Biocatalytic properties of thermolysin immobilized on polyvinyl alcohol cryogel

Article abstract of DOI:10.1134/S1068162008040079

Preparations with different contents of thermolysin were obtained by the immobilization of the enzyme on granulated polyvinyl alcohol cryogel. Their activity and stability in an aqueous medium and in mixtures of polar organic solvents of different composition were investigated. The catalytic properties of the preparations in reactions of peptide bond formation were studied, and the optimal amount of the biocatalyst, the concentrations of initial reagents, and the ratios of organic solvents and water necessary for effective enzymatic peptide synthesis catalyzed by immobilized thermolysin were determined. A series of peptides of the general formula Z-Ala-Ala-Xaa-pNA, where Xaa = Leu, Ile, Phe, Val, or Ala, were synthesized, and the immobilized enzyme was shown to retain substrate specificity in an organic medium.

Full text of DOI:10.1134/S1068162008040079

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2008, Vol. 34, No. 4, pp. 435–441. © Pleiades Publishing, Ltd., 2008.
Original Russian Text © A.V. Belyaeva, Yu.A. Smirnova, E.N. Lysogorskaya, E.S. Oksenoit, A.V. Timofeeva, V.I. Lozinskii, I.Yu. Filippova, 2008, published in Bioorganicheskaya
Khimiya, 2008, Vol. 34, No. 4, pp. 487–494.
Biocatalytic Properties of Thermolysin Immobilized
on Polyvinyl Alcohol Cryogel
A. V. Belyaevaa, Yu. A. Smirnovab, E. N. Lysogorskayac, E. S. Oksenoitc, A. V. Timofeevad,
V. I. Lozinskiia, and I. Yu. Filippovac,1
a Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, Moscow
b Faculty of Bioengineering and Bioinformatics, Moscow State University,
Vorob’evy Gory, Moscow, 119992 Russia
c Faculty of Chemistry, Moscow State University, Vorob’evy gory, Moscow, 119992 Russia
d Belozersky Research Institute of Physicochemical Biology, Moscow State University,
Vorob’evy gory, Moscow, 119992 Russia
Received June 14, 2007; in final form, October 22, 2007
Abstract—Preparations with different contents of thermolysin were obtained by the immobilization of the
enzyme on granulated polyvinyl alcohol cryogel. Their activity and stability in an aqueous medium and in mix-
tures of polar organic solvents of different composition were investigated. The catalytic properties of the prep-
arations in reactions of peptide bond formation were studied, and the optimal amount of the biocatalyst, the con-
centrations of initial reagents, and the ratios of organic solvents and water necessary for effective enzymatic
peptide synthesis catalyzed by immobilized thermolysin were determined. A series of peptides of the general
formula Z-Ala-Ala-Xaa-pNA, where Xaa = Leu, Ile, Phe, Val, or Ala, were synthesized, and the immobilized
enzyme was shown to retain substrate specificity in an organic medium.
Key words: polyvinyl alcohol cryogel, enzymatic peptide synthesis in water–organic mixtures, immobilized
thermolysin
DOI: 10.1134/S1068162008040079
INTRODUCTION
It is known that the efficiency of peptide synthesis in
polar organic solvents is substantially higher; however,
a fast inactivation of protease restricts the process. One
way of stabilizing the proteases to the inactivating
effect of polar organic solvents is covalent immobiliza-
tion [7−9]. We previously have shown that biocatalysts
Thermolysin, a typical bacterial zinc-dependent
neutral metalloprotease, is often used for the fragmen-
tation of proteins and peptides in structural studies.2 In
the last few years, this enzyme has been widely
employed as a catalyst at some stages of chemicoenzy- based on the serine protease subtilisin covalently
immobilized on PVA cryogel [10] feature stability and
a high synthase activity in anhydrous media [11]. PVA
cryogel is an inert polymeric carrier, which is obtained
by a simple procedure of freezing-thawing of a PVA
solution [12]. A unique feature of PVA cryogels is the
macroporous structure and stability in organic solvents.
In addition, cryogels are distinguished by a high hydro-
philicity, which enables them to hold water inside the
polymeric matrix even in anhydrous medium of polar
organic solvents. These properties make possible the
immobilization of large enzyme molecules on the car-
rier, by minimizing the restrained diffusion of the sub-
strate and the inactivation of the biocatalyst in organic
solvent media.
matic synthesis in the production of peptide hormones,
taste peptides, growth factors and their fragments, as
well as in the industrial manufacture of the low-calorie
sweetener aspartame [1−6]. ThL is involved only in
thermodynamically controlled processes, affecting the
rate of the attainment of equilibrium in the hydrolysis-
synthesis reaction. In water, the equilibrium of the reac-
tion is shifted toward hydrolysis. The shift of equilib-
rium toward synthesis is accomplished by (1) removing
the product from the reaction through precipitation or
extraction into another phase, (2) increasing the con-
centration of initial compounds, and (3) decreasing the
water content in the reaction system in reactions con-
ducted in organic solvent media.
The goal of this work was the synthesis of prepara-
tions of thermolysin covalently immobilized on PVA
cryogel, the examination of their stability in aqueous
medium and in media of polar organic solvents, includ-
1
Corresponding author; phone: +7 (495) 9395529; fax: +7 (495)
9328846; e-mail: irfilipp@genebee.msu.su
2
Abbreviations: Dnp, 2,4-dinitrophenyl; pNA, p-nitroanilide;
PVA, polyvinyl alcohol ThL, thermolysin; Z, benzyloxycarbonyl.
435

436
BELYAEVA et al.
Yield of the product, %
Residual activity %
100
100
90
80
70
60
50
40
30
20
10
80
60
40
20
0
25
40
60
80
95
1
2
3
4
5
6
7
8
0
Content of DMF, %
Content of the enzyme in the sample, nmol
Fig. 2. Dependence of the yield of Z-Ala-Ala-Leu-pNA on
the content of immobilized ThL in 300 µl of mixture DMF–
MeCN 1 : 3 (1 h, [S] 40 mM).
Fig. 1. Stability of immobilized ThL in DMF–MeCN of dif-
ferent composition (incubation 72 h).
bility of the immobilized enzyme in MeCN with differ-
ent content of DMF showed that the activity of
biocatalyst preparations did not change during the incu-
bation for 72 h (Fig. 1). It was found that, during a more
prolonged incubation for 3.5 days, the biocatalyst activ-
ity also remained unchanged. Keeping immobilized
ThL in mixtures containing 40–80% DMF led to a
slight inactivation of the enzyme; however, even after
10 days, the preparation retained no less than 60% of its
activity (not shown in the figure). A more abrupt loss of
activity was observed in 95% DMF/MeCN; however, in
this case, too, the catalyst retained more than 40% of
activity.
ing the study of their catalytic properties in reactions of
peptide bond formation.
RESULTS AND DISCUSSION
By the covalent fixation of the commercial ThL
preparation on the glutaraldehyde-activated matrix of
PVA cryogel, we obtained samples of the immobilized
enzyme (subsequently referred to as biocatalyst) with a
protein content of 2.4 (0.064 µmol), 4.4 (0.117 µmol),
and 11.6 mg (0.309 µmol)/g carrier (samples 1, 2, and
3, respectively). The amount of the protein on the car-
rier was determined from the data of amino acid analy-
sis after the hydrolysis of a biocatalyst sample by 5.7 N
hydrochloric acid.
The activity of biocatalysts was measured from the
hydrolysis of the chromophoric substrate Dnp-Gly-
Gly↓Ile-Arg. The specific activity of samples 1, 2, and
3 per 1 g of carrier differed and were 0.13, 0.18, and
0.50 µmol/min. However, the specific activity values
calculated per 1 mg of the enzyme were practically
equal, 0.050, 0.048, and 0.043 µmol/min. The stability
of the biocatalyst was estimated by measuring its spe-
cific hydrolytic activity after storage in media of differ-
ent composition.
The catalytic properties of ThL immobilized on
PVA cryogel were studied in a model reaction of the
synthesis of the chromogenic substrate of serine pro-
teases Z-Ala-Ala-Leu-pNA (I) in mixtures of polar
organic solvents DMF/MeCN and DMSO/MeCN:
Z-Ala-Ala-OH + Leu-pNA
(1)
immobilized.ThL
Z-Ala-Ala-Leu-pNA.
The amount of ThL sufficient for the effective syn-
thesis of tripeptide (I) (Fig. 2) was determined at the
equimolar ratio of the amino and carboxyl components
(experiments were carried out with sample 1). It is evi-
dent from Fig. 2 that initially (at ThL concentration up
to 3 nmol) the yield of the product was linearly depen-
dent on the amount of the enzyme in the biocatalyst
sample introduced into the reaction mixture, following
which it reached a plateau. A further increase in the
amount of the enzyme did not lead to an abrupt increase
The biocatalyst had a high stability at +4°C in an
aqueous 0.05 M Tris-HCl buffer, pH 7.2, containing
50 mM CaCl2 (buffer A) and retained no less than 95%
of initial activity even after 4 months of storage in this
buffer (specific activity of the immobilized enzyme
measured on the day of isolation was taken as 100%).
Of particular interest was the study of the stability of in the yield of the product. Therefore, further experi-
biocatalyst samples in practically anhydrous organic ments were carried out using biocatalyst preparations
solvent media suitable for peptide synthesis [13]. Pre- with a ThL content of 1.4 nmol. The effect of the con-
viously we have shown a high stability of ThL immobi- centrations of starting components on the yield of the
lized of PVA cryogel in MeCN [11]. A study of the sta- target product is shown in Fig. 3. It is seen that, as the
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 4 2008

BIOCATALYTIC PROPERTIES
Yield of the product, %
437
Yield of the product, %
100
100
90
80
70
60
50
40
30
20
10
90
80
70
60
50
40
30
20
10
0
20
40
60
80
100
0
20
40
60
80
100
120
[S], mM
Content of DMF, volume percent
Fig. 3. Dependence of the yield of Z-Ala-Ala-Leu-pNA on
the concentration of substrates in 300 µl of mixture DMF–
MeCN 1 : 3 (incubation for 24 h, the amount of ThL
1.4 nmol)
Fig. 4. Dependence of the yield of Z-Ala-Ala-Leu-pNA on
the content of DMF in a mixture with MeCN (1 h, [S] =
40 mM, E : S = 1 : 5000).
concentration of starting components increased to
30 mM, the rate of synthesis rose sharply, and begin-
ning from a concentration of 40 mM, it remained prac-
tically unchanged.
To determine how the DMF concentration in the
reaction mixture affects the catalytic activity of immo-
bilized ThL, the synthesis was conducted in
DMF/MeCN mixtures containing from 10 to 80% of
DMF (Fig. 4). It was shown that increasing the DMF
concentration leads to a monotonous decrease in the
synthase activity of the preparation throughout the
range of solvent concentrations used. In further experi-
ments, a mixture containing 20 volume percent of DMF
was used.
increased linearly; then it gradually reached some con-
stant value at a water content of 10% and remained
practically unchanged in the region corresponding to
high concentrations of water. The small decrease in the
yield observed at a 25% content of water is attributable
to the exceeding of the threshold of reaction product
solubility and the secondary hydrolysis of the resulting
product. Thus, 10–15% of water is necessary for the
occurrence of the maximum catalytic activity of immo-
bilized ThL.
We studied the dependence of the yield of Z-Ala-
Ala-Leu-pNA on the final concentration of added
DMSO (in the range of 10−80% volume percent) in a
system containing 10% aqueous buffer mixed with
The enzymatic synthesis catalyzed by ThL is a ther-
modynamically controlled process, which is the reverse
of the hydrolysis reaction [14]. Water (as a nucleophile)
is immediately involved in the cleavage of the peptide
bond, by attacking the hydrogen atom of the carbonyl
group. In heterogeneous reactions of peptide bond for-
mation, the amount of water in the reaction mixture
should be sufficient to provide the maximum catalytic
activity of the enzyme and, at the same time, it should
not lead to the precipitation of poorly soluble hydro-
phobic compounds and the reaction product from the
reaction mixture. Therefore, to find the conditions for
the most efficient use of the biocatalyst, it was neces-
sary to determine the amount of water in the reaction
mixture at which the yield is maximal, and the product
and starting substances are completely dissolved in the
reaction mixture. Figure 5 shows the dependence of the
yield of model peptide (I) on the water content in the
reaction mixture. The concentration range used was
restricted by the solubility of the reaction components
(hydrophobic substrates and reaction product). As the
volume of water in the reaction mixture increased from
0 to 7%, the yield of the product within 2 h first
Yield of the product, %
100
80
60
40
20
0
5
10
15
20
25
30
Content of H2O, volume percent
Fig. 5. Dependence of the yield of Z-Ala-Ala-Leu-pNA on
the concentration of water in a mixture 20% DMF−55−75%
MeCN−5−25% H O (2 h, [S] = 40 mM, E : S = 1 : 5000).
2
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 4 2008

438
BELYAEVA et al.
was also successfully tested as a substrate for subtilisin
Carlsberg.
Yield of the product, %
100
90
80
70
60
50
40
30
20
10
Using immobilized ThL under the above-described
conditions, we synthesized a series of peptides of the
general formula Z-Ala-Ala-Xaa-pNA, where Xaa = Ile,
Phe, Val [peptides (II)−(IV), respectively] with a yield
of 70–80% (table). The synthesis of Z-Ala-Ala-Ala-
pNA [peptide (V)] proceeded somewhat worse. Immo-
bilized ThL did not catalyze the formation of a peptide
bond between residues Pro [peptide (VIII)], Gly [pep-
tide (IX)], and those of dicarboxylic amino acids [pep-
tides (X) and (XI)]. These data suggest that ThL in
organic medium retained the substrate specificity inher-
ent in this enzyme in hydrolytic reactions [15]. It
should be noted that this regularity has been observed
earlier in studies of peptide bonding catalyzed by ThL
in water–organic media [16].
0
10 20 30 40 50 60 70 80 90
Content of DMSO, volume percent
Fig. 6. Dependence of the yield of Z-Ala-Ala-Leu-pNA on the
content of DMSO in a mixture 10% H O −10−80%
2
DMSO −10−80% MeCN (2 h, [S] = 40 mM, E : S = 1 : 5000).
It was found in the synthesis of Z-Ala-Ala-Leu-NH2
[peptide (VI)] that substituting amide for p-nitroanilide
did not influence the efficiency of peptide condensa-
tion: peptide (VI) was obtained with a quantitative
yield. The possibility of using Leu-OCH3 and Phe-
OCH3 as amino components was exemplified by the
synthesis of Z-Ala-Ala-Leu-OCH3 [peptide (VII)] and
Z-Asp-Phe-OCH3 (XII), a precursor of aspartame; the
yield of these peptides was 33 and 49%, respectively.
10−80% MeCN (Fig. 6). The high yields of the target
peptide (80–90%) were observed in a mixture contain-
ing up to 40% of this solvent; a further increase in its
concentration was accompanied by an abrupt decrease
in the yield.
The replacement of DMF by DMSO, the experi-
mental conditions being the same, did not substantially
affect the yield of the reaction product.
One obvious advantage of immobilized enzyme
preparations in the peptide synthesis, as compared with
native enzymes, is the possibility of their repeated uti-
lization. Previously in our laboratory we have shown
that serine protease preparations retain their catalytic
properties after at least three synthesis cycles with the
use of one and the same sample [11].
The catalytic properties of immobilized ThL on
repeated utilization were studied by conducting consec-
utive syntheses with one and the same sample of a bio-
catalyst without its intermediate reactivation. For this
purpose, between the synthesis cycles (reaction time
2 h), the preparation was washed with a 20%
DMF/80% MeCN mixture containing no water. The
amount of water required for the synthesis was intro-
duced to the reaction mixture by the addition of the cor-
responding aliquot of 0.05 M Tris-HCl buffer, pH 7.2,
containing 50 mM CaCl2 (buffer A). Even after six
cycles, the yield of the product remained high.
Thus, we showed that ThL covalently immobilized
on PVA cryogel is highly reactive and stable in media
of different composition. Optimal conditions for the
peptide synthesis catalyzed by immobilized ThL were
determined, and it was found that the biocatalyst for the
most part retains the primary substrate specificity of
native ThL in organic medium and can also be used for
the synthesis of esters and amides of peptides that are
the precursors of peptides of a more complicated struc-
ture. We showed that one biocatalyst sample can be
repeatedly used in the synthesis reaction.
EXPERIMENTAL
The following preparations were used: ThL with
M 37500 Da from Bacillus thermoproteoliticus (Fluka
Chemie AG, Switzerland, EC 3.4.24.27); PVA 16/1
(å 69000) (NPO Azot, Severodonetsk, Ukraine); SP
Sephadex C-25 (Pharmacia, Sweden); acetonitrile for
HPLC (extra pure grade, Lekbiofarm, Russia) contain-
ing no more than 0.01% of water; DMF (analytical
grade, Reakhim, Russia) additionally purified as
described in [17]; trifluoroacetic acid (analytical grade,
Fluka Chemie AG, Switzerland); acetic acid (Reakhim,
Russia); CaCl2 (Sigma, United States); Tris (ISN Bio-
Thus, immobilized ThL preparations can be repeat-
edly used as effective catalysts in the reactions of pep-
tide bond formation.
The potentialities of this immobilized biocatalyst
were exemplified by the preparative synthesis of Z-Ala-
Ala-Leu-pNA.
The reaction was conducted in a system 10% medicals, United States); triethylamine (analytical
DMSO/10% H2O/MeCN at the equimolar ratio of the grade, Reakhim, Russia) additionally distilled as
amino and carboxyl components (E : S ~ 1 : 5000). The described [17]; Leu-pNA (Sigma, NOA), and Phe-pNA
analytical yield of the product was 100%. After purifi- (Serva, Germany). The derivatives of other amino acids
cation, the yield was 71%. The product was character- and peptides were synthesized by standard methods
ized by the data of HPLC and amino acid analysis; it [18].
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BIOCATALYTIC PROPERTIES
439
Peptides synthesized at the catalysis by ThL immobilized on PVA cryogel
Carboxyl
component
Reaction time,
Retention time,
Amino component
Reaction product
Yield, %
h
min*
Z-Ala-Ala-OH
H-Leu-pNA
H-Phe-pNA
Z-Ala-Ala-Leu-pNA (I)
Z-Ala-Ala-Phe-pNA (II)
24
24
91
73
80
82
30
100
33
0
27
27.5
H-Val-pNA
H-Ile-pNA
Z-Ala-Ala-Val-pNA (III)
Z-Ala-Ala-Ile-pNA (IV)
Z-Ala-Ala-Ala-pNA (V)
Z-Ala-Ala-Leu-NH2 (VI)
Z-Ala-Ala-Leu-OCH3 (VII)
Z-Ala-Ala-Pro-pNA (VIII)
Z-Ala-Ala-Gly-pNA (IX)
Z-Ala-Ala-Asp-pNA (X)
Z-Ala-Ala-Glu-pNA (XI)
Z-Asp-Phe-OCH3 (XII)
24
24
24
24
24
72
72
72
72
48
26.0
27.5
22.1
17.0
22.6
H-Ala-pNA
H-Leu-NH2
H-Leu-OCH3
H-Pro-pNA
H-Gly-pNA
H-Asp-pNA
H-Glu-pNA
H-Phe-OCH3
0
0
0
Z-Asp-OH
22.5
49**
Notes: Reaction conditions: 20% DMF/10% buffer A/MeCN, [S] = 40−58 mM, +20°C, E : S = 1 : 5000.
*HPLC for all peptides in system M; for (XII) in system N (see the Experimental section).
** Reaction was carried out in tert-butanol containing 5% H2O.
The preparation of granulated (1–1.5 mm) PVA
cryogel and its reactive aldehyde-containing derivative
and the covalent linking of ThL to it were carried out in
the laboratory of cryochemistry of biopolymers (Insti-
tute of Organoelement Chemistry, Russian Academy of
Sciences) as described [11, 19]. Three samples of the
biocatalyst were obtained (2 g each). The biocatalyst
was stored in 0.05 M Tris-HCl buffer, pH 7.2, contain-
ing 50 mM CaCl2 (buffer A) The amount of ThL immo-
bilized on PVA cryogel was estimated from the amino
acid analysis data to be 2.4 (0.064 µmol), 4.4
(0.117 µmol), and 11.6 mg (0.309 µmol)/g carrier
(samples 1, 2, and 3, respectively).
The composition of a reaction mixture consisting of
initial components and a target peptide was calculated
without any correction for the difference of molar
absorption coefficients of the components.
Spectrophotometric measurements were carried out
on Specord UV VIS (Germany) and Genesys 10-S UV
spectrophotometers (Spectronics, United States).
The amino acid analysis was carried out on a Hitachi
835 automatic amino acid analyzer (Japan) after acid
hydrolysis by 5.7 N HCl at 105°ë in vacuumated
ampoules for 24 and 48 h.
Enzymatic activity of immobilized ThL in the
hydrolysis of Dnp-Gly-Gly-Ile-Arg. A solution of
substrate Dnp-Gly-Gly-Ile-Arg (1 ml, 0.5 mg/ml) in
0.05 M Tris-HCl buffer A was added to a biocatalyst
(10−15 mg) containing the enzyme (0.6−4.6 nmol), and
the reaction mixture was incubated at 37°ë. After
30−40 min, 900 µl of the solution was taken, 400 µl of
50% acetic acid was added, the solution was stirred,
and the mixture was applied to Sephadex SP-25 col-
umns (volume 3 ml), eluted with 2 ml of 0.1 N acetic
acid, and the optical absorption of the eluate (Dnp-Gly-
Gly) was measured at 360 nm.
The peptides were analyzed on an Altex Model
110A liquid chromatograph (United States). The elu-
tion was with 0.1% CF3COOH in a linear gradient of
acetonitrile concentration from 10 to 70% (flow rate
1 ml/min) for 30 min using Microsorb-MV C8 columns
(4.6 × 250 mm) (Rainin Instrument Company, Inc.;
United States) (system M) and Nucleosil C18 column
(4.6 × 250 mm; Biokhimmak, Moscow) (system N).
The detection of eluted peptides was at 220 and
280 nm.
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BELYAEVA et al.
Specific activity was calculated by the formula:
were preliminarily washed with MeCN (3 × 1.5 ml) and
a mixture MeCN–DMF 80 : 20 (volume percent) (2 ×
2.5 ml). Samples were taken and analyzed according to
the general method after 1, 2, and 24 h.
(A360 – Ac360) ⋅ Vtot
------------------------------------------
ε ⋅ t ⋅ m ⋅ Vsamp
,
where Ä360 and Ac360 is the absorption of an experimen-
Dependence of the yield of Z-Ala-Ala-Leu-pNA
on the content of DMSO was studied in syntheses car-
ried out by the general method in mixtures 10-80%
DMSO/10–80% MeCN/10% Tris-HCl buffer, pH 7.2,
and 50 mM CaCl2. Biocatalyst granules were prelimi-
narily washed with MeCN (3 × 1.5 ml) and a mixture
MeCN–DMSO 90 : 10 (volume percent) (2 × 2.5 ml).
Samples were taken and analyzed according to the gen-
eral method after 1, 2, and 24 h.
tal and control solution at 360 nm; Vtot is the total vol-
ume of the eluate, ml; ε is the molar absorption coeffi-
cient of the Dnp-group, 15 mM–1 cm–1; t is the reaction
time, min; m is the mass of a sample of the immobilized
enzyme, mg; Vsapm is the volume of the sample applied
to the column, ml. The specific activity was expressed
in µmol/mg × protein min.
Stability of immobilized ThL in aqueous buffer.
A biocatalyst preparation was stored under the layer of
buffer A at +4°ë. At regular intervals, biocatalyst sam-
ples (15–20 mg) were taken, and the activity of the
preparation was determined. The activity of a biocata-
lyst preparation obtained immediately after immobili-
zation was taken to be 100%.
Stability of immobilized ThL in organic solvent
mixtures was determined in a similar way by incu-
bating a biocatalyst sample (15–20 mg) in an
MeCN/DMF mixture of the corresponding composi-
tion. Before activity determination, the organic solvent
was decanted, and the preparation was washed by
buffer A (3 × 1.5 ml).
Synthesis of Z-Ala-Ala-Leu-pNA (general
method). A solution (300 µl) of 40 mM Z-Ala-Ala-OH
and Leu-pNA in the corresponding mixture of solvents
was added to 20 mg of a biocatalyst preparation [sam-
ple 2, content of ThL 0.09 mg (2.4 nmol)], and the mix-
ture was shaken on an orbital shaker at 20°ë. At regular
intervals, samples (5 µl) were taken for HPLC; elution
was in a gradient of M.
Dependence of the yield of Z-Ala-Ala-Leu-pNA
on the amount of immobilized ThL was studied after
the synthesis carried out by the general method in sys-
tem MeCN–DMF 75 : 25 (volume percent). Different
biocatalyst samples (sample 1) (10, 20, 30, 45, 60, and
110 mg; content of ThL 0.6–7.0 nmol) were introduced
into the reaction.
Dependence of the yield of Z-Ala-Ala-Leu-pNA
on the concentration of starting components was
studied after the synthesis carried out by the general
method in system MeCN/DMF 75/25 (volume percent)
at the equimolar ratio of starting components (Z-Ala-
Ala-OH and Leu-pNA) in the concentration range of
20–200 mM. The content of the enzyme was 1.4 nmol.
Possibility of repeated use of immobilized ThL
samples in the synthesis of Z-Ala-Ala-Leu-pNA, was
tested by the general method in a mixture MeCN–
DMF–H2O (70/20/10 per cent by volume), was studied
using one sample of immobilized ThL. After each syn-
thesis, the reaction mixture was separated (by a
pipette), and biocatalyst granules were washed with a
MeCN–DMF mixture 80 : 20 volume percent (4 ×
200 µl, 15 min) and introduced into the next synthesis
of this peptide, adding the solution of starting compo-
nents.
Preparative synthesis of Z-Ala-Ala-Leu-pNA. A
solution (3.75 ml) containing Z-Ala-Ala-OH (55.9 mg,
190 µmol) and Leu-pNA (47.7 mg, 190 µmol) in a mix-
ture MeCN–DMSO–H2O (80/20/10 volume percent)
was added to a biocatalyst sample (350 mg, content of
the protein 1.54 mg). The reaction mixture was shaken
on an orbital shaker at 20°ë, and 5 µl samples were
taken at regular intervals for the analysis by HPLC.
After 24 h, the reaction mixture was separated from the
biocatalyst by decantation, and biocatalyst granules
were washed with a MeCN–DMSO 90 : 10 (volume
percent) mixture (5 × 2 ml). The reaction mixture and
all washings were combined and evaporated on a rotor
evaporator. A solution of 0.1 N hydrochloric acid
(1.5 ml) was added dropwise to the residue (a yellowish
precipitate occurred), the solution was stirred, and cold
water (8 ml) was introduced in small portions. The yel-
lowish precipitate was transferred to a filter, washed
with cold water, and dried over NaOH in a vacuum exs-
iccator. Yield: 71 mg (71%).
Amino acid analysis of Z-Ala-Ala-Leu-pNA: Ala
16.7 nmol, Leu 9.9 nmol.
Condensation of Z-Ala-Ala-OH and Phe-pNA.
MeCN (100 µl), DMF (50 µl), a 250 mM solution of Z-
Ala-Ala-II (100 µl) and a 250 mM solution of Phe-pNA
in DMF were added to a biocatalyst preparation
(20 mg, the content of ThL 0.05 mg). The reaction mix-
ture was shaken on an orbital shaker at 20°ë, and 5 µl
Dependence of the yield of Z-Ala-Ala-Leu-pNA
on the content of DMF was studied in syntheses car-
ried out by the general method in MeCN–DMF mix-
tures containing from 10 to 80% DMF.
Dependence of the yield of Z-Ala-Ala-Leu-pNA samples were taken at regular intervals for the analysis
on the content of water in reaction mixture was stud- by HPLC. Before being applied to a column, samples
ied in syntheses carried out by the general method in were diluted with a starting eluent (20% solution of
mixtures 20% DMF/55–75% MeCN/5–25% Tris-HCl MeCN in water; 1.7 ml). The retention time of Z-Ala-
buffer, pH 7.2, and 50 mM CaCl2. Biocatalyst granules Ala-Phe-pNA in gradient (M) was 27.5 min.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 4 2008

BIOCATALYTIC PROPERTIES
441
The amino acid analysis of Z-Ala-Ala-Phe-pNA:
REFERENCES
Ala 3.4 nmol, Phe 2.2 nmol.
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Ala-pNA, Z-Ala-Ala-OH and Leu-OMe, Z-Ala-Ala-
OH and Leu-NH2 was carried out similarly to the con-
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Retention time in system I, min: Z-Ala-Ala-Ile-
pNA—27.5, Z-Ala-Ala-Val-pNA—26.0, Z-Ala-Ala-
Ala-pNA—22.1, Z-Ala-Ala-Leu-OMe—22.6, and
Z-Ala-Ala-Leu-NH2—17.0.
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Amino acid analysis, nmol: Z-Ala-Ala-Ile-pNA:
Ala 3.7, Ile 2.4; Z-Ala-Ala-Val-pNA: Ala 1.0, Val
0.53; Z-Ala-Ala-Ala-pNA: Ala 17.9; Z-Ala-Ala-Leu-
OMe: Ala 17.7, Leu 7.5; and Z-Ala-Ala-Leu-NH2 Ala
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Condensation of Z-Asp-OH and Phe-OMe.
Z-Asp-OH (50 µmol) and HCl · Phe-OMe (100 µmol)
were suspended in tert-butanol (500 µmol), then TEA
(14 µl, 100 µmol) was added, and the solution was
stirred for 20 min. A biocatalyst preparation (20 mg, the
content of the protein 0.2 mg) and 50 mM CaCl2 (30 µl)
were added to the resulting suspension. The reaction
mixture was shaken on an orbital shaker at 20°ë, and
5 µl samples were taken at regular intervals for the
analysis by HPLC. Before being applied to a column,
samples were diluted with a starting eluent (10% solu-
tion of MeCN in water; 200 µl). The retention time of
Z-Asp-Phe-OMe in system N was 22.5 min.
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ACKNOWLEDGMENTS
This work was supported by the Russian Foundation
for Basic Research (projects nos. 06-03-33056-a and
07-04-12025-ofi).
19. Lozinskii, V.I. and Zubov, A.L., RF Inventor’s Certificate
no. 2036095, Byull. Izobret., 1995, no. 15, p. 21.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 4 2008