groups.5 The enzyme content on the support depended
substantially on the mode of immobilization and varied
from 0.1 (for PVAC-E) to 4.5 mg (for PVAC-A) of the
protein per gram of the support. Immobilized thermolysin
was prepared only with the use of PVAC-A; the amount
of the immobilized enzyme was 3.5 mg g–1 (Table 1). In
the subsequent experiments, the target peptides were
synthesized with the use of the enzymes immobilized on
PVAC-A.
The catalytic activity of ISL was estimated spectro-
photometrically by hydrolysis of the highly specific
chromogenic peptide substrate of subtilisin, viz.,
Glp—Ala—Ala—Leu—pNA*.14 The specific activity of
ISL was 0.25—4% of the initial value. It should be
noted that the results obtained correlate with the pub-
lished data on immobilization of α-chymotrypsin (which
is similar in properties to subtilisin) on PVAC-A. After
immobilization, chymotrypsin retained approximately
Table 1. Characteristics of ISL and ITL
Sample
Enzyme content
αspec*
–
1
(support)
on the support/mg g
ISL (PVAC-E)
ISL (PVAC-A)
0.1
3.7
2.2
0.37
0.19
0.65
4
.5
ISL (PVAC-A) +
Ac—Trp—OH
ISL (PVAC-A) +
Bzl—Tyr—NH2
ITL (PVAC-A)
2.0
2.3
3.5
0.50
0.40
*
αspec is the specific activity per mg of the protein.
10% of the activity exhibited by the native enzyme with
respect to the highly specific substrate and more than
85% of the activity with respect to a less specific sub-
strate.7
To reduce inactivation of subtilisin (protection of its
active site during immobilization), the enzyme was at-
tached to PVAC-A in the presence of reversible competi-
tive inhibitors of serine proteinases, viz., Ac—Trp—OH
and Bzl—Tyr—NH . However, the specific activities of
2
the resulting specimens were only insignificantly higher
(see Table 1).
The enzyme activity of ITL was determined
based on hydrolysis of the highly specific chro-
mophoric peptide substrate of this enzyme, viz.,
Dnp*—Gly—Gly—Ile—Arg—NH .15 Immobilized ther-
2
molysin, unlike subtilisin, retained 60% of the initial
activity, which indicates that thermolysin is more stable
to unfavorable factors of immobilization.
Stability of immobilized enzymes. To ensure the
successful use of enzymes in synthetic processes over a
long period, it is necessary that the corresponding im-
mobilized biocatalysts possess high stability both in the
course of the reactions and upon storage. Hence, the
stabilities of ISL and ITL were estimated in the course
of their storage both in aqueous buffers and organic
solvents.
The data on the time-dependent measurements of
the enzyme activity of ISL in various media are pre-
sented in Fig. 1. The enzyme exhibited rather high
stability in an aqueous buffer (curve 1) and retained
more than 50% of the initial activity even after storage
in this medium for three months, whereas the activity of
native subtilisin decreased to 40% upon storage under
these conditions for only three days. After incubation in
MeCN and DMF—MeCN mixtures of various compo-
sitions, ISL partially lost hydrolytic activity during
two—three days (curves 2 and 3), but about 30% of the
initial activity was retained in the DMF—MeCN mix-
ture (60/40 v/v) even after two weeks. The effect of the
enhancement of the stability due to enzyme immobili-
zation on PVAC-A was particularly pronounced as the
*
Glp is the residue of pyroglutamic (L-5-pyrrolidone-2-car-
boxylic) acid, pNA = 4-NO C H NH-.
* Dnp is 2,4-dinitrophenyl.
2
6
4

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Russ.Chem.Bull., Int.Ed., Vol. 50, No. 10, October, 2001
Filippova et al.
Specific activity (%)
00
biocatalytic system in nonaqueous media cannot be
unambiguously judged from the results of the determi-
nation of hydrolytic activity because the activities of
proteinases were determined after the transfer of the
enzymes from an organic medium to water. Conse-
quently, the information obtained is circumstantial and
reflects the ability of the enzyme to undergo reactivation
rather than its true state in organic solvents. The valu-
able data on the properties of a biocatalyst can be
obtained in studies of esterification and transesterification
in organic media.4,16—18 However, in this approach to
the examination of the properties of proteinases in
nonaqueous media, no consideration is given to an
interesting function of proteolytic enzymes, viz., to
catalysis of the peptide bond formation. In our opinion,
the properties of biocatalytic systems in organic solvents
can be most adequately estimated using the data on the
synthetic activities of proteolytic enzymes in the peptide
formation. For this purpose, ISL and ITL were tested in
the enzymatic peptide synthesis in media with high
DMF and MeCN contents.
1
1
8
6
0
0
2
3
4
2
0
40
60
τ/h
Fig. 1. Measurements of the specific activity of ISL upon
incubation in media of various compositions: 1, a 0.05 M
Òris—HCl buffer (ðH 8.3); 2, MeCN; 3, DMF—MeCN
60/40 v/v); 4, DMF—MeCN (90/10 v/v); τ is the incuba-
tion time.
(
DMF content was increased. In the DMF—MeCN
system (90/10 v/v), ISL retained 60% of the initial
activity after two days (curve 4). At the same time,
powdered subtilisin suspended in the same mixture of
organic solvents lost more than 90% of hydrolytic activ-
ity upon storage over the same period of time.14 Appar-
ently, the higher stability of ISL compared to that of
suspended subtilisin arises from higher stability of the
immobilized enzyme with respect to the denaturing
action of polar organic solvents, for example, due to
multisite attachment of subtilisin macromolecules at
PVAC. Due to the highly organized supramolecular
Enzymatic peptide synthesis catalyzed by ISL and
ITL. The synthetic activity of ISL was examined in
DMF—MeCN systems with the DMF concentration
varying from 60 to 95 vol. % using a model reaction*:
ISL
Z—Ala—Ala—Leu—OMe + Phe—pNA
Z—Ala—Ala—Leu—Phe—pNA.
(1)
Reaction (1) was carried out using the equimolar ratio
of the amino and carboxyl components and the molar
enzyme : substrate ratio of 1 : 800. The ISL-catalyzed
synthesis proceeded most rapidly in a medium contain-
ing 60% of DMF (Table 2). Even after 2 h, the yield of
the target peptide was 95%. The rate of accumulation of
the product decreased as the concentration of DMF was
increased. However, the reaction in 95% DMF did not
cease even after 48 h**. After three days, the yield of the
product was 74%, whereas the synthesis, which was
carried out in an analogous system and catalyzed by
suspended subtilisin, was terminated after 3 h due to
complete enzyme inactivation.14 Therefore, noticeable
stabilization of the enzyme with respect to the denatur-
ing action of DMF was achieved by immobilization of
subtilisin on PVAC-A.
To elucidate the possibility of the use of im-
mobilized proteinases in organic media of various
compositions, a series of successive syntheses of
Z—Ala—Ala—Leu—Phe—pNA were carried out using
the same ISL sample in DMF—MeCN mixtures with
increasing DMF content (60, 80, and 95%, respec-
tively). After each cycle, ISL beads were washed with
the 0.05 M Tris—HCl buffer (pH 8.3). After three
cycles, the synthetic ability of the biocatalyst was tested
packing of polymer chains in the gel phase, which
strongly holds a particular amount of water,6 the sup-
port promotes the maintenance of the water balance
required for the enzyme functioning. It was demon-
strated by a special experiment that water is strongly
bound to the support in the solvent systems under study.
For this purpose, the ISL specimen was twice washed
with MeCN and incubated with a DMF—MeCN mix-
ture (60/40 v/v) for 48 h. It was established by Fischer
titration that the concentration of water in the organic
phase was only 0.16%.
The ITL specimens were characterized by somewhat
higher stability upon storage in aqueous buffers com-
pared to the ISL specimens. After storage in the
.05 M Tris—HCl buffer* (pH 7.6), which contained
.005 mol L–1 of Ca2+, at +2 °C for four months, the
specimen completely retained its activity. After storage
in MeCN for one year, the specimen of the biocatalyst
used in the synthesis showed 65% of the initial activity.
Hence, the results obtained are indicative of the
efficiency of covalent immobilization of subtilisin and
thermolysin on PVAC for the enhancement of stability
of the biocatalysts both in aqueous buffers and organic
solvents. However, in our opinion, the properties of the
0
0
*
*
Z is benzyloxycarbonyl.
* Each experiment was carried out with the use of a new
*
Tris is 2-amino-2-(hydroxymethyl)propane-1,3-diol.
portion of the biocatalyst.

Immobilized proteinases in the peptide synthesis
Russ.Chem.Bull., Int.Ed., Vol. 50, No. 10, October, 2001
1899
Table 2. Synthesis of Z—Ala—Ala—Leu—Phe—pNA catalyzed
by the same ISL specimen in MeCN—DMF media of various
compositions
zyme and provide evidence that covalent immobiliza-
tion on hydrophilic cryogels is an efficient procedure for
adapting proteinases to functioning in nonaqueous media.
It should be noted that activation of the carboxy
group is required for efficient catalysis of the peptide
bond formation under the action of serine proteinases in
aqueous-organic mixtures.1 We found that not only
ester of the tripeptide Z—Ala—Ala—Leu—OMe but also
its analog containing the free carboxy group, viz.,
Z—Ala—Ala—Leu—OH, can serve as an efficient
acylating agent in the ISL-catalyzed synthesis in a
nonaqueous medium:
Sequence
DMF—MeCN Reaction
Yield of
of experiments
(vol. %)
time/h
the product (%)
1
2
60/40
0.25
2
0.25
2
74
95
25
76
88
22
57
67
74
6
80/20
95/5
2
5
3
4
2.5
2
5
7
5
1
2
ISL
Z—Ala—Ala—Leu—OH + Phe—pNA
Z—Ala—Ala—Leu—Phe—pNA.
(2)
60/40,
repeatedly*
0.25
2
41
84
95
As can be seen from the data in Table 3, the yields
were virtually identical in both cases. Apparently, this is
determined by the fact that in nonaqueous organic
media, the carboxy group of the acylating component
remains nonionized and efficiently interacts with the
enzyme active site.
The above-mentioned characteristic feature of ca-
talysis of the peptide bond formation by ISL in an
organic medium with the minimum water content was
demonstrated by the synthesis of N-acylated tetrapeptides
containing basic and acidic amino acid residues. The
reactions were carried out without activation of the
carboxy component and protection of the side ionogenic
groups of polyfunctional amino acids:
2
7
4
2
*
See comments in the text.
once again in a DMF—MeCN mixture (60/40 v/v).
After 72 h, the final yield of the product was equal to
that obtained in the first cycle after 2 h (95%).
It was found that ISL is also rather efficient in the
synthesis without intermediate rehydration, at least within
several cycles (Fig. 2). The synthetic efficiency of one
portion of the immobilized biocatalyst was examined in
three successive synthetic cycles in DMF—MeCN
(
60/40 v/v) without intermediate washing with an aque-
ous buffer. After each cycle, the granules of the catalyst
were twice washed with a mixture of organic solvents to
extract the starting compounds and the products. The
reaction product was obtained in high yield (see Fig. 2).
Hence, the water quantity provided for the enzyme by
the supramolecular PVAC matrix is sufficiently large for
the biocatalyst to exhibit high synthetase activity in spite
of functioning in polar dehydrating organic solvents.
Compared to the results of investigations of the ISL
stability in organic media, the last-mentioned data even
better reflect the high biocatalytic potential of the en-
ISL
Z—Ala—Ala—Xaa—OH + Yaa—pNA
Z—Ala—Ala—Xaa—Yaa—pNA,
(3)
where Xaa = Leu, Lys, or Glu; Yaa = Phe or Asp.
p-Nitroanilides of protected tetrapeptides were pre-
pared in high yields, the only reaction product being
obtained in all cases (see Table 3). The purities of the
peptides synthesized were confirmed by the data from
HPLC and amino acid analysis.
The synthetic activity of ITL was examined in
DMF—MeCN (25/75 v/v) using the following reaction
as an example:
Y (%)
ISL
1
00
1
2
3
Z—Ala—Ala—OH + Leu—pNÀ
Z—Ala—Ala—Leu—pNÀ.
(4)
8
6
4
2
0
0
0
0
The reaction, which was carried out with the equimo-
lar ratio of the amino and carboxyl components and
with the enzyme : substrate ratio of 1 : 750 for 1 h,
afforded the product in 90% yield. Immobilized
thermolysin, which has been twice used in the synthesis
and stored in a buffer for six months, catalyzed the
synthesis of Z—Ala—Ala—Leu—pNA in organic sol-
vents in 80% yield. These data are indicative of the high
efficiency of ITL as a catalyst of the peptide synthesis in
organic media.
Fig. 2. Yield (Y) of Z—Ala—Ala—Leu—Phe—pNA in succes-
sive cycles (1—3) of the synthesis catalyzed by the same ISL
specimen in DMF—MeCN (60/40 v/v) without intermediate
rehydration of the biocatalyst. The yields after 1 and 2 h are
colored black and white, respectively.
To summarize, new biocatalysts, viz., PVAC-attached
subtilisin and thermolysin, were obtained. These immo-

1
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Russ.Chem.Bull., Int.Ed., Vol. 50, No. 10, October, 2001
Filippova et al.
Table 3. Peptides synthesized under the action of ISL in organic media
Acylating
component
Amino
component
Product
DMF/MeCN
(vol. %)
t
Y
τ
Amino acid
HPLC
/ha (%)b /minc composition/nmol
d
Z—Ala—Ala—Leu—OMe
Phe—pNA
Z—Ala—Ala—Leu—Phe—pNA
60/40
2
90
25.5e
Ala 10.4 (2),
Leu 5.5 (1),
Phe 5.2 (1)
e
Z—Ala—Ala—Leu—OMe
Z—Ala—Ala—Leu—OMe
Z—Ala—Ala—Leu—OH
Z—Ala—Ala—Lys—OH
Phe—pNA
Phe—pNA
Phe—pNA
Phe—pNA
Z—Ala—Ala—Leu—Phe—pNA
Z—Ala—Ala—Leu—Phe—pNA
Z—Ala—Ala—Leu—Phe—pNA
Z—Ala—Ala—Lys—Phe—pNA
80/20
95/5
60/40
60/40
25
74
2
90
72
90
90
25.5
25.5
25.5
e
e
2
25.2f
27.0f
24.0f
26.0f
31.5f
Ala 7.8 (2),
Lys 3.8 (1),
Phe 3.9 (1)
Ala 8.3 (2),
Phe 4.1 (1),
Glu 4.0 (1)
Ala 6.4 (2),
Lys 3.1 (1),
Asp 3.3 (1)
Ala 8.8 (2),
Glu 4.1 (1),
Asp 4.5 (1)
Ala 9.6 (2),
Leu 4.9 (1),
Asp 4.7 (1)
Z—Ala—Ala—Glu—OH
Z—Ala—Ala—Lys—OH
Z—Ala—Ala—Glu—OH
Z—Ala—Ala—Leu—OH
Phe—pNA
Asp—pNA
Asp—pNA
Asp—pNA
Z—Ala—Ala—Glu—Phe—pNA
Z—Ala—Ala—Lys—Asp—pNA
Z—Ala—Ala—Glu—Asp—pNA
Z—Ala—Ala—Leu—Asp—pNA
60/40
60/40
60/40
60/40
2
2
98
98
74
92
4
24
a
t is the reaction time.
Y is the yield.
τ is the retention time.
b
c
d
The number of the amino acid residues is given in parentheses.
HPLC on column 1 using the gradient À (see the Experimental section).
HPLC on column 2 using the gradient B (see the Experimental section).
e
f
bilized enzymes were tested for synthetase activity, which
demonstrated that covalent immobilization of protein-
ases on hydrophilic cryogels is a promising procedure
for adaptating the enzymes to functioning in nonaque-
ous media. The biocatalysts obtained are characterized
by high stability in water and polar organic solvents.
The advantages of new biocatalysts are their ability to
efficiently catalyze the synthesis of peptides, which are
soluble in organic media, without activation and protec-
tion of ionogenic groups of polyfunctional amino acids,
the possibility of the repeated use of biocatalysts without
essential loss of their activity, and the fact that they can
be readily removed from the reaction mixture.
Granules of poly(vinyl alcohol) cryogel (PVAC) approxi-
mately 1 mm in diameter were prepared from poly(vinyl alco-
hol) 16/1 (NPO Azot, Severodonetsk, Ukraine) according to a
known procedure.21 The preparation of reactive aldehyde- and
epoxide-containing PVAC derivatives and the covalent attach-
ment of the enzymes were carried out according to procedures,
which involved the treatment of granules of the initial cryogel
with glutaraldehyde in an acidic medium and epichlorohydrin
in an alkaline medium followed by incubation of the activated
support with an enzyme solution.5 The amount of the immobi-
lized enzyme was estimated based on the data from amino acid
analysis.
Peptide analysis by HPLC was carried out on an Altex
Model 100A liquid chromatograph (USA) equipped with
Microsorb-MV C8 (4.6½250 mm; Rainin Instrument Com-
pany, Inc., USA) (1) and Nucleosil C18 (4½250 mm;
Biochemmack, Russia) columns (2). Elution was performed
using a linear gradient of MeCN in water, which contained
Experimental
0
.1% ÒFA, from 10 to 70% during 26 min (A) and from 20 to
The experiments were carried out with the use of serine
proteinase from Bacillus subtilis (strain 72; subtilisin 72,
EC 3.4.21.14), which was isolated from a culture liquid accord-
ing to a known procedure,14 thermolysin (EC 3.4.24.1) (Fluka,
Switzerland), MeCN for HPLC (Lekbiopharm, Russia), DMF
80% during 35 min (B). The elution rate was 1 mL min–1. The
detection was carried out at 220 and 280 nm. In the calcula-
tions of the compositions of the reaction mixtures (based on
absorption at 220 nm), the difference between the extinction
coefficients of the components was ignored.
(
analytically pure grade, Reakhim, Russia), which was addi-
Amino acid analysis was performed on an automated
Hitachi-835 analyzer (Japan) after acid hydrolysis of peptides
and specimens of the immobilized enzymes in 5.7 M HCl at
105 °C for 48 h.
The optical absorption measurements for the solutions un-
der study were carried out on Specord UV VIS (Germany) and
Shimadzu UV-1601 (Japan) spectrophotometers.
1
9
tionally purified according to a procedure reported previously,
trifluoroacetic acid (TFA; analytically pure grade, Fluka
Chemie AG, Switzerland), Phe—pNA (Serva, Germany), and
Asp—pNA (Bachem Bioscience Inc., USA). Other derivatives
of amino acids and peptides were synthesized according to
standard procedures.20

Immobilized proteinases in the peptide synthesis
Russ.Chem.Bull., Int.Ed., Vol. 50, No. 10, October, 2001
1901
The activity of subtilisin was determined according to a
procedure described previously.14 The activity of thermolysin
was determined according to a known procedure.15
granules were washed with the 0.05 M Tris—HCl buffer (pH 7.3),
τHPLC = 28 min using the gradient B on a Microsorb-MV C8
column.
Determination of the activity of ISL. An ISL specimen
(
20—30 mg; the protein content was 0.08 mg) was suspended in
We thank M. M. Buzlanova (the Nesmeyanov Insti-
tute of Organoelement Compounds of the Russian Acad-
emy of Sciences) for performing tests for water content
according to the Fischer method.
a 0.05 M Tris—HCl buffer (pH 8.3, 2 mL) containing CaCl
2
(
1.5 mmol L–1) and then a solution of Glp—Ala—Ala—Leu—pNA
in DMF (50 µL, 5 mg mL–1) was added. The resulting mixture
was incubated with stirring at ∼ 20 °C for 10 min. The absorption
was measured at 410 nm (A410) at regular intervals. The specific
activity (µmol (min mg of the protein)–1) was calculated accord-
ing to the formula
This study was financially supported by the Russian
Foundation for Basic Research (Project Nos. 00-03-
3283a and 00-04-48455).
αspec = (A410 – Ac )V /8.9m t,
o
e
410
c
where A410 is the absorption of the mixture at 410 nm, A
is
410
the absorption of the reference solution, V î is the total volume
of the sample (mL), t is the reaction time (min), me is the
weight of immobilized subtilisin (mg), and 8.9 is the molar
References
absorption coefficient of p-nitroaniline (mmol L– cm ).
1
–1
1. V. M. Stepanov, Pure, Appl. Chem., 1996, 68, 1335.
. I. Gill, R. Lopez-Fandino, X. Jorba, and E. N. Vulfson,
Enzyme Microbiol. Technol., 1996, 18, 162.
. P. Adlercreutz, Eur. J. Biochem., 1991, 199, 609.
. A. K. Gladilin and A. V. Levashov, Biokhimiya, 1998, 63,
08 [Biochemistry, 1998, 63, 345 (Engl. Transl.)].
. V. I. Lozinsky, F. M. Plieva, and A. L. Zubov, Bio-
tekhnologiya, 1995, No 1—2, 32 [Russ. Biotechnology, 1995,
No 2, 1 (Engl. Transl.)].
. V. I. Lozinsky, Usp. Khim., 1998, 67, 641 [Russ. Chem.
Rev., 1998, 67, 573 (Engl. Transl.)].
7. Yu. N. Belokon´, K. A. Kochetkov, F. M. Plieva, N. S.
Ikonnikov, V. I. Maleev, V. S. Parmar, R. Kumar, and V. I.
Lozinsky, Appl. Biochem. Biotechnol., 2000, 88, 97.
8. F. M. Plieva, K. A. Kochetkov, I. Singh, V. S. Parmar,
Yu. N. Belokon´, and V. I. Lozinsky, Biotechnol. Lett.,
2000, 22, 551.
2
Determination of the activity of ITL. An ITL specimen
(
a
35—50 mg; the protein content was 0.16 mg) was suspended in
0.05 Tris—HCl buffer (pH 7.3, mL) containing
3
4
M
1
Dnp—Gly—Gly—Ile—Arg—NH2 (0.5 mg). The resulting mix-
ture was incubated with stirring at 37 °C for 30—60 min. Then
the solution was decanted from granules of the biocatalyst and
4
5
6
5
0% AcOH (0.2 mL) was added. The resulting solution was
passed through a column with a SP Sephadex C-25 ion-
exchanger (3 mL) and the column was eluted with 0.5 M AcOH
(
(
2 mL). The absorption of the eluate was measured at 360 nm
A
3
6
0
)
.
T
h
e
s
p
e
c
i
f
i
c
a
c
t
i
v
i
t
y
(
µ
m
o
l
(
m
i
n
m
g
o
f
t
h
e
p
r
o
t
e
i
n
)
–
1
)
was calculated according to the formula
αspec = (A360 – Ac ) V /15m t,
where A360 is the absorption of the mixture at 360 nm, A
î
e
360
c
is
360
the absorption of the reference solution, V î is the total volume
of the sample, t is the reaction time (min), me is the weight of
the immobilized thermolysin (mg), and 15 is the molar ab-
sorption coefficient of 2,4-dinitrophenylethylenediamine
9
. H. Kise and A. Hayakawa, Enzyme Microb. Technol.,
991, 13, 584.
10. P. Clape´s, P. Adlercreutz, and B. Mattiasson, Biotechnol.
Appl. Biochem., 1990, 12, 376.
1
mmol L–1 cm–1).
(
Synthesis of Z—Ala—Ala—Leu—Phe—pNA (typical proce-
dure). An ISL specimen (80 mg; the protein content was
.3 mg, 15 nmol), which has been preliminarily washed with
1
1
1
1
1
1. V. M. Stepanov, E. Yu. Terent´eva, T. L. Voyushina, and
M. Yu. Gololobov, Bioorg. Med. Chem., 1995, 3, 479.
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Microbiol. Technol., 1999, 24, 480.
3. T. L. Voyushina, J. V. Potetinova, E. I. Milgotina, and
V. M. Stepanov, Bioorg. Med. Chem., 1999, 7, 2953.
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E. S. Oksenoit, J. Mol. Catal. B: Enzym., 2000, 11, 89.
5. A. L. Osterman, G. N. Rudenskaya, and V. M. Stepanov,
Biokhimiya, 1984, 49, 292 [Biochemistry USSR, 1984, 49,
31 (Engl. Transl.)].
6. C. R. Wescott and A. M. Klibanov, Biochim. Biophys. Acta,
994, 1206, 1.
7. V. Cerovsky and H.-D. Jakubke, Enzyme Microbiol. Technol.,
994, 16, 596.
8. O. Almarsson and A. M. Klibanov, Biotechnol. Bioeng.,
996, 49, 87.
0
MeCN (1 mL) and then twice washed with a MeCN—DMF
mixture (1 mL) of the corresponding composition, was added
to a solution of Z—Ala—Ala—Leu—OMe (5.1 mg, 12 µmol)
and Phe-pNA (3.4 mg, 12 µmol) in a MeCN—DMF mixture
(
400 µL). The reaction mixture was stirred at 20 °C and the
aliquots (5 µL) were taken at regular intervals and analyzed by
HPLC. After completion of the synthesis, the reaction mixture
was separated and the cryogel granules were washed with either
the buffer or a DMF—MeCN mixture (40/60 vol. %).
Syntheses of Z—Ala—Ala—Lys—Phe—pNA, Z—Ala—Ala—
Glu—Phe—pNA, Z—Ala—Ala—Lys—Asp—pNA, Z—Ala—Ala—
2
1
1
1
1
2
1
Glu—Asp—pNA,
and
Z—Ala—Ala—Leu—Asp—pNA
1
were carried out analogously to the synthesis of
Z—Ala—Ala—Leu—Phe—pNA.
1
Synthesis of Z—Ala—Ala—Leu—pNA. An ITL specimen
9. A. Gordon and R. Ford, in The Chemist´s Companion,
J. Wiley and Sons, Inc., New York, 1974, 393.
0. A. A. Gershkovich and V. K. Kibirev, Khimicheskii sintez
peptidov [Chemical Synthesis of Peptides], Naukova Dumka,
Kiev, 1992, 360 pp. (in Russian).
(
100 mg; the protein content was 0.35 mg, 9.3 nmol), which
has been preliminarily washed with MeCN (1 mL) and then
twice washed with a DMF—MeCN mixture (25/75 vol. %;
1
7
mL), was added to a solution of Z—Ala—Ala—OH (2.1 mg,
µmol) and Leu—pNA (1.8 mg, 7 µmol) in a DMF—MeCN
2
1. Russ. Inventor's Certificate No. 2036095, Byul. Izobr., 1995,
mixture (25/75 vol. %; 300 µL). The reaction mixture was
stirred at 20 °C and the aliquots (5 µL) were taken at regular
intervals and analyzed by HPLC. After completion of the
synthesis, the reaction mixture was separated and the cryogel
1
5 (in Russian).
Received March 5, 2001