Biochemical characterization of the cutinases from Thermobifida fusca

Article abstract of DOI:10.1016/j.molcatb.2010.01.001

Thermobifida fusca produces two cutinases which share 93% identity in amino acid sequence. In the present study, we investigated the detailed biochemical properties of T. fusca cutinases for the first time. For a better comparison between bacterial and fungal cutinases, recombinant Fusarium solani pisi cutinase was subjected to the similar analysis. The results showed that both bacterial and fungal cutinases are monomeric proteins in solution. The bacterial cutinases exhibited a broad substrate specificity against plant cutin, synthetic polyesters, insoluble triglycerides, and soluble esters. In addition, the two isoenzymes of T. fusca and the F. solani pisi cutinase are similar in substrate kinetics, the lack of interfacial activation, and metal ion requirements. However, the T. fusca cutinases showed higher stability in the presence of surfactants and organic solvents. Considering the versatile hydrolytic activity, good tolerance to surfactants, superior stability in organic solvents, and thermostability demonstrated by T. fusca cutinases, they may have promising applications in related industries.

Full text of DOI:10.1016/j.molcatb.2010.01.001

Journal of Molecular Catalysis B: Enzymatic 63 (2010) 121–127
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Biochemical characterization of the cutinases from Thermobifida fusca
Sheng Chena,b, Lingqia Sub, Susan Billigc, Wolfgang Zimmermannc, Jian Chenb,∗, Jing Wua,b,∗∗
a State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Ave., Wuxi, Jiangsu 214122, China
b School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Ave., Wuxi, Jiangsu 214122, China
c Institute of Biochemistry, Department of Microbiology and Bioprocess Technology, University of Leipzig, Johannisallee 21-23, Leipzig D-04103, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Thermobifida fusca produces two cutinases which share 93% identity in amino acid sequence. In the
present study, we investigated the detailed biochemical properties of T. fusca cutinases for the first time.
For a better comparison between bacterial and fungal cutinases, recombinant Fusarium solani pisi cuti-
nase was subjected to the similar analysis. The results showed that both bacterial and fungal cutinases are
monomeric proteins in solution. The bacterial cutinases exhibited a broad substrate specificity against
plant cutin, synthetic polyesters, insoluble triglycerides, and soluble esters. In addition, the two isoen-
zymes of T. fusca and the F. solani pisi cutinase are similar in substrate kinetics, the lack of interfacial
activation, and metal ion requirements. However, the T. fusca cutinases showed higher stability in the
presence of surfactants and organic solvents. Considering the versatile hydrolytic activity, good toler-
ance to surfactants, superior stability in organic solvents, and thermostability demonstrated by T. fusca
cutinases, they may have promising applications in related industries.
Received 18 June 2009
Received in revised form
27 December 2009
Accepted 4 January 2010
Available online 11 January 2010
Keywords:
Thermobifida fusca
Cutinase
Characterization
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
Cutinases have been found in both fungi and bacteria [9]. Fun-
gal cutinases, such as from Fusarium solani pisi, Monilinia fructicola
Cutin is a major component of the plant cuticle which consti-
tutes an efficient barrier against desiccation and pathogens [1].
Cutinases are inducible extracellular enzymes secreted by microor-
ganisms capable of catalyzing the cleavage of ester bonds in cutin
[2]. They display hydrolytic activity not only towards cutin but
also a variety of soluble synthetic esters, insoluble triglycerides,
synthetic fibers (polyethylene terephthalate fibers), plastics (poly-
caprolactone) and others [3,4]. Therefore, cutinases have been
recognized as versatile lipolytic enzymes in laundry and dishwash-
ing detergent formulations, and for other applications in the textile,
food and chemical industries [5–7]. In addition to their hydrolytic
activity, cutinases also show synthetic activity and have potential
use for the synthesis of structured triglycerides, polymers, pharma-
ceuticals and agrochemicals [8].
[10], Botrytis cinerea [11], and Aspergillus oryzae [4] have been
studied comprehensively. Especially, the cutinase from F. solani
pisi has been extensively investigated, including gene identifica-
tion, cloning, expression, characterization, structure elucidation,
and applications [12]. In contrast, limited studies have been per-
formed on cutinases from bacterial sources until recently when
the genes encoding cutinase from the thermophilic actinomycete
Thermobifida fusca were identified in our laboratory [13].
T. fusca has two cutinase-encoding open reading frames,
Tfu 0882 and Tfu 0883. The two isoenzymes are 93% identical
in their amino acid sequence. Initial characterization showed
that Tfu 0882 and Tfu 0883 share similar temperature depen-
dence profiles and thermostability, but display higher temperature
optimum and greater thermostability compared to fungal cuti-
nases. Although both bacterial and fungal cutinases belong to
the a/␤ hydrolase fold superfamily, the bacterial sequences are
significantly longer and demonstrate no similarity to the fungal
sequences. In this respect, it has been suggested that the bacte-
rial and fungal enzymes should be classified into prokaryotic and
eukaryotic cutinase subfamilies, respectively [13]. A more in-depth
analysis of the enzymatic properties of bacterial cutinases will fur-
ther our understanding on cutinase subfamilies. In addition, such
studies will further explore their potential applications in biotech-
nology.
Abbreviations:
FspC, Fusarium solani pisi cutinase; TDOC, sodium tau-
rodeoxycholate; pNPB, p-nitrophenyl butyrate; pNPP, p-nitrophenyl palmitate;
TPA, terephthalic acid; MHET, mono(2-hydroxyethyl terephthalate); BHET, bis(2-
hydroxyethyl terephthalate); EMT, 1,2-ethylene-mono-terephthalate-mono(2-
hydroxyethyl terephthalate).
∗
Corresponding author. Tel.: +86 510 85329031; fax: +86 510 85918309.
Corresponding author at: State Key Laboratory of Food Science and Technology,
∗∗
School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of
Education, Jiangnan University, 1800 Lihu Ave., Wuxi, Jiangsu 214122, China. Tel:
+86 510 85327802; fax: +86 510 85327802.
E-mail addresses: jchen@jiangnan.edu.cn (J. Chen), jingwu@jiangnan.edu.cn
(J. Wu).
In the present study, we investigated the detailed biochem-
ical properties of T. fusca cutinases. For a better comparison
between the two subfamilies of cutinases, recombinant F. solani pisi
1381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.01.001

122
S. Chen et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 121–127
cutinase (FspC) was subjected to the same analysis. The results
showed that these enzymes are similar in substrate specificity, sub-
strate kinetics, interfacial activation, and metal ion requirement.
However, they differed significantly in their stability in the pres-
ence of surfactants and organic solvents.
of emulsion and enzyme. The reaction was initiated by adding
the enzyme to the reaction solution, incubated for 15 min and
quenched by adding 7.5 ml of ethanol. The released fatty acids
were quantified by titration with 0.05N NaOH. One unit of lipase
activity was defined as the release of 1 ␮mol of fatty acid per
minute.
2. Materials and methods
2.6. Degradation of cutin by the cutinases
2.1. Chemicals
1 mg of Tfu 0882, Tfu 0883, and FspC were incubated with 1%
(w/v) apple cutin in 25 mM potassium phosphate buffer (pH 8.0).
The incubation temperature was 60 ◦C for Tfu 0882 and Tfu 0883,
and 40 ◦C for FspC. At various time intervals, aliquots were removed
and assayed for released fatty acids by titration with 0.02N NaOH.
Sodium taurodeoxycholate (TDOC), p-nitrophenyl butyrate
(pNPB), p-nitrophenyl palmitate (pNPP), triolein, tributyrin, tereph-
thalic acid (TPA), mono(2-hydroxyethyl terephthalate) (MHET),
bis(2-hydroxyethyl terephthalate) (BHET) and 1,2-ethylene-mono-
terephthalate-mono(2-hydroxyethyl terephthalate) (EMT) were
obtained from Sigma. Other chemicals were obtained from
Sinopharm Chemical Reagent Co. Ltd.
2.7. Degradation of cyclic PET trimer by the cutinases
0.3 g of a cyclic PET trimer preparation and 10 ml 25 mM phos-
phate buffer (pH 8) were added to a Erlenmeyer flask. The reaction
was initiated by the addition of 40 U pNPB activity corresponding
to 37 ␮g, 280 ␮g, and 94 ␮g protein of FspC, Tfu 0882 and Tfu 0883,
respectively. The incubation was performed at 60 ◦C for Tfu 0882
and Tfu 0883, and 40 ◦C for FspC in an incubator (150 rpm) over
72 h. During the incubation, aliquots were removed and prepared
for the analysis by RP-HPLC as previously described [6].
2.2. Enzyme preparation
Recombinant cutinases Tfu 0882 and Tfu 0883 were purified
from E. coli BL21 (DE3) cells harboring the plasmids pET20b(+)-
Tfu 0882 or pET20b(+)-Tfu 0883. Recombinant FspC was purified
from Bacillus subtilis harboring the plasmid pBSMuL3 as previously
reported [13].
The amounts of released soluble hydrolysis products were
calculated based on the enzyme concentrations employed
(mM/mg protein). The hydrolysis products were detected as
previously reported by Hooker et al. [18] for terephthalic
acid (TPA), mono(2-hydroxyethyl terephthalate) (MHET) and
bis(2-hydroxyethyl terephthalate) (BHET). 1,2-Ethylene-mono-
terephthalate-mono(2-hydroxyethyl terephthalate) (EMT) was
detected and confirmed by LC–MS analysis. The hydrolysis prod-
ucts were calculated as the sum of the amounts of TPA, MHET, BHET
and EMT produced.
2.3. Molecular mass determination
The molecular masses of Tfu 0882 and Tfu 0883 were deter-
mined under denaturing conditions by SDS-PAGE. The native
molecular weight of the protein was determined by analytical gel
filtration chromatography using a Superose 12 column (HR 10/30;
Pharmacia). Chromatography was performed at ambient tempera-
ture on a Pharmacia FPLC system monitoring the eluent at 280 nm.
The running buffer was 0.15 M sodium chloride, 50 mM Tris (pH
7.5) and a flow rate of 0.5 ml/min was used. A standard curve
was generated using proteins from a molecular weight marker kit
(MWGF-200, Sigma). Standards and samples were used at concen-
trations of 5–10 mg/ml and were loaded onto the column using
a 50 ␮l sample loop. The elution volume, Ve, was determined in
triplicate for all samples and standards.
2.8. Triglyceride position specificity of the cutinases
The reaction solution contained 2.5 ml of 25 mM potassium
phosphate buffer (pH 8), 2 ml of triolein emulsion and enzyme.
Analysis of the regioselectivity of triolein hydrolysis by cuti-
nase was carried out by incubating the reaction solution at 60 ◦C
(Tfu 0882 and Tfu 0883) or 40 ◦C (FspC) for 15 min. The reaction
mixture was extracted by isopropanol and n-hexane and analyzed
by HPLC [19].
2.4. Esterase assay
Esterase activity was determined by a continuous spectrophoto-
metric assay using p-nitrophenyl butyrate (pNPB) as the substrate
[14]. The standard assay contained in a final volume of 1 ml pNPB
(1 mM, stock in acetonitrile), enzyme solution, and buffer (20 mM
Tris–HCl containing 10 mM NaCl, pH 8.0) at 37 ◦C. The reaction was
initiated by the addition of pNPB.
The activity against p-nitrophenyl palmitate (pNPP) was deter-
mined as previously reported [15].
The hydrolysis of pNPB and pNPP was spectrophotometrically
monitored for the formation of p-nitrophenol at 405 nm. One unit
of enzyme activity was defined as the production of 1 ␮mol p-
nitrophenol per minute [16].
2.9. Kinetic analysis of the cutinases
Kinetic studies were performed with pNPB (100–2000 ␮M)
as substrate using the continuous spectrophotometric assay as
described above. Initial reaction velocities were calculated from the
linear region (60 s) of the reaction progress curve and measured in
triplicate by varying the concentration of the substrate. Apparent
kinetic constants Km were calculated from a double-reciprocal plot
(1/v vs. 1/[pNPB]) of the initial rate data. Results are the average of
triplicate assays [20].
2.5. Lipase assay
2.10. Interfacial activation of the cutinases
Lipase activity was measured as previously reported [17] with
the following modifications. Triolein or tributyrin was used as
the substrate. Triolein or tributyrin emulsion was prepared by
emulsifying Triolein/tributyrin in 25 mM potassium phosphate
buffer (pH 8) and 0.5% (w/v) gum arabic for 2 min at maxi-
mum speed in a Waring blender. The reaction solution contained
2.5 ml of 25 mM potassium phosphate buffer (pH 8) and 2 ml
Tributyrin emulsion (3–35 mM) was prepared by mixing a given
amount of tributyrin in 30 ml of 0.5% gum arabic and 0.15 M NaCl
in a Warring blender. The reaction solution contained 2.5 ml of
25 mM potassium phosphate buffer (pH 8) and 2 ml of emulsion
and enzyme. The reaction was initiated by adding the enzyme to
the reaction solution, incubated in 37 ◦C for 15 min and quenched

S. Chen et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 121–127
123
by adding 7.5 ml of ethanol. The released fatty acids were quantified
via titration by 0.05N NaOH.
2.11. Effect of metal ions on enzyme activity
Various metal ions (CaCl2, CuSO4, MgCl2, MnCl2, ZnSO4, CoCl2,
NiCl2, BaCl2, PbCl2, CrCl2, HgCl2 and FeSO4) at a concentration of
1 mM were added to 5 nM cutinase (20 mM Tris–HCl buffer, pH
8), and the solution was preincubated at 37 ◦C for 5 min and then
assayed for esterase activity against pNPB. Esterase activity of the
enzyme without added metal ion was defined as 100% [21].
2.12. Effect of surfactants on enzyme activity
Various surfactants (SDS, Triton X-100, Tween 20 and TDOC)
at different concentrations were added to 5 nM cutinase (20 mM
Tris–HCl buffer, pH 8), and the solution was preincubated at 37 ◦C
for 5 min and then assayed for esterase activity against pNPB.
Esterase activity of the enzyme without added surfactants was
defined as 100% [22]. The SDS inhibition constant was determined
by a double-reciprocal plot (1/v vs. 1/[pNPB]) of the initial rate data
at three concentrations of SDS [23]. The inhibition data were ana-
lyzed using a Lineweaver-Burk plot.
Fig. 1. Degradation of cutin by the cutinases. The enzymes were incubated with 1%
(w/v) apple cutin in 25 mM potassium phosphate buffer (pH 8.0) and the amount
of released fatty acids was measured by titration with 0.02N NaOH. (ꢀ) Tfu 0883;
(ꢁ) Tfu 0882; (ꢂ) FspC. Error bars correspond to the standard deviation of three
determinations.
Subsequently, the chain length specificity of esters was inves-
tigated using p-nitrophenyl-fatty acyl esters and triglycerides
(Table 1). For all three enzymes, their activities against the C4 p-
nitrophenyl-fatty acid ester pNPB were significantly higher than the
corresponding C16 ester pNPP. Similarly, their activities against the
C4 triglyceride tributyrin were significantly higher than the corre-
sponding C18 ester triolein. Therefore, all three enzymes appeared
to have a preference for a shorter carbon chain of the acyl moiety.
It is notable that FspC exhibited highest specific activity toward
all these four substrates. When comparing the T. fusca cutinases,
both Tfu 0883 and Tfu 0882 showed similar activities toward
triglyceride but differential activities toward p-nitrophenyl-fatty
acid ester which was hydrolyzed at a significantly higher rate by
Tfu 0883.
2.13. Stability in organic solvent
The stability in organic solvents was tested in a buffer (20 mM
Tris–HCl, pH 8) containing 75% (v/v) of various solvents and
5 nM cutinase. After 18 h of incubation at 20 ◦C, aliquots were
removed for determination of residual esterase activity against
pNPB. Esterase activity of the enzyme without solvents was defined
as the 100% level [24].
3. Results and discussion
3.1. Molecular mass determination
Recently, it has been reported that FspC can hydrolyze syn-
thetic polyesters such as PET and improve the surface properties
of PET fibers in an environmentally friendly way [6,26]. The ability
of cutinases to hydrolyze cyclic PET trimers has also been evaluated
[18]. As shown in Fig. 3, FspC exhibited the highest activity toward
this polyester, while Tfu 0883 exhibited less activity. Surprisingly,
Tfu 0882 was virtually inactive towards this polyester despite its
high sequence similarity with Tfu 0883, suggesting that the minor
sequence differences may be mainly located at the substrate bind-
ing site. Further studies, including crystallographic studies may
shed light on this dramatic difference between the two T. fusca
enzymes.
The subunit molecular masses of the purified Tfu 0882 and
Tfu 0883 were both 29 kDa as determined by SDS-PAGE, which is
consistent with their calculated molecular masses of 29.220 kDa
and 28.997 kDa, respectively. Their native molecular masses were
both 32 kDa as determined by analytical gel filtration chromatog-
raphy. Therefore, both Tfu 0882 and Tfu 0883 were predicted to
be monomeric proteins in solution. The FspC also determined as a
monomeric protein in solution (data not shown) which confirmed
the previous results [25].
3.2. Substrate specificity
The above results revealed that the T. fusca cutinases have
a broad substrate specificity against cutin and other polyesters,
insoluble triglycerides, and soluble esters. They could therefore be
described as intermediate enzymes between lipases and esterases
as has been suggested for their fungal counterparts [12]. Such broad
specificity is consistent with their open active sites previously pre-
dicted [13].
The catalytic efficiency of Tfu 0882, Tfu 0883, and FspC toward
cutin was evaluated by measuring the released fatty acid products.
As shown in Fig. 1, all three enzymes hydrolyze cutin in a similarly
effective way, which is consistent with their physiological function
as a cutin hydrolase.
Given the broad substrate specificity of fungal cutinases, we
further evaluated the hydrolytic activity of the T. fusca cutinases
towards additional esters. Previously, it has been shown that
Tfu 0882, Tfu 0883 and FspC hydrolyze the insoluble triglyceride
triolein [13]. To gain more insight into the position specificity for
triolein, the reaction products were analyzed by HPLC. As shown in
Fig. 2, the molar concentration of 1,2(2,3)-diolein obtained was not
very different from 1,3-diolein for the three enzymes. The cutinases
not only hydrolyzed the ester bond of triacylglycerids in the sn-1,3-
position, but also in the sn-2 position. None of the three cutinases
showed 1,3-position specificity.
Table 1
Specificity of the cutinases towards the acyl chain length of different esters. Values
are means SD (n = 3).
Substrate
Tfu 0882 cutinase
499 10
Tfu 0883 cutinase
1016 15
FspC
pNPB
2083 23
pNPP
tributyrin
triolein
269
237 11
137
5
383
217 12
125
6
1041
643 15
321 10
9
7
8

124
S. Chen et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 121–127
Fig. 2. HPLC chromatogram of triolein hydrolyzed by the cutinases. The enzymes were incubated with triolein emulsion in 25 mM potassium phosphate buffer (pH 8) for
15 min. The reaction mixture was extracted by isopropanol and n-hexane and analyzed by HPLC. (A) Tfu 0882; (B) Tfu 0883; (C) FspC. (a) Triolein; (b) oleic acid; (c) 1,3-diolein;
(d) 1,2(2,3)-diolein. The molar ratio of c/d for A, B and C were 0.55, 0.68 and 1.33, respectively.
3.3. Kinetic analysis
FspC exhibited the highest catalytic efficiency with a kcat/Km of
3.214 s−1 ␮M−1. Interestingly, the catalytic efficiency (kcat/Km) of
Tfu 0883 is twice as much as that of Tfu 0882 even though they
showed a 93% identity in amino acid sequences and an almost
identical structure of their active sites (data not shown).
The kinetic constants of Tfu 0882, Tfu 0883 and FspC were
determined for the commonly used esterase substrate pNPB. The
results showed that all three cutinases showed Michaelis–Menten
kinetics with FspC exhibiting the highest affinity (lowest Km value)
for the substrate (Table 2). In addition, among the three enzyme,
3.4. Evaluation of an interfacial activation of the cutinases
Interfacial activation has been observed with most lipases [27].
Tributyrin, a suitable substrate to test the interfacial activation phe-
nomenon of lipases [28] was selected to evaluate the a possible
interfacial activation of the cutinases. As shown in Fig. 4, the specific
activity of the three cutinases as a function of tributyrin concentra-
tion followed normal Michaelis–Menten kinetics. The activity did
not sharply increase when the solubility limit of tributyrin (12 mM)
was reached. Therefore, Tfu 0882, Tfu 0883 and FspC did not pos-
sess interfacial activation phenomenon.
FspC has been previously reported not to exhibit an interfacial
activation, which sets it apart from most of the true lipases. The
Table 2
Kinetic parameters of the cutinases. Kinetics parameters of Tfu 0882 and Tfu 0883
were determined at their optimal temperature of 60 ◦C and FspC at its optimal
temperature of 40 ◦C. Values are means SD (n = 3).
Kinetic parameter
Tfu 0882 cutinase
673 32
Tfu 0883 cutinase
505 29
FspC
KmpNPB (␮m)
272 10
837 25
Fig. 3. Degradation of cyclic PET trimers by the cutinases. The enzymes were
incubated with cyclic PET trimers in 10 ml 25 mM phosphate buffer (pH 8) and
the hydrolysis products were analyzed by RP-HPLC and LC–MS. (ꢀ) Tfu 0883; (ꢁ)
Tfu 0882; (ꢂ) FspC. Error bars correspond to the standard deviation of three deter-
minations.
kcat (s−1
kcat/Km (s−1 ␮M−1
)
483 19
742 24
)
0.7
1.5
3.2

S. Chen et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 121–127
125
Table 4
Effect of surfactants on cutinase activity. Cutinase was preincubated with the sur-
factant at 37 ◦C for 5 min, and then assayed for esterase activity against pNPB. Values
are means SD (n = 3).
Surfactant
Tfu 0882
cutinase
Relative
Tfu 0883
cutinase
Relative
FspC
Relative
activity (%)
activity (%)
activity (%)
Triton X-100
1 mM
10 mM
42
42
3
1
72
77
3
3
102
100
4
4
Tween 20
1 mM
10 mM
34
33
1
1
73
84
4
4
91
94
3
3
SDS
1 mM
10 mM
87
75
4
4
69
43
2
1
22
24
1
1
TDOC
1 mM
10 mM
Fig. 4. Evaluation of interfacial activation of the cutinases. The enzymes were incu-
bated with 3–35 mM of tributyrin emulsion at 37 ◦C for 15 min. The released fatty
acids were quantified by titration with 0.05N NaOH. (ꢀ) Tfu 0883; (ꢁ) Tfu 0882; (ꢂ)
FspC; vertical dot, the critical micelle concentration (CMC) of tributyrin (12 mM).
107
89
3
2
97
111
4
3
123
174
4
5
a medium inhibitory effect. Cr, however, inhibited most enzyme
activity, whereas Hg completely inactivated the cutinases.
absence of a lid structure and an exposure of the nucleophilic ser-
ine in this enzyme has been described as the main reason for this
behaviour [29]. In many true lipases, a lid structure burying the
nucleophilic serine has been reported to be involved in interfacial
activation. It undergoes a conformational change in response to an
adsorption of the enzyme at the oil–water interface [30]. A lid inser-
tion was also absent in the previously developed homology models
of Tfu 0882 and Tfu 0883 [13], supporting the results obtained. The
absence of interfacial activation in these cutinases further confirms
that they are different from true lipases although all the enzymes
belong to the ␣/␤ hydrolase fold superfamily.
3.6. Effect of surfactants on cutinase activity
Application of industrial enzymes often involves relatively harsh
conditions such as the presence of surfactants and organic sol-
vents. The activity of the cutinases was tested in the presence of
the nonionic surfactants Triton X-100, Tween 20 and the anionic
surfactants SDS and TDOC. At concentrations of 1 mM and 10 mM,
Triton X-100 and Tween 20 inhibited both cutinases from T. fusca
but did not significantly reduce FspC activity. SDS inhibited all three
enzymes. TDOC, on the other hand, stimulated FspC activity with a
23.11% increase at 1 mM and a 73.65% increase at 10 mM. It did not
have a significant effect on the T. fusca cutinase activity (Table 4).
The effects of TDOC on cutinase activity were further tested
at concentrations up to 100 mM (Fig. 5). Tfu 0882 appeared to be
fairly stable in the presence of this surfactant with 71.43% activity
remaining at 100 mM. Tfu 0883 was slightly stimulated at TDOC
concentrations between 1 mM and 50 mM and fully retained its
activity at 100 mM. FspC was significantly stimulated by TDOC with
the highest stimulatory effect at 10 mM and almost full activity
remaining at 100 mM. TDOC is an anionic surfactant with a bulky
side chain which may bind to the hydrophobic sites of proteins
3.5. Metal ion requirement
To determine whether the cutinases requires a metal cofactor
for activity, they were incubated with the metal chelator EDTA or
metal ions and then assayed for esterase activity against pNPB.
EDTA (1 mM or 10 mM) did not affect their activities (Table 3),
suggesting that the cutinases did not require divalent cations for
their activity. When they were incubated with 1 mM of divalent
metal ions, Mn, Co, Ni, Mg, Ba, Cu, or Ca did not exhibit a signifi-
cant effect on the enzyme activity, whereas Zn, Fe, or Pb showed
Table 3
Effect of metal ions and metal chelator on cutinase activity. The cutinase were prein-
cubated with metal ions (1 mM) or metal chelator at 37 ◦C for 5 min, and then assayed
for esterase activity against pNPB. Values are means SD (n = 3).
Metal ion or chelator
Tfu 0882
cutinase
Relative
Tfu 0883
cutinase
Relative
FspC
Relative
activity (%)
activity (%)
activity (%)
Control
MnCl2
CoCl2
NiCl2
BaCl2
CuSO4
CaCl2
MgCl2
ZnSO4
100
109
104
105
93
72
72
66
50
1
4
2
3
2
2
1
2
1
3
2
1
100
124
124
107
107
94
82
111
44
2
5
4
2
2
3
3
5
2
1
3
1
100
136
107
93
101
83
94
89
82
46
2
5
3
3
2
2
4
2
3
2
3
1
FeSO4
PbCl2
CrCl2
55
52
11
64
45
10
61
28
HgCl2
EDTA (1 mM)
EDTA (10 mM)
N.D.
101
101
N.D.
101
102
N.D.
102
103
Fig. 5. Effect of TDOC on cutinase activity. The enzyme was incubated with TDOC
at 37 ◦C for 5 min and then assayed for esterase activity against pNPB. (ꢀ) Tfu 0883;
(ꢁ) Tfu 0882; (ꢂ) FspC. Error bars correspond to the standard deviation of three
determinations.
2
2
1
2
3
2
N.D.: no detectable activity.

126
S. Chen et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 121–127
Fig. 6. Inhibition kinetics of the cutinase by SDS. (A) Tfu 0882; (B) Tfu 0883; (C) FspC. Double-reciprocal plots (1/v vs. 1/[pNPB]) of the initial rate data were determined at
three concentrations of SDS. (ꢃ) SDS 1 mM; (ꢀ) SDS 0.5 mM; (ꢀ) SDS 0 mM.
Table 5
and metal ion requirement. However, they differ significantly in
their sensitivity to surfactants and dramatically in their sensitivity
to organic solvents. Considering their versatile hydrolytic activity,
Stability of the cutinase in organic solvents. The cutinase were incubated with 75%
(v/v) of organic solvents in assay buffer at 20 ◦C for 18 h. Aliquots were removed for
determination of residual activity. Values are means SD (n = 3).
good tolerance to surfactants, superior stability in organic sol-
vents, and superior thermostability the T. fusca cutinases may have
promising applications in related industries.
Organic solvent (75%)
Tfu 0882
cutinase
Relative
Tfu 0883
cutinase
Relative
FspC
Relative
activity (%)
activity (%)
activity (%)
Control
Methanol
Ethanol
Isopropanol
Butanol
Acetone
100
87
100
27
44
97
2
1
4
3
5
3
4
4
100
86
98
65
20
99
84
92
2
3
2
3
3
3
3
4
100
6
4
1
9
31
70
32
2
1
1
1
1
2
4
1
Acknowledgements
This work was supported by the Program for the National
High-tech Research and Development Program of China
(2009AA02Z204); the National Natural Science Foundation of
China (No. 30970057), the Key Program of National Natural Sci-
ence Foundation of China (No. 20836003), New Century Excellent
Talents in University (NCET-06-0486), the National Outstanding
Youth Foundation of China (No. 20625619), Research Program of
State Key Laboratory of Food Science and Technology (No. SKLF-
MB-200802), Program of Innovation Team of Jiangnan University
(2008CXTD01), and the Program for Advanced Talents within Six
Industries of Jiangsu Province (08-B-WU Jing).
n-Hexane
Dimethyl sulfoxide
96
94
preventing their aggregation and rendering them more stable [31].
FspC is less stable than Tfu 0882 and Tfu 0883 in solution, so that
TDOC showed more effective in stimulating FspC.
In addition, inhibition kinetics was performed for SDS to
evaluate its inhibitory efficiency on the three cutinases. The inhi-
bition constants were determined by a double-reciprocal plot
(1/v vs. 1/[pNPB]) of the initial reaction rate at varying concentra-
tions (Fig. 6). SDS appeared to be a competitive inhibitor for all
three enzymes with a KiSDS of 1385.6 ␮M for Tfu 0882, 944.4 ␮M
for Tfu 0883 and 657.5 ␮M for FspC. Compared with the other two
enzymes, Tfu 0882 appeared to be more resistant to SDS and may
be more favorable for applications in detergent formulations.
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