Acyl-peptide releasing enzyme from Sulfolobus tokodaii (ST0779) as a novel promiscuous biocatalyst for aldol addition

Article abstract of DOI:10.1016/j.catcom.2015.03.030

This work, for the first time, demonstrated the catalytic promiscuity of an acyl-peptide releasing enzyme from Sulfolobus tokodaii (ST0779) for aldol addition reaction, which shows accelerated activity at elevated temperature. The turnover number k_ca

Full text of DOI:10.1016/j.catcom.2015.03.030

Catalysis Communications 66 (2015) 111–115
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Short communication
Acyl-peptide releasing enzyme from Sulfolobus tokodaii (ST0779) as a
novel promiscuous biocatalyst for aldol addition
c
b,
Rong Li a,b, Bianca Perez b, Hui Jian a, Renjun Gao a, , Mingdong Dong , Zheng Guo
⁎
⁎
a
Key Laboratory for Molecular Enzymology and Engineering, The Ministry of Education, Jilin University, Changchun, 130021 Changchun, China
Department of Engineering, Faculty of Science and Technology, Aarhus University, Gustav Wied Vej 10, Aarhus 8000, Denmark
b
c
Interdisciplinary Nanoscience Center, Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus C, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
This work, for the first time, demonstrated the catalytic promiscuity of an acyl-peptide releasing enzyme from
Sulfolobus tokodaii (ST0779) for aldol addition reaction, which shows accelerated activity at elevated tempera-
ture. The turnover number kcat (s−1) of this thermostable enzyme at 55 °C is 7.78-fold higher than that of porcine
pancreatic lipase (PPL) at its optimum temperature of 37 °C, which is one of the best reported enzymes for pro-
miscuous catalysis of aldol reaction; and the molecular catalytic efficiency kcat/Km (M−1 s−1) adds up to 140
times higher than PPL.
Received 4 February 2015
Received in revised form 14 March 2015
Accepted 23 March 2015
Available online 25 March 2015
Keywords:
Aldol reaction
© 2015 Elsevier B.V. All rights reserved.
Sulfolobus tokodaii peptidase (ST0779)
Hydrolase
Catalytic promiscuity
Biocatalysis
1. Introduction
catalytic properties, including antibody, BSA, Subtilisin E, lipase, prote-
ase, aminopeptidase and tautomerase, etc. [2,3,9–16]. In the attempts
Catalytic promiscuity is one of the most exciting discoveries in enzy-
mology field after non-/micro-aqueous enzymology [1–3], which is of
particular interest for in vitro biosynthesis and organic synthesis by pos-
sibly improving existing catalysis or creating novel synthesis pathways
that are currently not available [1,4,5]. Enzyme promiscuity (commonly
known as catalytic promiscuity) is defined as the ability of an enzyme to
catalyze multiple chemical transformations by the same active site
distinctly different from its primary activity [6,7]. The biological value
of enzyme promiscuity has been well reviewed [8,9], which the formal-
ized idea is “under changing environments, promiscuous activity in an
existing protein can endow the organism with a selective advantage
and thereby enable its survival and further evolution”. While, the specif-
ic value of catalytic promiscuity fascinating synthetic chemists is to add
new dimensions to our understanding of protein structure–activity re-
lationship; [10–12] it thus makes “evolution of new functions from
existing protein scaffolds” more practical; which will greatly expand
the scientific toolbox of bio-synthetic methodologies [13–15].
to exploit the potential of enzyme promiscuity, two themes are prevail-
ing, namely, identifying novel promiscuous activities from existing en-
zymes; [1,5,16–19] and engineering the existing enzyme/protein
scaffolds for improved promiscuous activities [12–16]. In the present
work we report a successful example in identifying new enzyme species
for promiscuous catalytic synthesis.
Aldol reaction is the reaction when the enolate of an aldehyde or a
ketone reacts at the α-carbon with the carbonyl of another molecule
under basic or acidic conditions to obtain β-hydroxy aldehyde or ketone
[20–22]. Aldol reaction is one of the most important carbon–carbon
bond forming reactions in organic synthesis [23,24]. In the efforts to-
wards the identification and engineering of promiscuous enzymes to
catalyze aldol addition reactions, several successful cases have been re-
ported, among which most of them belong to α/β hydrolases [16–20,23,
24]. The α/β hydrolase fold includes lipase, esterase, protease/peptidase
and dehalogenase etc., [16–31] which has been widely identified as an
important source of the enzymes that have promiscuous activities to
catalyze a variety of C–C and C–N formation reactions [1,5,6,16–19]. Re-
cently, we have cloned an enzyme acyl-peptide releasing enzyme
(AARE) ORF0779 (ORF, open reading frame) from thermophilic archaea
Sulfolobus tokodaii (ST0779) and expressed in Escherichia coli BL21 [25].
This enzyme having a molecular weight of 63 kDa (Fig. S1), belongs to a
serine peptidase family and catalyzes the NH2-terminal hydrolysis of
Nα-acylpeptides to release Nα-acylated amino acids; however, ST0779
has also been observed to possess esterase activity [25]. Keeping in
Since the pioneer work by Thorn et al. [2] and Hollfelder et al., [3]
promiscuous catalysis has been applied for different reactions such as
Aldol addition, Michael addition, Markovnikov addition, Henry reaction,
Kemp elimination, Morita–Baylis–Hillman reaction, etc. [16–19]. A vari-
ety of enzymes/proteins have been identified for their promiscuous
⁎
Corresponding authors.
E-mail addresses: gaorj@jlu.edu.cn (R. Gao), guo@mb.au.dk (Z. Guo).
http://dx.doi.org/10.1016/j.catcom.2015.03.030
1566-7367/© 2015 Elsevier B.V. All rights reserved.

112
R. Li et al. / Catalysis Communications 66 (2015) 111–115
mind that many of hydrolases which ST0779 belongs to, have promiscu-
ous activity, we thus surmised that ST0779 might have promiscuous ac-
tivity as well. Another interesting feature of ST0779 is its natural origin
from S. tokodaii, a hydrothermophilic archaeon growing optimally at
80 °C. Consequently, this enzyme demonstrated excellent thermostabil-
ity, as evidenced that about 70% of its acylpeptide releasing activity was
retained after being incubated at 70 °C for 55 h [25]. This feature enables
ST0779 an applicability for biocatalysis at elevated temperature, which
has been rarely addressed in enzyme promiscuity area [1,5,6,12–16].
In the present work, for the first time, we attempt to testify the follow-
ing hypothesis: 1) The molecular feature of ST0779 might also contrib-
ute to promiscuous catalysis; [25] 2) being a thermo-stable enzyme,
ST0779 mediated reaction could take place at enhanced temperature;
as a result, the reaction could be accelerated kinetically. Thus the pro-
miscuous reaction can be greatly accelerated by changing intrinsic reac-
tion kinetics. To our surprise, in our primary trials, ST0779 showed
superior activity in catalyzing aldol addition in comparison with porcine
pancreatic lipase (PPL), one of the best enzymes for promiscuously cat-
alyzed aldol addition [23,24]. Therefore, to shed a light on the catalytic
promiscuity of ST0779, this work focuses on a comparative assessment
of catalytic efficiency and kinetics with PPL.
The reaction was initiated by addition of ST0779/PPL with a typical
concentration of 0.032 μM calculated on the basis of protein content
(commercial PPL with a protein content of 21% measured by Lowry
method) at desired temperatures with magnetic agitation; and
the reaction progress was monitored by periodically sampling at
set time and then subjected to TLC-FID analysis with diethyl ether/
petroleum ether (1:2, v/v) as developing solvents. The conversion
of 4-nitrobenzaldehyde was quantified as the formation of aldol
product (%) in the sum of unreacted 4-nitrobenzaldehyde and its aldol
product with the corresponding aliphatic ketone with the area percent-
age treated as weight.
3. Results and discussion
In the present work, we chose an aldol reaction (nucleophilic addi-
tion of a ketone enolate (nucleophile) to an aldehyde) as a model sys-
tem (Scheme 1), one of which could be promiscuously catalyzed by
lipases and has also been intensively investigated elsewhere [23,24,
26–29]. APE1547 is a thermophilic esterase, which has activity to cata-
lyze the asymmetric aldol addition reaction [29]. We examined the con-
version of the aldol additions catalyzed by three different enzymes and
the results are summarized in Table 1. The results indicate that ST0779
achieves 96.1% conversion in 4 h (entry 3), while PPL obtained only
26.3% conversion after 24 h reaction (entry 1) and APE1547 achieved
just 14.3% after 4 h. This implies that, as an acyl-peptide releasing
enzyme, ST0779 also demonstrates excellent promiscuous activity.
The contribution from the background reactions could be excluded as
almost no conversion of 4-nitrobenzaldehyde (b0.5%) was observed
when denatured PPL/ST0779 was used as catalyst or the reaction was
conducted with the absence of any catalyst (Table 1, entries 4–6). It is
worth to note that a few other lipases (Candida antarctica lipase B,
Rhizopus delemar lipase and so on) have also been examined for cataly-
sis of aldol reaction between 4-nitrobenzaldehyde and acetone; yet
none of them achieved a better catalytic efficacy than PPL (data not
shown). Thus in this context PPL was used as the sole control for com-
parison with ST 0779.
As depicted in Table 2, ST0779 achieved comparable conversion of
4-nitrobenzaldehyde in the reaction with acetone as PPL when the
same enzyme concentration was employed under other identical condi-
tions (entries 1 & 7; entries 2 & 8). The effect of temperature was also
investigated at 37 °C for PPL (entry 3) and 55 °C for ST0779 (entry 9).
37 °C is the optimal temperature for PPL as a decreased activity was ob-
served at higher temperatures (data not shown); and 55 °C was used
because the boiling point of acetone is 56 °C. Excitingly, ST0779
achieved 96.1% conversion in 4 h (entry 9), while PPL obtained only
26.3% conversion after 24 h reaction (entry 3). If comparing both con-
versions at 4 h (PPL obtained only 2.8% conversion), the catalytic effi-
ciency of ST0779 is 34.3-fold higher than PPL. In essence, this result
somewhat verified our hypothesis that enhanced promiscuous activity
could be achieved by the catalysis of thermostable ST0779 at elevated
temperature.
2. Experimental
2.1. Expression and purification of ST0779
The gene (ST0779) from the archaeon S. tokodaii (Japan) was ampli-
fied by PCR using the primers: 5′-ATATTAGGATCCATGACGCCGGAAGA-
3′ and 5′-ACGGAACTCGAGTC AAGACTTCAGAT-3′ and constructed with
pET-28a. Recombinant plasmid was transformed into the expression
host E. coli BL21 (DE3) and the resulting E.coli cells containing ST0779
gene was cultured at 37 °C until OD600 reached 1.0. After ultrasonic
cell disruption, the target acyl-peptide releasing enzyme (APRE)
ST0779 was purified by a two-step process: 1) The cell debris was re-
moved by centrifugation at 12,000 rpm for 20 min; and the supernatant
was heated at 70 °C for 20 min to remove impure proteins by thermo-
denaturation and further subjected to centrifugation. 2) After the re-
moval of thermo-denatured protein by centrifugation at 12,000 rpm
for 20 min, the resulting supernatant was loaded to a Hi-Trap Q-
Sepharose column to obtain APRE ST 0779 with purity of 92.1%
(Fig. S1.). The protein power of ST0779 was obtained by lyophilisation.
PPL in powder form is purchased from Sigma-Aldrich (St. Louis, MO,
USA).
2.2. ST0779/PPL catalyzed aldol reactions
A typical reaction of ST0779/PPL mediated aldol reaction between
4-nitrobenzaldehyde and different aliphatic ketones was conducted in
a reaction mixture of 4 ml ketone and 1 ml deionized water (20%
water content) containing 0.03 mM 4-nitrobenzaldehyde. The aliphatic
ketones in excessive dosage functioned as both substrate and solvent.
OH
O
*
CH
CHO
O
R2
ST 0779 or PPL
R1
+
R1
R2
O2N
O2N
1. R1=H, R2=CH3 (acetone);
2. R1=CH3, R2=CH3 (2-butanone)
3.-R1-R2-== -CH2CH2CH2CH2- (cyclohexanone)
Scheme 1. Aldol reactions catalyzed by ST0779 or PPL.

R. Li et al. / Catalysis Communications 66 (2015) 111–115
113
Table 1
Comparison of the different enzyme catalyzed aldol additions between 4-
nitrobenzaldehyde and acetone.
Entry
Enzyme
Temperature (°C)
Time (h)
Conversion (%)
1
2
3
4
5
6
PPL
APE1547
ST0779
Denatured PPL
Denatured ST0779
Control/no enzyme
30
55
55
30
55
30
24
4
4
24
4
24
21.0
14.3
96.1
b0.5
b0.5
b0.5
To confirm the generality and broad scope of substrates of the enzy-
matic asymmetric aldol reactions mediated by ST0779, we extended the
reaction to 2-butanone and cyclo-hexanone (Table 2, entries 10–13).
For the reaction with 2-butanone at 30 °C after 48 h, ST0779 produced
slightly higher conversion (8.3%, entry 10) than PPL (5.2%, entry 4). Fur-
ther prolongation of reaction time till 72 h, the conversion by PPL in-
creased to only 12.0% (entry 5); while at 55 °C in 24 h the conversion
by ST0779 added up to 24.6% (entry 11). To better view the reaction
progress, the time course of ST0779/PPL catalyzed aldol reaction of 4-
nitrobenzaldehyde and 2-butanone was examined (Fig. 1a). ST0779
produced only slightly higher conversion at 30 °C than PPL at its optimal
temperature of 37 °C. However, the conversion at 55 °C is enhanced sig-
nificantly, for instance, in 24, 36 and 48 h, the conversion difference in
comparison with PPL at 37 °C by the folds of 20.1, 7.1 and 5.4, respec-
tively (Fig. 1a). A more illustrative representation was showed in
Fig. 1b, where the relative conversion (%) could be seen. This indicated
that the influence of temperature on reaction kinetic is spectacular. As
presented in Table 1, for cyclohexanone at 30 °C in 72 h ST0779 obtain-
ed 3.3% product conversion (entry 12) higher than PPL of 1.4% (entry 6).
At 55 °C in 24 h the product conversion by ST 0779 was further in-
creased to 5.7%. Even though the enhancement of reaction rate by
ST0779 at 55 °C compared with the reaction at 30 °C by PPL (b1%) (by
fold of 6) is marketed; the conversion of 4-nitrobenzaldehyde is still
very low. This indicates that the reactivity of a substrate is largely deter-
mined by its molecular feature; thereby the large steric hindrance of
cyclohexanone plays a decisive role [23].
Fig. 1. (a) Time course of ST 0779/PPL catalyzed aldol addition with 2-butanone at differ-
ent temperatures. (b) The relative conversion and ee% of the aldol product of PPL at 30 °C
(A) and 37 °C (B), and ST0779 at 30 °C (C) and 55 °C (D) with 2-butanone at different tem-
peratures for 48 h. The conversion of PPL at 30 °C is defined as 100%. The relative conver-
sion is calculated as the percentage of the conversion of other enzymes relative to that of
PPL at 30 °C.
Stereoselectivity is a measure of a reaction that creates an optically
active product from an achiral starting material, which is also treated
as an important index to assess a catalyst to synthesize preferable enan-
tiomer [32]. The results in Table 1 indicated that PPL gives slightly
higher ee% value than ST0779 when the reaction of acetone is conducted
at the same temperature with similar conversion, e.g., entries 1 and 7;
entries 2 and 8; however, for the reaction with 2-butanone the ee%
values are almost the same (entries 4, 82.7% and 10, 82.5%). This
suggested that the stereoselectivity of an enzymatic reaction is not
only determined by the property of enzyme but also depended largely
on molecular recognition of individual substrate [33,35]. A marked
decrease of stereoselectivity is seen when the reaction temperature is
increased from 30 °C (entry 8, 77.0%) to 55 °C (entry 9, 22%) for acetone;
likewise for 2-butanone, entry 10, 82.5% vs entry 11, 20.0%. Similar but
smaller decrease of ee% for PPL mediated reaction was also observed,
when reaction temperature was increased from 30 °C (entry 1, 93.4%)
to 37 °C (entry 3, 89.2%) (intuitively because the increase of temperature
Table 2
Comparison of acyl-peptide releasing enzyme (ST0779) and porcine pancreatic lipase (PPL) catalyzed aldol additions between 4-nitrobenzaldehyde and different aliphatic ketones.
Entry
Enzyme
PPL
Substrate
Temperature (°C)
Time (h)
Conversion (%)a
eeb [%]
dr (anti/syn)c
1
2
3
4
5
6
7
8
Acetone
Acetone
Acetone
2-Butanone
2-Butanone
Cyclohexanone
Acetone
Acetone
Acetone
2-Butanone
2-Butanone
Cyclohexanone
Cyclohexanone
30
30
37
30
30
30
30
30
55
30
55
30
55
24
48
24
48
72
72
24
48
4
48
24
72
24
21.0
39.2
26.3
5.2
12.0
1.4
19.7
34.1
96.1
8.3
24.6
3.3
93.5
94.5
89.2
82.7
85.3
63.6
73.5
77.0
22.0
82.5
20.0
65.8
45.3
36:64
37:63
84:16
ST0779
9
10
11
12
13
38:62
33:67
78:22
65:35
5.7
a
The products were identified by 1H NMR, 13C NMR and HR-MS (see detail in Supporting information).
ee (enantiomeric excess) (anti) was determined by HPLC analysis.
b
c
dr (diastereomeric ratio) was determined by 1H NMR.

114
R. Li et al. / Catalysis Communications 66 (2015) 111–115
Table 3
increased to 37 °C, only unremarkable decrease of Km value was ob-
served for PPL (0.96 M to 0.85 M). However, when ST 0779 catalyzed
aldol reaction was conducted at 55 °C, the Km value markedly decreased
15.7 times compared to the reaction at 30 °C (0.737 to 0.047 M), imply-
ing a significant enhancement of enzyme affinity to the substrate as the
response of temperature change. Moreover, the corresponding turnover
number increased by 8.7-fold (0.025 to 0.217 s−1) as well.
Comparison of kinetic parameters of ST 0779 and PPL mediated aldol reaction between ac-
etone and 4-nitrobenzaldehyde.
⁎
Enzyme
Temperature
(°C)
Km
(mM)
kcat
kcat/Km
Ea
(s−1
)
(M−1 s−1
)
(kJ/mol)
ST0779
PPL
30
55
30
37
7.37
0.47
9.60
8.50
0.023
0.217
0.024
0.028
3.12
461.70
2.50
51.1a
31.9b
The constant kcat/Km is a measure of the catalytic efficiency of an en-
zyme to convert a substrate into product. As pointed out by Thorn et al.
[2] the constant kcat/Km (catalytic efficiency) of the primary activity of an
enzyme can vary wildly depending on enzyme sources. Nevertheless,
the catalytic efficiency of promiscuous activity is generally lower in
two orders of magnitude than its primary activity [2,3]. As we could cal-
culate that the ratio of (kcat/Km)ST0779 at 55 °C/(kcat/Km)PPL at 37 °C is
139.9, which means that the catalytic efficiency can be enhanced by two
orders of magnitude, when the aldol reaction is catalyzed by ST0779 at
elevated temperature compared to the reaction by PPL at its optimal
temperature. This is a really exciting observation, which presents a
promising perspective that the promiscuous activity of enzyme can be
evolved to achieve comparable value as primary activity if a correct
strategy (e.g., choosing enzyme from thermostable host) is deter-
mined. The effect of temperature on the reaction activity of ST0779
shows a spectacular increase of initial rates against temperature
3.29
⁎
The kinetic parameters were determined by measuring reaction velocity change as a
function of 4-nitrobenzaldehyde concentration (0.01–0.10 mM) and fitting into
Michaelis–Menten equation at the set temperature. Acetone was in large excess and used
as substrate and solvent, and thus its concentration was treated as a constant in data fitting
and estimation of kinetic parameters.
a
Ea of ST0779 was measured at 30–55 °C.
Ea of PPL was measured at 20–37 °C.
b
in PPL case is less than in ST 0779 case, by 7 °C vs by 25 °C). This observa-
tion is also evident in Fig. 1b. Conceptually at elevated temperature the
difference in the differential free energy of activation ΔΔG directed to
the two stereoisomers is eliminating and the reaction is progressing
towards racemization (when ΔΔG = 0, the temperature of which the
product is a complete mixture of two equal isomers is called “racemic
temperature”) [32]. This implied that, “racemic temperature” being a
critical variable, is an important factor for consideration when designing
or optimizing an enzyme for a preferable enantioselective synthesis [32].
Study of enzyme kinetics helps to reveal the catalytic mechanism,
specifically, through measurements of kinetic constants to analyze
the affinity with which enzyme binds substrates and turnover rate
[33–35]. The kinetic parameters for ST0779 and PPL catalyzed aldol re-
actions were presented in Table 2. As shown in Table 2, the Km value
at 30 °C of PPL catalyzed reaction (0.960 M) was only slightly higher
than that of ST0779 (0.737 M) at the same temperature, indicating
that there is no significant difference between ST0779 and PPL in
terms of the affinity to substrate at 30 °C. In coincidence there is only
marginal difference between their corresponding reaction rate constant
kcat (turnover number) 0.023 vs 0.024 s−1. When temperature
(Fig. 1), yielding a significantly high activation energy (51.1 KJ·mol−1
)
than PPL (31.9 KJ·mol−1) estimated by Arrhenius equation (Table 3).
The high Ea of ST0779 might be ascribed to the rigid molecular structure
of thermostable ST0779. At elevated temperature the high activation
energy barrier can be easily overcome, and an enhanced reaction activity
is obtained. This could explain at lower temperature that the reaction ac-
tivity of ST0779 is no better than that of PPL (Table 3 and Fig. 1); however,
the activity is spectacularly increased at 55 °C.
As presented in Scheme 2, we proposed a ST0779 catalyzed aldol re-
action with model reaction of acetone and 4-nitrobenzaldehyde based
on simulated structure of ST0779 [25]. Due to thermodynamical prefer-
ability and high affinity, acetone is firstly accessing to the active sites,
and then accommodated by active site residues Ser439 and His555
OH
O
*
O2N
C
CH3
O
O2N
O
H2
Ser439
Ser439
O
O
H
Asp523
O
O
H
H
O
N
H
N
Asp523
O
H2C
CH3
H
N
Free enzyme
H
N
His555
His555
O
H
O
H3C
CH3
O2N
Ser439
O
Ser439
O
O
H
O
O
O
O
H
Asp523
O
H
N
H2C
CH3
Asp523
H
N
C
CH3
H
N
H2
H
N
His555
His555
Scheme 2. Proposed mechanism for ST0779 catalyzed aldol reaction between acetone and 4-nitrobenzaldehyde.

R. Li et al. / Catalysis Communications 66 (2015) 111–115
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115
(Scheme 2). A proton transfer takes place to form an enolate transition
state which is stabilized by Ser439. Asp523 participates in stabilizing the
positive charge of protonated imidazoyl ring of His555. In the following
step the oxygen of carbonyl group in 4-nitrobenzaldehyde is protonated
by the proton from imidazoyl ring of His555, and the carbon atom in the
same carbonyl group receives neuclophilic attack from the enolate
carbon to form new C–C bond. Finally the aldol product is released
from the enzyme and the enzyme is freed for a new reaction.
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