ISSN 0006-2979, Biochemistry (Moscow), 2026, Vol. 91, No. 8, pp. 1470-1479 © Pleiades Publishing, Ltd., 2026.
1470
Structural and Functional Analysis
of the Recombinant Aureobasidium pullulans
Cutinase Variants for Efficient
Polycaprolactone Degradation
Filipp K. Ermilov
1
, Elena V. Eneiskaya
1
, Svetlana A. Panasenko
1
,
Elena V. Zhurishkina
1
, Kirill S. Bobrov
1
, Mikhail G. Petukhov
1
,
Anna A. Kulminskaya
1
, and Irina A. Sizova
1,a
*
1
Konstantinov Petersburg Nuclear Physics Institute, National Research Center “Kurchatov Institute”,
188300 Gatchina, Russia
a
e-mail: sizova_ia@pnpi.nrcki.ru
Received May 4, 2026
Revised July 27, 2026
Accepted July 29, 2026
Abstract— Polycaprolactone (PCL) is a biodegradable polyester widely used in industry, but it degrades slowly
in the environment. Enzymatic hydrolysis catalyzed by cutinases represents a promising approach for PCL
waste utilization. Through screening, we selected the Aureobasidium pullulans VKM 1116 strain capable of
degrading PCL. We amplified the ApCUT1 cutinase gene from the A. pullulans VKM 1116 genomic DNA.
Heterologous expression in Komagataella phaffii yielded ~60 mg/L of the target protein. The recombinant
enzyme exhibited maximum activity at pH 6.0-6.5 and 30-40°C. Using site-directed mutagenesis, we generated
single mutants (Y58W and L186F) and a double mutant (Y58W/L186F). The double mutant demonstrated
the highest catalytic efficiency toward the model substrate 4-nitrophenyl 16-methylsulfonyl hexadecanoate
(4-NP-(16-MS-C16)), exhibiting a two-fold increase in the hydrolysis rate compared to the wild-type enzyme.
During PCL hydrolysis, the Y58W mutant showed the highest activity, providing a 1.8-fold increase in the
suspension degradation rate and complete polymer granule degradation at a rate of 1 mg/h per 1 mg of
protein. Our molecular modeling suggests that the hydrophobic bridge at the entrance to the active site
functions as a “molecular trap.” Forthe low-molecular-weight substrate (4-NP-(16-MS-C16)), enzymatic activity
is limited by the kinetics of the substrate retention step, which is consistent with the maximum catalytic
efficiency of the Y58W/L186F mutant with a closing trap. Conversely, activity toward PCL is limited by the
initial adsorption stage, for which an open conformation of the free enzyme – as observed in the Y58W
variant – is critically important. These results demonstrate the potential of the ApCUT1_Y58W cutinase for
developing advanced PCL recycling technologies.
DOI: 10.1134/S0006297926601449
Keywords: cutinase, polycaprolactone, enzymatic degradation, Aureobasidium pullulans, heterologous
expression, site-directed mutagenesis, molecular modeling
* To whom correspondence should be addressed.
INTRODUCTION
Cutinases (EC 3.1.1.74) are esterases with a char-
acteristic Ser-His-Asp catalytic triad in their active
sites. They catalyze hydrolysis of ester bonds of natu-
ral and synthetic polymers. Location of the active site
on the surface of most cutinases allows substrates to
enter the catalytic cycle quickly without requiring
additional rearrangement of the protein molecule [1].
Consequently, cutinases demonstrate broad substrate
specificity. Their catalytic plasticity enables applica-
tions in various fields, such as textile industry, deter-
gent production, biomass processing, food production,
and detoxification of environmental pollutants [2, 3].
POLYCAPROLACTONE-DEGRADING CUTINASE AND ITS VARIANTS 1471
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026
The primary natural substrate for cutinases is
cutin, an aliphatic polyester that serves as a protec-
tive barrier in many plants [2]. However, cutinas-
es can also hydrolyze synthetic polymers, including
polycaprolactone (PCL) [4]. Due to its biocompatibil-
ity, mechanical strength, and elasticity, PCL is wide-
ly used in medicine, cosmetology, 3D printing, and
packaging materials [5]. Therefore, developing envi-
ronmentally friendly technologies for PCL utilization
and recycling using cutinases is of particular interest.
Accordingly, it is necessary to search for new highly
efficient natural enzymes and/or modify known cuti-
nases to increase their catalytic activity and expand
their substrate range.
Genomic databases currently contain over 3000
genes encoding potential cutinases. Sequence identi-
ty between these potential enzymes is low, and pre-
sumably, only some are active. A detailed structural
and functional analysis of five phylogenetically dis-
tant cutinases by Novy et al. [1] revealed that they
differ in their modes of interaction with substrates,
which include binding to an open active site exposed
to the solvent, interaction via a lid typical for true
lipases, and binding with an active site with its acces-
sibility controlled by the conformational rearrange-
ment of the flexible loops. Activity of the enzymes
towards bulky hydrophobic substrates is determined
by combination of structural factors: configuration
of the catalytic cleft, length and hydrophobicity of
the loops flanking the catalytic cleft, and bulkiness
of amino acid residues at the entrance to the ac-
tive site. When flexible loops are closed, these res-
idues form a steric barrier for the ligand binding.
Examples include Phe52 and Ile176 in the cutinase
CLE1, as well as Leu81 and Val184 in the cutinase
FsCUT [1, 6, 7]. However, it remains unclear how ami-
no acid substitutions in the substrate-binding region
affect the dynamics of the steric barrier formation
at the entrance to the catalytic cleft, both in the
free state of the enzyme and when bound to various
ligands.
This study aimed to identify and obtain a new
cutinase, characterize its biochemical and structur-
al features, and engineer mutant variants with en-
hanced catalytic activity. Through screening of the
PCL-degrading cutinase producers among the micro-
organisms from the laboratory collection, the strain
A. pullulans VKM 1116 was selected. Genetic con-
structs for heterologous expression of the genes en-
coding the ApCUT1 cutinase and its mutant variants
in the K. phaffii cells were created and optimized.
Using molecular modeling and molecular dynamics
(MD) methods, structural features of the proteins
were examined, and dynamics of the steric barrier
formation and its impact on the efficiency of synthet-
ic substrate hydrolysis were analyzed.
MATERIALS AND METHODS
Materials. Enzymes for molecular work were
obtained from SibEnzyme and Helicon (Russia).
Salts and other reagents were from Dia-M (Russia).
4-Nitrophenyl 16-methylsulfonyl hexadecanoate (4-NP-
(16-MS-C16)) was synthesized according to a pub-
lished method [8]. PCL was obtained from Sigma-
Aldrich (USA).
Strains and media. Screening of strains secreting
PCL-degrading enzymes was conducted among the mi-
croorganisms from the collection of the Biotechnology
Laboratory of the NRC “Kurchatov Institute” – PNPI.
Screening was performed on an agarized medium
(g/L: peptone – 1.2; yeast extract – 0.6; glucose – 0.6;
NaCl– 0.6; agar – 7.5) containing 1%PCL suspension.
For plasmid construction and plasmid isolation, a
E. coli DH5α strain was used. The culture was grown
at 37°C in an LB medium. If necessary, ampicillin or
zeocin (InvivoGen, USA) were added at concentrations
of 100 μg/mL or 25 μg/mL, respectively. A K. phaffii
GS115 (his4-) strain was kindly provided by
Dr.J. M. Cregg from the Keck Institute (USA) and used
for protein expression. The yeast culture K. phaffii
was grown in YPD and BMMY media, as described
in the Invitrogen manual [9]. Selection of K. phaffii
transformants was performed on an agarized YPD
medium containing 1000 μg/mL of zeocin.
Cloning. Genomic DNA of A. pullulans VKM
1116 was isolated using a Thermo Scientific GeneJET
Plant Genomic DNA Purification Mini Kit. The genes
ApCut1, ApCut2, and ApCut3 were amplified from ge-
nomic DNA using a Fusion DNA polymerase (Helicon,
Russia) and specific primers. PCR was performed as
follows: 95°C for 3 min, followed by 30 cycles (95°C
for 10 s, 67°C for 8 s, 72°C for 1 min), and a final
elongation at 72°C for 3 min. Gene sequences were
determined by Sanger sequencing and analyzed us-
ing MycoCosm-Blast [10] and BLASTN [11]. Lengths of
the signal peptides determined using SignalP-6.0 [12].
Coding sequences without signal peptides were cloned
into a pPICZαA vector at the EcoRI and XbaI sites
with corresponding primer pairs (Table S1 in the
Online Resource 1). Restriction, ligation (T4 DNA li-
gase), and DNA fragment purification were performed
according to the manufacturers’ protocols (SibEnzyme,
Dia-M, Russia). Yeast transformation was performed
via electroporation according to the Invitrogen proto-
col [9]. Bacterial transformation was performed using
the standard method [13]. Sequencing was performed
using a Nanophor-05 device with a BigDye™ Termina-
tor v3.1 kit (Thermo Fisher Scientific, USA).
Cultivation and enzyme preparation. K. phaffii
cells were cultured for 48 h at 28°C and 250 rpm
in a YPD medium, next transferred to a BMMY me-
dium containing 1.0% (V/V) methanol, and grown
ERMILOV et al.1472
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026
for 3 days, with 1% (V/V) methanol added daily. Cells
were separated by centrifugation, and the superna-
tant was dialyzed against a 10 mM sodium acetate
solution (pH 4.1, Buffer A) and loaded onto a Bio-
Suite SP column (Waters, USA). Protein elution was
performed using a linear gradient (0-0.5 M) of NaCl
solution in Buffer A. Final purification of the enzymes
was performed by gel filtration on a Sephacryl S-200
HR column (GE Healthcare, USA), equilibrated with
10 mM sodium acetate solution, pH 5.5, containing
0.1 M NaCl. Preparations were stored at 5°C. Protein
concentration was determined using the molar extinc-
tion coefficient, calculated based on the amino acid
sequence using ProtParam [14]. Purity and molecular
weight were determined using denaturing electropho-
resis in 12% SDS-PAGE [15].
Characterization of cutinase and its mutant
forms. Cutinase activity was assessed in the reac-
tion of hydrolysis of a chromogenic substrate 4-NP-
(16-MS-C16). The reaction was conducted at 37°C in a
50mM citrate-phosphate buffer solution, pH6.0, con-
taining 8 mM 4-NP-(16-MS-C16), previously dissolved
in dimethyl sulfoxide. The reaction was stopped by
adding a 10% Na
2
CO
3
solution, and optical density of
4-nitrophenol was measured at 405 nm. One unit of
cutinase activity was defined as an amount of enzyme
releasing 1 μmol of 4-nitrophenol per minute under
assay conditions. Michaelis constant(K
m
) and catalyt-
ic constant (k
cat
) were determined by approximating
an initial section of kinetic curves with the Michae-
lis-Menten equation using nonlinear regression. Mean
values and standard deviations were calculated for
at least three replicates.
Hydrolysis of PCL suspension was assessed tur-
bidimetrically. A PCL suspension (2 mg/mL) was pre-
pared according to Lang et al. [16] with subsequent
dialysis against distilled water. The hydrolysis reaction
was conducted at 25°C in a 50 mM citrate-phosphate
buffer solution, pH 6.5, with initial suspension con-
centration of 0.5 mg/mL. The rate of hydrolysis was
evaluated from the decrease in A
600
, calculated from
the linear section of the kinetic curve, and expressed
in mg of substrate hydrolyzed by 1 mg of enzyme
per minute. The rate of degradation of PCL gran-
ules (10 mg) by the cutinase mutant ApCUT1_Y58W
(0.4 mg) was determined based on the residual weight
of the sample after incubation at 25°C for 2-24 h in
the same buffer.
Effect of pH on enzyme activity was studied in
0.2 M citrate-phosphate buffer in the pH range 2.6-7.6
and 0.2 M Tris-HCl buffer in the pH range 7.1-8.9. Sta-
bility was assessed following 24 h incubation at 37°C
in the same pH range, followed by measuring resid-
ual activity at pH 6.0. Temperature optimum was de-
termined by incubating the enzyme in the tempera-
ture range 25-60°C for 10 min. Thermal stability was
determined by measuring the residual activity of the
enzymes after incubation for 5-30 min at 45-60°C.
Molecular modeling of proteins and their com-
plexes with ligands. Spatial structures of ApCUT1
and the mutants Y58W, L186F, and Y58W/L186F were
built using homology modeling (ICM-Pro [17]) based
on the structures of cutinases from Cryptococcus sp.
(PDB: 2CZQ) and Pseudozyma antarctica (PDB: 7CC4).
The constructed models were subjected to regulariza-
tion followed by energy minimization to relieve steric
strains. Docking of substrates (tricaprolactone (TCL)
and 4-NP-(16-MS-C16)) was performed using ICM-Pro
virtual screening algorithms with the ICM-Score func-
tion [18].
MD modeling of free enzymes and their complexes
with ligands was performed in the Amber-20 package
using the ff14SB force field for proteins and GAFF for
ligands [19]. MD modeling of cutinase ApCUT1 from the
wild-type strain and its three mutant forms in the free
state, as well as with eight complexes with ligands and
two ligands in the free state (a total of 14 molecular
systems), was performed in a periodic water box using
TIP3P water molecule models. Thecharge of the system
was neutralized by adding counterions, after which MD
trajectories of 100 ns duration (integration step 2 fs)
were calculated in the NPT ensemble at 298 K and
1 atm. Conformational stability was assessed from the
dynamics of the root-mean-square deviation (RMSD) of
the Cα-atom coordinates relative to the starting struc-
ture of the simulation, and local mobility of amino
acid residues by their root-mean-square fluctuations
(RMSF). The RMSD graph reaching a plateau indicated
that the system had reached equilibrium and conver-
gence of the molecular dynamics trajectory.
To test the “molecular trap” hypothesis [20], the
distance between the residue 58 (Tyr/Trp) and Ile181
was analyzed. A closed conformation was defined as
a state where the distance was ≤7 Å. MD trajectories
were used to determine the fraction of time in the
closed state.
Entropic contribution of the hydrophobic inter-
actions was assessed from the change in the num-
ber of low-entropy water molecules (LEW molecules)
in the active site (displacement of each provides
~2 kcal/mol) [21]. Hydration analysis was performed
in ICM-Pro software for free proteins and complexes
with ligands (10 replicates). Molecules of the first hy-
dration shell were determined by a distance of ≤3.2 Å
from the heavy atoms of the protein [22].
The number of LEW molecules displaced upon
binding (ΔN
LEW
) was calculated using the formula (1):
ΔN
LEW
= N
AC
free
+ N
ligand
free
− N
complex
. (1)
where N
AC
free
is the total number of LEW molecules
in the active site of the free enzyme, N
ligand
free
is the
POLYCAPROLACTONE-DEGRADING CUTINASE AND ITS VARIANTS 1473
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026
number of LEW molecules around the free ligand,
and N
complex
is the number of LEW molecules in the
active site of the complex.
RESULTS
Obtaining cutinase ApCUT1 and its mutant
forms. During strain screening on agar plates con-
taining PCL, out of 13 examined strains, A. pullulans
VKM 1116, along with the control (Fusarium solani
VTTD 93918), demonstrated the largest clearance zones
(Fig. S1 in the Online Resource 1). Genomic analysis
of A. pullulans revealed at least eight genes encod-
ing putative cutinases [10, 23]. We amplified three
genes (ApCUT1, ApCUT2, and ApCUT3) via PCR using
VKM 1116 genomic DNA as a template. Their prod-
ucts demonstrated a high degree of identity (62-70%)
with the cutinase CLE1 from Cryptococcus sp. S-2 [6]
and 96% with the similar enzymes from A. pullulans
var. namibiae (NCBI: XP_013423757.1, XP_013423111.1,
XP_013431735.1, respectively). Alignment of the ami-
no acid sequences of these proteins (without signal
peptides) and CLE1 is shown in Fig. S2 in the Online
Resource 1.
All three proteins contained the canonical cata-
lytic triad Ser-His-Asp, the consensus sequence Gly-
Tyr-Ser-Gln-Gly, the oxyanion hole (Thr, Gln), and two
disulfide bonds. Following expression in K. phaffii
GS115 cells, ApCUT1 and ApCUT2 were produced
predominantly, whereas ApCUT3 was detected only
in trace amounts. Hydrolytic activity against PCL
was detected only for the ApCUT1 cutinase, so fur-
ther studies focused on this enzyme (sequence depos-
ited in GenBank, PV834916.1). To assess the effect of
amino acid substitutions similar to mutations F52W
and L181F in the cutinase CLE1 [6], the mutant vari-
ants of ApCUT1 with substitutions Y58W, L186F, and
the double mutant Y58W/L186F were constructed.
Thewild-type and mutant forms of the gene were ex-
pressed in the K. phaffii system providing a protein
yield of 50-60 mg/L. The proteins were purified to a
homogeneous state, and their molecular weight was
22 kDa (Fig. S3 in the Online Resource 1).
Characterization of cutinase ApCUT1 and its
mutant forms. The effect of pH and temperature on
hydrolytic activity of the ApCUT1 cutinase and the
mutants was assessed using two substrates: chromo-
genic substrate 4-NP-(16-MS-C16) and PCL polymer.
ApCUT1 exhibited maximum activity toward 4-NP-
(16-MS-C16) at pH 6.0, while the activity towards PCL
was observed in a wider range of pH 5.0-6.5 (Fig. 1a).
The enzyme was stable in the pH range 4.0-7.5, re-
taining at least 80% activity after 24 h incubation
at 37°C (Fig. 1b). Temperature optimum was 45°C
in the reaction with 4-NP-(16-MS-C16) and 30-40°C –
with PCL (Fig. 1c). The enzyme retained 90% activi-
ty after 30 min incubation at 50°C, dropping to 50%
after 5 min at 55°C and to 10% after 5 min at 60°C
(Fig. 1d). Biochemical properties of the mutant cuti-
nases did not differ significantly from those of the
wild-type cutinase (Fig. S4 in the Online Resource 1).
Kinetic parameters of the 4-NP-(16-MS-C16) hy-
drolysis reaction are summarized in Table 1. The K
m
values for all enzymes varied slightly, but the k
cat
and catalytic efficiency (k
cat
/K
m
) were higher for the
mutants. The double mutant ApCUT1_Y58W/L186F
demonstrated more than a twofold increase in k
cat
and k
cat
/K
m
.
PCL depolymerization was evaluated using both
the polymer suspension and granules. The rate of PCL
suspension hydrolysis by ApCUT1 was 62 ± 3 mg/min
per 1 mg of protein. The Y58W substitution increased
the rate of hydrolysis 1.8-fold, while the L186F substi-
tution and the double substitution had no significant
effect (Fig. 2a). The rate of degradation of PCL granules
under the action of ApCUT1_Y58W was 1 ± 0.05 mg/h
per 1 mg of protein with complete destruction of the
granules occurring within 24 h (Fig. 2b).
Spatial structure and molecular dynamics of
cutinase ApCUT1 and its mutant forms. Spatial
alignment of the structures of the three closely relat-
ed cutinases is presented in Fig. 3. With the exception
of two mobile loops (Fig. 3a, residues 120-125 and
167-175), the RMSD between the spatial positions of
Cα-carbon atoms of the main chain of the cutinases
is in the range of 0.2-0.3 Å. MD modeling of enzymes
showed that all systems reach a stable plateau within
Table 1. Kinetic parameters of the recombinant cutinases ApCUT1, ApCUT1_Y58W, ApCUT1_L186F, and
ApCUT1_Y58W/L186F in the reaction of 4-NP-(16-MS-C16) hydrolysis
Enzyme K
m
, mM k
cat
, s
−1
k
cat
/K
m
× 10
3
, M
−1
s
−1
ApCUT1 1.9 ± 0.1 2.6 ± 0.1 1.4 ± 0.1
ApCUT1_Y58W 2.0 ± 0.1 4.8 ± 0.2 2.4 ± 0.2
ApCUT1_L186F 2.1 ± 0.1 4.1 ± 0.2 2.0 ± 0.2
ApCUT1_Y58W/L186F 2.3 ± 0.1 6.6 ± 0.3 2.9 ± 0.3
ERMILOV et al.1474
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026
Fig. 1. Biochemical properties of the ApCUT1 cutinase: pH optima in the hydrolysis of 4-NP-(16-MS-C16) (curve 1) and PCL
(curve 2) (a); pH stability (b); temperature optima in the hydrolysis of 4-NP-(16-MS-C16) (curve 1) and PCL (curve 2) (c);
thermal stability at 45-60°C (d).
Fig. 2. PCL depolymerization kinetics: comparison of relative hydrolysis rates of PCL suspension (the rate of ApCUT1 set
to 100%) (a); residual mass of PCL granules during hydrolysis catalyzed by ApCUT1_Y58W (b).
POLYCAPROLACTONE-DEGRADING CUTINASE AND ITS VARIANTS 1475
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026
Fig. 3. Models of the spatial structure of cutinase ApCUT1 and its mutants: spatial superposition of the crystal structures of
cutinases PDB 2CZQ and PDB 7CC4 with the homology-built structure of ApCUT1 structures (colored blue, green, and light
blue, respectively). Red rectangles mark the positions of two mobile loops exhibiting low sequence conservation(a); active
site of cutinase ApCUT1. Key hydrophobic residues (Tyr58, Ile181, Leu186), highlighted in green, adopt an “open” confor-
mation, providing access for solvent and substrate molecules to the active site(b); complex of cutinase ApCUT1_Y58W with
TCL (yellow), generated via molecular docking of the flexible substrate. The “closed” conformation shows Trp58 approach-
ing Ile181 to form tight contacts with the ligand (c); Complexes of ApCUT1 with TCL and 4-NP-(16-MS-C16). Orientation of
the −(C=O)−C− bond is shown by arrows (d).
the first 20 ns of modeling. For the wild-type cuti-
nase, the RMSD value of 1.80 ± 0.19 Å was recorded.
Stable trajectories were also observed for the mutant
forms (Fig. S5 in the Online Resource 1). Analysis of
the flexibility of the Cα-atoms of the catalytic triad
(Ser91, Asp170, His185) confirmed that the active-site
architecture remained preserved across all studied
systems. The RMSF values for all proteins were in the
range of 0.2-1.5 Å, indicating stability of the catalytic
residues during modeling.
Flexible-ligand molecular docking methods were
used to determine the structures of ApCUT1 complex-
es with model substrates, 4-NP-(16-MS-C16) and TCL.
TheTCL molecule was chosen as a minimal fragment
of the PCL polymer potentially capable of binding in
the active site of the cutinase. In the complex with
TCL and 4-NP-(16-MS-C16), side chains of the catalytic
residues Ser91 and His185 are located at a distance of
3-4 Å from the carbon of the carbonyl group in the
ester bond of the substrate chains (Fig. 3d). Hydro-
phobic –CH
2
– and hydrophilic –COO– groups of the
substrates interact with the complementary hydropho-
bic or polar amino acid residues in the cavity of the
active site. In the resulting complexes, the substrates
adopt orientations where the ester bonds face oppo-
site directions regarding the carbonyl carbon targeted
for nucleophilic attack by the oxygen atom of Ser91,
which maintains its position. The calculated binding
energy score (ICM-Score ≈ −60) indicates high affinity
of the ligands for the cutinase active site [18]. This
suggests that the elongated cavity of the ApCUT1 can
accommodate substrates in different binding modes.
MD modeling of the proteins and their complexes
revealed periodic approach of the residues at posi-
tions 58 and 181 to each other at the entrance to the
active site (Fig. 3, b and c), similar to that described
for other cutinases[1]. Inthe presence of a bulky tryp-
tophan at position 58 (Y58W substitution), this interac-
tion results in the formation of a hydrophobic bridge.
DISCUSSION
Cutinases are enzymes belonging to the family of
serine hydrolases that possess the unique ability to
catalyze cleavage of both soluble esters and insoluble
polyesters, including PCL [1]. A promising direction
for increasing catalytic activity of cutinases is rational
ERMILOV et al.1476
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026
Table 2. Hydration properties and conformational dynamics of the cutinase active site in the free state and in
complexes with TCL and 4-NP-(16-MS-C16): insights from molecular modeling and molecular dynamics
Enzyme
The fraction
of MD time of the
APO-form enzyme
spends in the closed
conformation, %
The fraction of MD
time the enzyme–
ligand complex
spends in the closed
conformation, %
The average number of LEW molecules
displaced from the AS when substrate
binding, units (σ)
Open conformation
Closed
conformation
4-NP-(16-MS-C16)
ApCUT1 2.33 1.3 51.4 (5.8) 51.8 (8.8)
ApCUT1_Y58W 1.64 14.55 52.5 (7.3) 56.2 (7.2)
ApCUT1_L186F 0.18 1.38 50.5 (7.5) 51.9 (7.2)
ApCUT1_Y58W/L186F 7.68 26.99 48.2 (7.0) 50.8 (7.4)
Tricaprolactone
ApCUT1 2.33 0 38.2 (7.1) 43.0 (5.6)
ApCUT1_Y58W 1.64 0.62 40.0 (6.9) 47.6 (8.7)
ApCUT1_L186F 0.18 2.32 42.1 (4.0) 39.1 (6.7)
ApCUT1_Y58W/L186F 7.68 14.21 37.6 (7.7) 46.5 (8.7)
design aimed at optimizing the substrate-binding re-
gions and direct ester bond hydrolysis. Kodama et al.
demonstrated that the mutations F52W, L181F, and
their combination in the cutinase CLE1 increase the
efficiency of hydrolysis of the long-chain substrates,
such as palmitate derivatives. This effect is due to
the presence of hydrophobic and/or aromatic amino
acid residues in the binding sites of the enzyme [6].
Similar substitutions at positions Y58 and L186 in the
ApCUT1 sequence also led to an increase in catalyt-
ic efficiency. As observed for cutinase CLE1 during
the hydrolysis of the poorly water-soluble palmitate
derivative 4-NP-(16-MS-C16), the maximum effect was
seen for the double mutant ApCUT1_Y58W/L186F: the
rate and efficiency of hydrolysis reactions increased
by more than two-fold compared to the wild-type en-
zyme (Table 1). However, the effect was substrate-de-
pendent. In the reaction of PCL hydrolysis, the Y58W
mutant showed the highest activity, resulting in a 1.8-
fold increase in the hydrolysis rate compared to the
wild-type enzyme. Theamino acid substitutions L186F
and Y58W/L186F did not lead to significant changes
in the process efficiency. The ApCUT1_Y58W mutant
also ensured complete depolymerization of the sol-
id polymer granules. Notably, unlike the low-molec-
ular-weight substrate 4-NP-(16-MS-C16), in the case
of PCL, the limiting stage may be not only cleavage
of the ester bond but also adsorption of the enzyme
on the hydrophobic surface of the plastic [24, 25].
Thedegradation rate of PCL granules was significant-
ly lower than that of the particle suspension, likely
due to the reduced surface area available for enzyme
binding and subsequent catalysis.
To explain the observed differences in the activi-
ty between the two substrate types, we analyzed con-
formational dynamics of the active site and changes
in its hydration using molecular modeling and MD
(Table 2). The results indicated that the active site
could transition to a closed state through approach
of the amino acid residues at positions 58 and 181 to
each other, forming a hydrophobic bridge. This mech-
anism underlies functioning of the well-known “mo-
lecular trap,” which retains the ligand and prevents
its premature exit from the reaction cleft in many
other proteins [1, 20]. Additionally, binding efficiency
is driven by the hydrophobic effect associated with
the displacement of low-entropy water (LEW) mole-
cules upon complex formation between the enzyme
and the ligand.
To identify molecular mechanisms determining
the substrate-dependent activity of the mutants, we
first assessed the thermodynamic contribution of the
hydrophobic effect (Table 2). Statistical analysis of
the number of displaced LEW molecules using the
non-parametric Mann–Whitney criterion showed that
at the stage of open complex formation, this contri-
bution remained comparable for all studied enzyme
variants (p > 0.05). However, upon transition to the
POLYCAPROLACTONE-DEGRADING CUTINASE AND ITS VARIANTS 1477
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026
closed conformation in the complex with the poly-
meric substrate (TCL), the mutants Y58W and Y58W/
L186F demonstrated a statistically significant increase
in the number of displaced LEW molecules (p = 0.03
and p = 0.02, respectively). This indicates that intro-
duction of a bulky indole radical of the tryptophan
residue forms a maximally dense hydrophobic con-
tact upon trap closure, providing additional energy
for stabilization of the polymer chain in the produc-
tive conformation. In the wild-type and in the L186F
mutant, no reliable entropic gain was observed upon
closure of the active site.
Based on the MD data, we analyzed the influence
of the kinetic factor– stability of the “molecular trap”
conformations (Table 2). Upon binding of the low-mo-
lecular-weight substrate 4-NP-(16-MS-C16), the highest
fraction of the closed conformation in the complex
was demonstrated by the double mutant Y58W/L186F
(26.99% of the time vs. 1.3% in the wild-type). Since
no significant differences in the number of displaced
LEW molecules for this substrate between the mu-
tant forms were detected (p > 0.05), we can exclude
a change in the contribution of the hydrophobic ef-
fect as the primary cause of their different activities.
Therefore, the observed increase in the catalytic ef-
ficiency of the double mutant (Table 1) is achieved
primarily through kinetic control, namely, high fre-
quency of the trap closure that effectively sterically
retains the small ligand within the catalysis zone.
During polycaprolactone (PCL) degradation, the
overall efficiency is likely limited by the stage of pri-
mary PCL chain capture. Despite high stability of the
closed complex (14.21%), the double mutant was infe-
rior to the Y58W variant. Thereason is likely the ste-
ric barrier at the entrance: the enzyme of the double
mutant spends 7.68% of the time in the closed confor-
mation (three times longer than the wild-type), which
hinders binding of the massive polymer chain. For
the low-molecular-weight substrate 4-NP-(16-MS-C16),
this limitation at the entrance is less pronounced. The
best result in the hydrolysis of PCL was shown by
the enzyme with Y58W substitution, providing an op-
timal balance between accessibility and affinity. The
free enzyme maintains an open architecture at the
entrance, favorable for primary chain capture (closed
1.64% of the time). Hydration analysis revealed that
the subsequent closure of the trap in the complex
(0.62%) is accompanied by a statistically significant
increase in the number of displaced LEW molecules
(p = 0.03), indicating additional stabilization of the
polymer chain in the productive conformation. The
significance of this specific interaction is confirmed
by the behavior of the enzyme with the L186F sub-
stitution, which contributes minimally to PCL degra-
dation efficiency. Although in the complex this mu-
tant closes more often (2.32%) than the Y58W mutant
(0.62%), absence of the indole ring of the Trp58 pre-
vents the formation of a dense hydrophobic contact.
As a result, transition to the closed conformation is
not accompanied by the reliable change in the hydro-
phobic contribution (p > 0.05), reducing the efficiency
of polymer fixation.
Thus, in this work we obtained a recombinant
strain of K. phaffii producing the ApCUT1_Y58W cuti-
nase, which is a promising biocatalyst for developing
environmentally friendly technologies for PCL utiliza-
tion and recycling. Molecular modeling indicated that
the hydrophobic bridge at the entrance to the active
site of cutinase ApCUT1 could function as a “molec-
ular trap.” Activity towards the low-molecular-weight
substrate 4-NP-(16-MS-C16) is governed by the kinetic
control at the substrate retention stage, as evidenced
by the maximum catalytic efficiency of the rigidly
closing Y58W/L186F mutant. Activity towards PCL is
limited by the initial adsorption stage, for which an
open architecture of the free enzyme, such as in the
Y58W mutant, is critically important. These results
highlight the intrinsic complexity of the molecular
mechanisms by which amino acid substitutions af-
fect enzymatic activity. These factors include not only
changes in the static intermolecular interactions in
their active sites but also dynamic characteristics of
the enzymes and active-site hydration.
Abbreviations
4-NP-(16-MS-C16) 4-nitrophenyl 16-methylsulfonyl
hexadecanoate
MD molecular dynamics
LEW molecules low-entropy water molecules
PCL polycaprolactone
RMSD root-mean-square deviation
TCL tricaprolactone
Supplementary information
The online version contains supplementary material
available at https://doi.org/10.1134/S0006297926601449.
Contributions
I. A. Sizova, A. A. Kulminskaya, and M. G. Petukhov:
concept and supervision of the work. F. K. Ermilov,
E. V. Eneiskaya, E. V. Zhurishkina, K. S. Bobrov, and
S. A. Panasenko: conducting experiments. I. A. Sizova,
A. A. Kulminskaya, F. K. Ermilov, E. V. Eneiskaya,
M. G. Petukhov, and S. A. Panasenko: discussion of
the research results. I. A. Sizova, A. A. Kulminskaya,
F. K. Ermilov, E. V. Eneiskaya, and M. G. Petukhov:
writing of the text. I. A. Sizova, A. A. Kulminskaya,
and K. S. Bobrov: Editing of the article text.
Funding
The experimental part of the work was carried out
within the framework of the state assignment of
ERMILOV et al.1478
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026
the Ministry of Science and Higher Education of the
Russian Federation (no. 1024011100004-4-1.6.8; 1.6.4;
1.6.7; 1.6.1. Functional and structural organization
of complex, multicomponent biological systems and
their dynamics. Molecular and cellular components
of the pathogenesis of socially significant diseases).
Development of the methods for studying mechanisms
of the enzyme-ligand binding was carried out within
the framework of the state assignment of the Minis-
try of Science and Higher Education of the Russian
Federation (no. 1024011000015-3-1.6.7. Development
of new computational methods in interdisciplinary
studies of the structural and functional properties of
biomacromolecules and biomacromolecular systems).
Molecular modeling and molecular dynamics of the
enzyme and its mutant forms in the free state and
in complex with ligands were supported by the grant
from the Ministry of Science and Higher Education
of the Russian Federation in the form of a subsidy
for implementation of a scientific project under the
leadership of a leading scientist (Project no. 075-15-
2025-012).
Ethics approval and consent to participate
This work does not contain any studies involving hu-
man and animal subjects.
Conflict of interest
The authors of this work declare that they have no
conflicts of interest.
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