Biochemical Engineering Journal 101 (2015) 99107

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Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Regular article

Lipase-catalyzed Knoevenagel condensation in waterethanol solvent

system. Does the enzyme possess the substrate promiscuity?
Weina Li a,b,c , Rong Li b,d , Xiaochun Yu a , Xuebing Xu b , Zheng Guo b, , Tianwei Tan a, ,
Sergey N. Fedosov b,
a
College of Life Science and Technology, Beijing University of Chemical Technology, Beisanhuan East Road 15, Beijing 100029, China
b
Department of Engineering, Faculty of Science and Technology, Aarhus University, Gustav Wied Vej 10, Aarhus 8000, Denmark
c
Department of Chemical Engineering, Northwest University, Taibaibeilu 229, Xian 710069, China
d
Key Laboratory for Molecular Enzymology and Engineering, The Ministry of Education, Jilin University, Changchun 130021, China

a r t i c l e i n f o a b s t r a c t

Article history: Lipase-catalyzed Knoevenagel condensation of a ketone/aldehyde (e.g., benzaldehyde 1) and the active
Received 30 January 2015 hydrogen compound (e.g., ethyl cyanoacetate 2 or malononitrile 3) is often regarded as catalytic promis-
Received in revised form 8 April 2015 cuity. The alternative mechanism suggests partial enzymatic hydrolysis of 2, whereupon the products
Accepted 26 April 2015
initiate fusion 1 + 2. Three lipases (porcine pancreatic, Mucor javanicus and Yarrowia lipolytica) did not
Available online 8 May 2015
hydrolyze 2, but signicantly accelerated condensations 1 + 2 and 1 + 3 (3 is not hydrolyzable), thereby
corroborating promiscuous enzymatic activity. Main conversion took place within the active site (based
Keywords:
on competitive inhibition by caffeic acid). Yet, the active Ser residue of lipases was unimportant, because
Knoevenagel condensation
Lipase
its covalent modication did not affect condensation. The reaction (particularly 1 + 3 condensation) was
Kinetic parameters to some extent promoted by unspecic residues of lipase, as well as albumin and simple proton acceptors.
Promiscuity Spontaneous condensation in water/ethanol surprisingly revealed kinetics with substrate saturation. We
Enzyme biocatalysis explained this depart from linearity by a two-step steady state mechanism including deprotonation of the
Ethanol active hydrogen substrate 3H by polar solvent, followed by direct collision of a temporary complex sol-
vent H+ 3 with 1. Similar mechanism with a more sophisticated binding of substrates was conjectured
for the lipases.
2015 Elsevier B.V. All rights reserved.

1. Introduction In this process the carbonyl group belongs to an aldehyde or a

Knoevenagel condensation is a modied aldol condensation,
which goes via nucleophilic addition of an active hydrogen com-
pound to a carbonyl group followed by elimination of one water
molecule (hence condensation) [1,2]. The product is often an ,-
conjugated enone. The Hantzsch pyridine synthesis, the Gewald
reaction and the FeistBenary furan synthesis all include Knoeve-
nagel condensation as an intermediate reaction step. The relevant
examples of reaction are shown in Fig. 1.
Abbreviations: 1, benzaldehyde; 2, ethyl cyanoacetate; 3, malononitrile; BSA,
bovine serum albumin; CA, caffeic acid; CALB, Candida antarctica lipase B; GC, gas
chromatography; THL, tetrahydrolipstatin; pNPB, p-nitrophenyl butyrate; pNP, p-
nitrophenol; PPL, porcine pancreatic lipase; YlLip2, Yarrowia lipolytica lipase LIP2.
Corresponding authors.
E-mail addresses: zhencheng874125@163.com (W. Li), rongli@eng.au.dk (R. Li),
yes yuxiaochun@163.com (X. Yu), xuxuebing@wilmar-intl.com (X. Xu),
guo@mb.au.dk (Z. Guo), twtan@mail.buct.edu.cn (T. Tan), snf@mb.au.dk
(S.N. Fedosov).
ketone (substrate 1). Another reactant is called the active hydrogen
component [2], whose typical examples include diethyl malonate,
Meldrums acid, ethyl acetoacetate, ethyl cyanoacetate (2), malonic
acid, malononitrile (3), nitromethane etc. These compounds have a
general structure of Z1 CH2 Z2 , Z1 CHRZ2 or Z1 CHR1 R2 , where
Z1 and Z2 are the electron withdrawing groups [2]. These groups
destabilize the methylene bridge and make possible dissociation
of its proton in the presence of a proton acceptor. The conven-
tional catalysts of Knoevenagel reaction are weakly basic amines,
whereas the stronger bases induce self-condensation of aldehydes
(or ketones).
Two mechanisms are suggested to explain activity of an amine
as a catalyst. One of them postulates formation of a Schiff base
between the amine-containing catalyst and the aldehyde sub-
strate 1. Afterward, the obtained imine intermediate condensates
with carbanions. Exactly this mechanism was originally reported
by Knoevenagel [1]. The alternative pathway (HannLapworth
mechanism [3]) starts with deprotonation of the active hydrogen
compound in the presence of a basic catalyst. The formed enol

http://dx.doi.org/10.1016/j.bej.2015.04.021
1369-703X/ 2015 Elsevier B.V. All rights reserved.
100 W. Li et al. / Biochemical Engineering Journal 101 (2015) 99107
Fig. 1. The lipase-catalyzed Knoeveagel reaction of our study. Substrate B benzalde-
hyde (1) undergoes condensation with substrate A (active hydrogen component),
represented by either ethyl cyanoacetate (2) or malononitrile (3), giving the respec-
tive products of 4 or 5.
intermediate reacts with the carbonyl group, and the produced
aldol undergoes subsequent elimination. Currently, there is no clear
answer, which mechanism takes place in reality (though the accu-
mulating data point to higher probability of the second scheme).
It was shown that some enzymes can mediate Knoevenagel con-
densation, for example lipase from porcine pancreas [4], acylase
[5], papain [6] and alkaline protease from Bacillus licheniformis [7].
Lipases (triacylglycerol acylhydrolases, E.C. 3.1.1.3) are a subclass
of esterases (enzymes acting on ester bonds, E.C. 3.1). They usually
catalyze hydrolysis of natural lipids, as well as a variety of syn-
thetic compounds. All lipases investigated so far exhibit a surprising
degree of structural and functional similarity [8]. The catalytic triad
of the active site is composed of SerHis and Asp/Glu residues,
which are also found in the respective triads of proteases, esterases
and thioesterases [9]. During catalysis, the negatively charged car-
boxylic group of Asp or Glu interacts with His residue and helps it
to deprotonate Ser [9]. The highly nucleophilic SerO attacks the
carbonyl group of the acylglycerol forming an acylenzyme inter-
mediate, stabilized within so called oxyanion hole adjacent to
the active Ser. The deacylation step is controlled by nucleophilic-
ity of the second substrate (e.g., H2 O, methanol etc.), which attacks
the acylated enzyme. The reaction cycle ends by release of a prod-
uct (free fatty acid, its methyl ester etc.) and regeneration of the
catalytic groups [8,9].
Apart from the normal catalytic mechanism, several exam-
ples of unusual lipase activities are mentioned in the literature
describing formation of carbon carbon, carbon heteroatom and
heteroatom heteroatom bonds, as well as oxidative processes
[1014]. For example, Li et al. [11] reported asymmetric aldol
reaction between acetone and different aromatic aldehydes cat-
alyzed by porcine pancreatic lipase (PPL) under aqueous reaction
conditions. Later on, the same group of authors described decar-
boxylative aldol reaction and Knoevenagel condensation between
derivatives of benzaldehyde and -ketoesters [12]. A mutant
(Ser105Ala) of Candida antarctica lipase B (CALB) exhibited catalytic
activity under aldol addition [13,15] or Michael Addition [14]. The
authors suggested His residue of active site to be the basic catalyst
in the absence of mutated Ser [13].
Despite a number of well-documented examples of catalytic
promiscuity of lipases, some controversy still exists. For example,
condensation of derivatives of benzaldehyde and -ketoesters was
interpreted as the true promiscuous catalysis by Feng et al. [12].
This interpretation was questioned by Evitt and Bornscheuer [15],
who pointed out that the ester substrates can be hydrolyzed by
lipases in a conventional way. The generated products (most specif-
ically carboxylic acid as stated in Ref. [15]) mediate a spontaneous
condensation outside the active site of enzyme. A non-enzymatic
test reaction included, however, not a carboxylic acid but its salt
(lithium acetoacetate [15]), providing thereby basic conditions
instead of acidic.
In this work we investigate whether lipases exhibit true
substrate promiscuity under Knoevenagel condensation of alde-
hydes and ester substrates, or this process goes via conventional
hydrolytic pathway. For this purpose we have used different sub-
strates (with and without ester bond). Three lipases (from porcine
pancreas, Mucor javanicus and Yarrowia lipolytica LIP2) were tested,
as well as several unspecic catalysts (bovine albumin, sodium
methoxide, sodium acetate, and water). The performed exami-
nation showed that lipases do perform the abnormal reaction,
but do not hydrolyze ester substrate in a conventional way. Main
conversion took place within the active site, but its active Ser
residue was not involved into Knoevenagel condensation. The
kinetic mechanism was suggested providing quantitative interpre-
tation of velocity and its unusual depart from linearity in the case
of simple catalysts.
2. Materials and methods
2.1. Materials
Lipase from porcine pancreas (Type II) and Amano lipase M
from M. javanicus were purchased from SigmaAldrich Co. Crude
lipase Y. lipolytica (YlLip2) was prepared in our laboratory accord-
ing to previously published procedures [16]. Commercial reagents
benzealdehyde, ethyl cyanoacetate, malononitrile, caffeic acid,
tetrahydrolipstatin (as well as salts and buffers) were obtained from
SigmaAldrich Co. Lipase suicide inhibitor methyl 4-nitrophenyl
hexylphosphonate (or ELSIMC) was purchased from EUCODIS
Bioscience (Austria). Pierce BCA Protein Assay Kit (biuret with bicin-
choninic acid) was obtained from Thermo Scientic.
2.2. GC method
The yield was calculated by GC as described previously [17]. In
short, the sample (1 L) was injected into a GC system (Thermo
scientic Trace GC Ultra system) equipped with an Omegawax TM
250 fusedsilica capillary column (30 m 0.25 mm 0.25 mm lm
thickness) using hydrogen used as the mobile phase. The injector
temperature was set to 250 C.
2.3. Reaction conditions
Standard condensation of 1 + 2 was performed at 0.25 M of
substrate 1, 0.375 M of substrate 2, 10 mg/mL of lipase prepara-
tion (total mass including proteins, carbohydrates and salts), all
dissolved in 4 mL of ethanol with 3.23% (v/v) water added, 50 C,
500 rpm. Reactions progress was recorded over 30 min for PPL and
90 min for MJL and YlLip2 (6080% of conversion was covered).
Standard condensation of 1 + 3 was performed at 0.17 M of
substrate 1, 0.25 M of substrate 3, 2.5 mg/mL of lipase (total mass),
all dissolved in 6 mL of anhydrous ethanol, 25 C, 200 rpm. Reac-
tions progress was recorded over 10 min (6080% of conversion
was covered).
Hydrolysis of substrate 2 was performed at 0 M or 0.25 M of
substrate 1 and 0.375 M of substrate 2, standard conditions.
Reaction conditions with simple catalysts included standard
substrate concentrations, no catalyst, or 0.041 M of sodium acetate,
or 0.062 M of sodium methylate, or BSA 2.5 mg/mL added, all
dissolved in 6 mL of anhydrous ethanol, 25 C, 200 rpm, 5 min incu-
bation.
Reaction conditions of kinetic study. Condensation of 1 + 2
involved 10 mg/mL of lipase (total mass) dissolved in 4 mL of the
optimal medium ethanol + water (0% or 5% v/v for PPL, 45% for MJL
 W. Li et al. / Biochemical Engineering Journal 101 (2015) 99107 101

and YlLip2), 50 C, 500 rpm, reaction time of 1560 min. Conden- Here k+ is the rate constant of efcient collisions, and Y is the cata-
sation of 1 + 3 involved 2.5 mg/mL of lipase (total mass), dissolved lyst. The catalyst concentration y can be either added to or excluded
in 6 mL of ethanol + water (45% v/v), 25 C, 200 rpm, reaction time from the velocity equation.
of 10 min. The substrate concentrations were changed according (ii) Mechanism with complex-formation (Eq. (2)) implies that A
to the following schemes: (i) substrate 1 was maintained constant and B produce a fast equilibrating temporary complex AB with the
at 0.04 M, 0.2 M or 0.6 M, whereas substrate 2 (or 3) varied in the dissociation constant Kab , whereupon AB undergoes a slow catalytic
range of, 0.031.5 M; (ii) substrate 2 (3) was kept constant (0.06 M, conversion (k+ ), optionally mediated by an efcient collision with
0.3 M, 0.6 M), whereas 1 varied in the range of 0.041.5 M. a simple catalyst Y:
Kab k+
A + B AB (+Y) P (+Y)
2.4. Enzyme and protein concentrations.
   (2)
The content of active lipase/esterase in different preparations k ( y)
v= + a + b + Kab
2
(a + b + Kab ) 4 a b
was determined by active site titration using the suicide lipase 2
inhibitor methyl 4-nitrophenyl hexylphosphonate [18] according
(iii) Mechanism with sequential collisions between a catalyst Y
to recommendations of the manufacturer (EUCODIS Bioscience).
and the reactants A, B (Eq. (3)) goes in two steps (both at comparable
The titration medium contained 10 mg/mL of the test catalyst (total
rates). The efcient encounter between Y and A produces a tempo-
mass including all compounds) mixed with 0.1 mM of the titration
rary complex AY, which afterward undergoes a direct collision with
reactant in 50 mM TrisHCl buffer, pH 8.0, 1.5% sodium dodecyl
the substrate B:
sulfate, 1% CH3 CN, total volume of 1 mL. The molar absorbance
of 14280 M1 cm1 was used to recalculate the burst phase of k+a

absorbance change to the active site concentration. k+b

The total protein content in lipase preparations was measured
by PierceTM BCA Protein Assay Kit [19] as recommended by the
manufacturer (Thermo Scientic).
2.5. Inhibition of lipase activity
The lipase activity was suppressed using a competitive inhibitor
caffeic acid [20], taken at the concentration of 0.1 M and added
to the standard reaction mixture of 1 + 2. Reactions were recorded
over 1540 min.
In the alternative setup we used an irreversible inhibitor
tetrahydrolipstatin (THL) [21]. This compound covalently modies
Ser residue of the active site [22], which is responsible for esterase
activity. Here a lipase preparation of 80 mg was dissolved in 1 mL
of the standard reaction medium (without substrates 1 and 2)
and mixed with THL (1.8 mg in 20 L DMSO) or DMSO (control).
Each sample was incubated overnight, whereupon the enzyme was
mixed with the substrates to get the standard nal concentrations
of all compounds (see Section 2.3). The remaining enzymatic activ-
ity was tested in the condensation of 1 + 2 or in the hydrolysis of
p-nitrophenyl butyrate (pNPB), see next paragraph.
Esterase activity was measured via hydrolysis of 0.14 mM
pNPB added from a stock solution (50 mM pNPB in DMSO) to
3 mL of 25 mM TrisHCl pH 7.5, 44 mM NaCl. The reaction was
started immediately after mixing of pNPB with aqueous sol-
vent by adding 20 L of 80 mg/mL lipase solution (either native
or inactivated sample). The reaction progress was recorded at
400 nm over 5 min. Concentration of the accumulated colored prod-
uct pNP was calculated from its coefcient of molar absorbance
400 = 10538 M1 cm1 , determined for the standard compound
pNP (0.0080.1 mM) at pH 7.5. Comparison of the initial veloci-
ties (native versus inactivated lipase) was done for the same initial
pNPB concentrations.
2.6. Theory of simple bisubstrate reactions
Reactions between two substrates A and B (taken at the con-
centrations of a and b) can follow the mechanisms of different
complexity, often involving a simple catalyst Y (a solvent molecule,
acid, base etc.). Several relevant examples of irreversible reactions
with the corresponding velocity equations are presented.
(i) Mechanism of a simple collision (Eq. (1)) is shown below:
k+
A + B (+Y) P (+Y) ; v = k+ ( y) a b (1)
A+Y AY + BP + Y;
ka
(3)
k+a a k+b y0 k+a k+b y0
v= =
k+a a + k+b b + ka k+b k+a ka
+ +
a b ab
here y0 is the total concentration of the catalyst (y0 = y + ay, y0 < a0
and b0 ) and k+a , ka and k+b are the rate constants of the correspond-
ing steps (ka might be relatively small). The opposite sequence of
collisions does not affect the general appearance of the rate equa-
tion in Eq. (3) but changes k a to k b . More details about similar
mechanisms can be found elsewhere [23].
The reaction velocity depends on the concentration of each indi-
vidual substrate either linearly (Eq. (1)) or shows a clear depart from
linearity called saturation by the substrates (Eqs. (2) and (3)).
2.7. Theory of enzymatic bisubstrate reactions
A bisubstrate reaction catalyzed by an enzyme E can follow vari-
ous mechanisms differing in the order of ligand binding (substrates
A and B) and the number of enzymesubstrate complexes [24].
(i) General model of such reaction (in the absence of products)
can be presented as shown in Scheme 4, where the sequence of sub-
strate binding and presence of a ternary complex are not specied.
The model is described by the overall equation Eq. (4) [24]:
k+
E + A. . . + B. . . E (A) (B) P + E;
(4)
V+
v= ; V+ = K+ e0
KmA KmB KsA KmB
1+ + +
a b a b
Notation in Eq. (4) is as follows: v is the initial velocity of for-
ward reaction; V+ is the maximal velocity of forward reaction; a
and b correspond to the concentrations of free substrates A and B,
respectively; KmA and KmB are the Michaelis constants of ligands A
and B, respectively; KsA is the substrate constant of A (often iden-
tical to the dissociation constant of E + A EA); k+ corresponds to
the turnover number, i.e., maximal number of catalytic events per
time per active site; e0 is the total concentration of active site E. The
product of two constants KsB KmA might substitute for KsA KmB
in Eq. (4) if required/allowed by the specic mechanism. The exact
meaning of the Michaelis and substrate constants varies depending
on the model. The general form of Eq. (4) can be used to discriminate
between different mechanisms depending on the set of none-zero
elements in the denominator obtained after tting [24].
102 W. Li et al. / Biochemical Engineering Journal 101 (2015) 99107
(ii) Another relevant mechanism of ordered binding (a variant
TheorellChance model) has one equilibrium step and two steady
state stages:
k+ea
Ka
k+b
E + AE A EA + B P + E;
kea
k+ea k+b e0
v=
Ka (k+ea + kea ) /k+b Ka kea /k+b
1+ + +
a b ab
In Scheme 5, the substrate A binds to the enzyme in a fast equi-
librium manner producing a temporary complex E A. Then, a
signicantly slower transition to a tighter complex takes place. This
process might be essentially shifted to the right. Finally, an efcient
collision of the low-afnity substrate B with the activated complex
EA gives the product P without (or very little) formation of EAB
complexes.
The equation Eq. (5) is identical in its form to Eq. (4) except for
the meaning of the constants. If the rate constant of backward tran-
sition (kea ) is relatively small in comparison to two other steady
state rate constants (k+ea and k+b ), the last element of denominator
in Eq. (5) can be canceled.
2.8. Fitting and statistics
The data tting was performs by computer software KyPlot 5
(Keyens Inc., Japan). The error in parameters of best approximation
is given as the standard error of mean. Goodness of t was evaluated
by coefcient of determination R2 adjusted for the number of tting
parameters.
3. Results
3.1. Lipase preparations
The concentration of active site in 10 mg/mL solution of each
catalyst was assessed according to the specic titration method,
see Section 2.4. The results were 44.3 M for PPL, 27.4 M for
MJL and 12.1 M for YlLip2. The active site concentrations corre-
sponded to the lipase protein concentrations of 2.2 mg/mL (PPL),
0.57 mg/mL (MJL) and 0.41 mg/mL (YlLip2) based on mass esti-
mates of their protein cores: Mw = 50 kDa for PPL (UniProtKB,
P00591); Mw 20.5 kDa for MJL excluding 0.5 kDa of carbo-
hydrates [25]; and Mw 34 kDa for YlLip2 excluding 5 kDa of
carbohydrates [16]. The total protein content in 10 mg/mL solution
of the catalyst was equal to 3.29 mg/mL (PPL), 2.39 mg/mL (MJL)
and 2.30 mg/mL (YlLip2). The main non-protein component in PPL
Fig. 2. Effect of water concentration on the condensation velocity expressed as % of conversion in 15 min for 1 + 2; or 5 min for 1 + 3). (A) Reaction 1 + 2, none or 10 mg/mL of
lipases, 50 C; (B) Reaction 1 + 3, 25 C, none or 2.5 mg/mL of lipases.
and MJL was apparently dextrin, based on the product specica-
tion sheets. The main non-protein component of our preparation
of YlLip2 [26] was maltodextrin [27].
3.2. Effect of water on the velocity of enzymatic reaction
(5) Water is the obligatory component of any hydrolytic reaction
(suspected to be crucial under condensation of 1 + 2 according to
Ref. [15]). It is also an important effector of 3D structure of macro-
molecules in different organic solvents. Therefore, we have tested
the inuence of increasing water concentration on the velocity of
Knoevenagel condensation for both 1 + 2 (2 is hydrolyzable) and
1 + 3 (3 is resistant to hydrolysis) using the ethanol medium in the
presence and absence of lipases. In all cases, the time curves of prod-
uct accumulation tended to 90100% (Supplementary material, Fig.
S1). In other words, the condensation process could be regarded as
nearly irreversible.
We observed a relatively weak spontaneous condensation in the
mixture of 1 + 2 (the bottom curve in Fig. 2A). On the contrary, this
process was very noticeable in the mixture of 1 + 3 (the bottom
curve in Fig. 2B). The difference was attributed to the number of
strong electron withdrawing CN-groups: one in substrate 2, two in
substrate 3 (Fig. 1). Kinetics of the spontaneous condensation was
examined in further details in Section 3.6.
Addition of lipases (PPL, MJL or YlLip2) noticeably accelerated
the condensation, especially in the medium with optimal aqueous
ethanol (standard conditions). The maximal specic velocity was
reached at 1050% of water (Fig. 2A, 1 + 2) or 1025% of water
(Fig. 2B, 1 + 3). Application of purely aqueous solutions deceler-
ated conversion, probably, because of a diminished solubility of
the substrates. The overall catalytic efciency of lipases under con-
densation of 1 + 2 (Fig. 2A) was signicantly lower if compared to
1 + 3 (Fig. 2B), where shorter incubation times, lower enzyme con-
centrations and lower temperatures were used to bring the data
of both panels within a similar scale (see standard conditions in
Section 2.3).
3.3. Test of hydrolytic activity under Knoevenagel condensation
Potential hydrolytic cleavage of the ester compound 2 was
examined rst without substrate 1 but in the presence of lipases.
The GC proles indicated that the content of substrate 2 did not
change over 7 h of incubation (slope of +1.9 2.2% per hour based
on linear tting). Incubation of 2 in a basic medium (0.1 M NaOH in
ethanol +45% v/v water) also did not cause any changes over 20 h
(+0.4% 0.4% per h).
Additional tests were conducted by measurement of pH, which
is expected to decrease if hydrolysis of esters takes place in a non-
 W. Li et al. / Biochemical Engineering Journal 101 (2015) 99107 103

Fig. 3. Tests of side reactions and reaction specicity. (A) Test of hydrolytic cleavage of ester compound 2 by lipases. The values of pH were recorded over 40 min in the
mixtures 1 + 2 + lipase (standard conditions) and in the mixtures 1 + 2 + lipase + acetic acid. (B) Test of reaction specicity under Knoevenagel condensation. Catalysts (BSA
and lipases) were used with or without treatment by inhibitors (CA and THL).

buffered solution. The values of pH were recorded over 40 min in Table 1

the mixtures 2 + lipase or 1 + 2 + lipase (standard conditions). No
reliable change of pH was observed in any of the experiments (mean
initial value of pH 5.7 0.1, mean slope of +0.23 0.1 pH unit
per hour), see Fig. 3A. At the same time, addition of acetic acid to
the mixture 1 + 2 + enzyme caused a very noticeable decrease of pH
(Fig. 3A). In other words, even a 0.1% hydrolysis of 2 (0.375 M) would
have caused a drop of pH by one unit. While pH measurements were
conducted, the reaction proceeded normally and reached 7590% of
conversion after 40 min of incubation (in the absence of acetic acid).
We concluded that hydrolysis of ester-substrate 2 did not occur
under the experimental conditions and was not required for the
efcient Knoevenagel condensation 1 + 2 in the presence of lipases.
3.4. Test of reaction specicity under Knoevenagel condensation
Specicity of enzymes in regard to Knoevenagel reaction was
tested by comparison to bovine serum albumin (BSA), as well as
using inhibitors targeted at the lipase active site. The inhibitors
in question were caffeic acid (CA) and tetrahydrolipstatin (THL).
The rst one represents a competitive ligand, which occupies the
active site of lipase and hinders access of the substrates [20]. The
second one is a specic covalent modier of the active Ser-residue
of lipase [21]. The results of specicity test are shown in Fig. 3B,
Here the rate of spontaneous conversion (initiated by solvent) was
subtracted, and the remaining protein-related velocity was nor-
malized to 1 mg/mL of the total protein. The reaction 1 + 2 in the
presence of BSA was slow and remained unaffected by inhibitors.
The lipase-mediated catalysis was noticeably faster than that of
Condensation reaction catalyzed by simple basic catalysts.
Reaction Sodium acetate Sodium methoxide No catalyst
0.041 M 0.062 M (control)
1+2 68.4% 28.4% 8.4%
1+3 26.6% 74.0% 18.7%
Conditions: anhydrous ethanol, 25 C, 5 min of incubation
BSA, indicating importance of the lipase active site. The competitive
inhibitor CA signicantly decreased activity of all enzymes, where
the highest effect was observed for MJL and YlLip2. The Ser-targeted
reagent THL had nearly no effect on the velocity of enzymatic Kno-
evenagel condensation. Yet the esterase activity of pNPB hydrolysis
was essentially reduced indicating successful modication of Ser
residue (see the bar labeled as X expected in Fig. 3B).
A visible rate of Knoevenagel condensation was discovered for
the unspecic catalyst BSA, as well as in pure polar solvents with-
out amine compounds (Fig. 2). This attracted our attention, and we
tested two organic salts not related to amines.
3.5. Reaction performed by simple basic catalysts
Sodium acetate and sodium methoxide were used as basic cat-
alysts incapable to from a Schiff bases. Both of them facilitated
Knoevenagel conversion (Table 1), though the nature of substrates
affected the efciency of reaction. Sodium acetate was a more
potent catalyst in the mixture of substrates 1 + 2 if compared to
1 + 3. The situation was opposite with sodium methoxide. Efcient
conversion in the presence of basic salts extends the number of

Fig. 4. Spontaneous condensation of benzaldehyde 1 (ligand B) and malononitrile 3 (ligand A), 25 C. Reaction velocities are plotted versus the respective substrate concen-
trations. The dark and light circles are the experimental points situated below and above the tting surface, respectively. (A) Reactions in anhydrous ethanol. (B) Reactions
in ethanol with 45% v/v water. Parameters of best approximation are presented in the main text.
104 W. Li et al. / Biochemical Engineering Journal 101 (2015) 99107

Table 2
The kinetic data of lipase-catalyzed Knoevenagel condensation.

Enzyme additive reaction PPL H2 O 1 + 2 PPL + H2 O MJL + H2 O YlLip2 + H2 O 1 + 2 YlLip2 YlLip2

1+2 1+2 H2 O 1 + 3 +H2 O 1 + 3

e0 , M 4.43 105 4.43 105 2.74 105 1.21 105 3.02 106 3.02 106
Fig. Fig. 5A Fig. 5B Fig. 5C Fig. 5D

Eq. (4)
V+ , M/min 0.16 0.34 0.080 0.023 0.23 4000
KmA , M 0.94 0.58 0.60 0.036 6.4 20 00
KmB , M 0.55 0.81 0.29 0.10 7.6 0.54
KsA , M 0.014 0.009 0.026 0.17 0.015 1300
k+ , s1 a 60 127 49 34 1270
Adj. R2 0.793 0.822 0.859 0.673 0.713 0.777

Eq. (5)
Ka , M 0.9 0.5 0.5 0.2 1.0 0.2 0.05 0.02 1.0 0.7 0.06 0.04
k+ea , s1 45 20 68 22 50 18 15 3 122 79 1800 4200
kea , s1 0.05 k+ea b 0.05 k+ea b 0.02 k+ea b 0.2 k+ea 0.2 k+ea 1.0 k+ea
k+b , M1 s1 81 10 102 9 87 7 138 16 128 0.5 390 37
Adj. R2 0.797 0.820 0.852 0.674 0.712 0.767

Substrates: benzaldehyde 1 and either ethyl cyanoacetate 2 or malononitrile 3.

a
The turnover number was derived from k+ = V+ /e0 .
b
A change of kea from the shown value to zero provides only a marginal improvement of the goodness of t.

protein residues with potential relevance to Knoevenagel conden- (y0 ) and provided slightly higher rate constants of all conversion
sation. steps, see Section 4.2 for further details.

3.6. Kinetics of spontaneous condensation 1 + 3 3.7. Kinetics of lipase-catalyzed condensations 1 + 2 and 1 + 3

A noticeable spontaneous condensation was observed in polar
solutions of ethanol/water, especially in the case of benzaldehyde
1 and malononitrile 3, see Fig. 2B. Therefore, we decided to exam-
ine kinetics of this process in further details to better understand
its nature and subtract the unspecic conversion from the specic
enzymatic reactions.
The 3D charts in Fig. 4 show the reaction velocities mea-
sured at different substrate concentrations in the absence of water
(Fig. 4A) or with 45% v/v water added. Quite surprisingly, both pan-
els showed a variant of saturation kinetics inconsistent with a
simple collision mechanism (Section 2.6, Scheme 1 and Eq. (1)).
Such deviation from linearity is usually interpreted in terms of a
complex-formation (e.g., Eq. (2)) or a steady state scheme with
two-steps (e.g., Eq. (3)). Either of the models could be used for
approximation of the data in Fig. 4. Yet formation of a temporary
complex between A and B (according to Scheme 2) lacked physical
basis, and this mechanism was abandoned.
We therefore, examined the steady state Scheme 3, where the
catalytic events can be interpreted as follows. At the rst step,
the active hydrogen compound 3 (written here as 3H) produces a
temporary complex with the activated solvent molecule (Y) char-
acterized by an increased dipole moment. This causes transition of
proton from 3H to Y and formation of an ionic complex YH+ . . 3 .
The Scheme 3 treats these two events as a simultaneous process
(though other variants are also feasible). At the second step, the
aldehyde compound 1 (substrate B) collides with the complex YH+ .
. 3 . The rate constant (k+b ) reects frequency of the productive
encounters between the negatively charged 3 and the positively
charged part of the aldehyde leading to formation of their conjugate
and elimination of water.
Fitting of the reaction velocity in ethanol (Fig. 4A) by
Eq. (3) gave the following parameters of best approx-
imation: y0 = 0.0064 0.002 M, k+a = 3.1 0.4 M1 min1 ,
ka 0.01 0.3 min1 , k+b = 3.0 0.4 M1 min1 , R2 = 0.930. The
best approximation of the reaction in ethanol + water medium
(Fig. 4B) gave y0 = 0.16 0.05 M, k+a = 6.6 0.7 (M)1 min1 ,
ka 0.46 0.45 min1 , k+b = 5.0 0.4 M1 min1 , R2 = 0.942. These
results indicate that a more polar mixture of ethanol + water
contained a higher concentration of catalytically active solvent
Kinetic analysis of the reaction 1 + 2 was performed for all three
lipases in ethanol containing some water (0% or 5% for PPL; 45%
for MJL and 45% for YlLip2). We used the optimal water con-
centrations, to guarantee the highest enzymatic activities (Fig. 2A).
PPL had the lowest demand for water and allowed exploration of
the reaction in anhydrous ethanol or 5% H2 O, which contrasted
this enzyme with two other lipases. All reaction velocities lin-
early depended on the concentrations of enzymes (Supplementary
material, Fig. S2) implying absence of any complicating effects, like
enzyme dimerization and mass transfer limitations. Kinetic analy-
sis of the conversion for another substrate combination 1 + 3 was
done only for YlLip2 in either anhydrous ethanol or the aqueous
mixture ethanol + 45 % water. Velocities of spontaneous conden-
sation 1 + 3 determined earlier in Fig. 4 were subtracted from the
corresponding enzymatic measurements.
Reaction rates in question were plotted versus substrate concen-
trations as 3D charts, and the full list of examined reactions is shown
in Table 2. The most relevant data are depicted in Fig. 5 including the
condensations of 1 + 2 at optimal water for all three lipases (panels
A, B, C) and the alternative reaction 1 + 3 at optimal water for YlLip2
(panel D). The experimental points were approximated by bivari-
ate functions (Eqs. (4) and (5)). It should be mentioned that these
equations do not consider presence of the products at the start of
reaction. Yet water (both the product and the structural effector)
was, in fact, added in a number of setups. This means that water is
a hidden element of rate coefcients, which might change if the
conditions are noticeably altered. The effect can be marginal (see
Table 2, columns PPL, 1 + 2, H2 O and +H2 O) or very signicant
(YlLip2, 1 + 3, H2 O and +H2 O).
All sets of tting parameters are shown in Table 2. The upper part
of this table presents the coefcients of unconstraint tting using
Eq. (4) (general equation of a bisubstrate reaction). The major pur-
pose was to assess the turnover numbers by extrapolating velocities
to innite substrate concentrations. The approximating procedure
was, however, unstable and occasionally gave negative or innitely
large half-saturation coefcients (which make no sense in terms of
chemistry). Such result means that the strayed parameter has
low impact on the goodness of t and any value below or above
certain level provides a satisfactory approximation of the data.
 W. Li et al. / Biochemical Engineering Journal 101 (2015) 99107 105

Fig. 5. Enzymatic condensation of benzaldehyde 1 (ligand B) and either ethyl cyanoacetate 2 or malononitrile 3 (ligands A) in ethanol. Reaction velocities are plotted versus
the respective substrate concentrations. The dark and light circles are the experimental points situated below and above the tting surface, respectively. The lipases and
conditions are as follows: (A) PPL, substrates 1 + 2 with 5% water, 50 C; (B) MJL, substrates 1 + 2 with 45% water, 50 C; (C) YlLip2, substrates 1 + 2 with 45% water, 50 C; (D)
YlLip2, substrates 1 + 3 with 45% water, 25 C.

Therefore, a hypothetical Scheme 5 was used as one of several pos-
sible mechanisms, where a plausible constraint of parameters could
be introduced based on a general reasoning. This alternative model
resembles Scheme 3 (used for pure solvents in Fig. 4). To make
Eqs. (4) and (5) identical in their layout, we added to Scheme 5 an
additional substrate binding step described as a fast equilibrium
(e.g., E + 2 E . . 2). We constrained the ratio of constants kea /k+ea
at the next reaction step (interpreted as deprotonation, e.g., E . .
2H EH+ 2 ). This ratio is expected to be relatively low, based on
the results of the unconstraint tting trials (Table 2, upper part). The
values of kea /k+ea were assumed to be similar for the same enzyme
in all reactions. In other words, the value of 0.1 in one setup pre-
cluded acceptance of 1000 in another. The trade-off variants were
found in course of several ts to make a best possible approach to
coefcient of determination in the unconstraint ts (Table 2, upper
part). The results of the best alternative ts are shown in the lower
part of Table 2. They should be regarded as plausible speculations
about the reaction kinetics if considering the mechanism of Eq. (5).
Additional interpretations are presented in the Section 4.
4. Discussion
Analysis of enzymatic Knoevenagel condensations 1 + 2 and 1 + 3
(Fig. 1) indicated presence of the following components of the reac-
tion kinetics: (i) spontaneous condensation in a polar solvent; (ii)
unspecic reaction associated with amino acid residues outside the
active site; (iii) specic reaction performed within the active site of
lipase. We sequentially discuss these issues below. We also exam-
ine whether the enzymatic reaction goes via promiscuous catalysis
or articially evolves from the normal esterase activity of lipases,
as was suggested in Ref. [15].
4.1. Spontaneous condensation in ethanolwater
It is generally believed that Knoevenagel condensation requires
a catalyst of amine (imine) nature [1,2]. Yet the data from literature
[28] and of the current work indicate that polar solvents alone can
promote this reaction if the active hydrogen compound contains
strong electron withdrawing group(s). For example, spontaneous
condensation of 1 and Meldrums acid (with a highly dissociative
proton of methylene bridge) was observed in pure aqueous solution
at room temperature [28].
In our assay, pure ethanol and ethanol + water mixture also
enhanced the spontaneous condensation (Figs. 2 and 4). Reac-
tion velocity was much higher for the substrate combination 1 + 3,
because of higher mobility of H+ in 3 (pK 11 for 3 versus pK 13
for 2). Presence of water at an optimal concentration accelerated
condensation 1 + 3 if compared to both anhydrous ethanol and pure
water (lowest curve in Fig. 2B). Approximate content of the catalyti-
cally active anhydrous ethanol was assessed as 0.0064 M (or 0.04%),
see Fig. 4A and Section 3.6. This active part of solvent apparently
has a higher dipole moment, sufcient to abstract proton from 3
and initiate its condensation with 1. Addition of water signicantly
increased the apparent concentration of active solvent, as well
as provided elevated rate constants of conversion (see Fig. 4B and
Section 3.6). Yet any direct interpretation is difcult when working
with liquid capable of various patterns of intermolecular hydrogen
bonding. The deduced concentration of catalytically active solvent
(0.16 M) can be, therefore, ascribed to both individual molecules
and their mixed combinations.
106 W. Li et al. / Biochemical Engineering Journal 101 (2015) 99107
4.2. Unspecic condensation (simple catalysts and BSA)
Amines are not the only simple catalysts capable to promote
Knoevenagel condensation. One can nd in the literature exam-
ples with application of ionic liquids, salt of metals under solid
catalysis ([2] and references thereof) and an organometallic cage
in water [28]. A peculiar example concerns condensation of 4-
nitrobenzaldehyde and lithium acetoacetate in aqueous solution
of MeCN + cyclen [15]. The authors interpreted this reaction as an
example of acid catalysis because trace quantities of acetoacetic
acid (conjugated inactive acid according to BrnstedLowry) are
formed in the presence of water. Yet their medium was obviously
basic as expected for any salt of a weak acid in an aqueous solu-
tion (e.g. A + Li+ + H2 O AH + Li+ + OH ). In the produced mixture
both the high excess of RCOO and/or a small amount of OH
could act as basic acceptors of proton from the methylene bridge
of acetoacetate.
In this regard, we have tested two simple basic catalysts (sodium
acetate and sodium methoxide). Both accelerated Knoevenagel
condensation (1 + 2 and 1 + 3) though to a different degree (Table 1).
Formation of Schiff base was obviously impossible in both cases.
This experiment adds Asp and Glu residues (RCOO at neutral
pH), as well as highly nucleophilic Ser of lipase active site (RO
at neutral pH under catalytic transition), to the list of amino acid
residues capable to initiate Knoevenagel condensation. If salts
alone could catalyze the reaction, the non-specic amino acid
residues were also fair candidates to do this job, as was tested on
BSA.
Some unspecic activity was indeed recorded for a non-catalytic
protein BSA (Fig. 3B). We can ascribe this effect mostly to basic
amino acid residues (ArgLys and His) situated on the sur-
face of BSA. We have earlier demonstrated that separate amino
acids of basic nature promote Knoevenagel reaction at neu-
tral pH, though the catalytic efciency is not very high [17].
Existence of a background protein catalysis (associated with
unspecic surface residues) immediately raised a question about
the relation between specic and unspecic components of the
enzymatic Knoevenagel condensation. The enzymes clearly out-
performed BSA if making comparison of activity per 1 mg of
each protein (Fig. 3B). We ascribe the acceleration of conver-
sion to presence of the catalytic site in each lipase molecule.
The specic nature of enzymatic condensation was addition-
ally demonstrated, when the competitive inhibitor CA noticeably
suppressed lipase activity (which was decreased nearly to the
level of unspecic protein catalysis). On the other hand, the Ser-
targeted specic covalent modier THL had practically no effect
on Knoevenagel condensation, though esterase activity of lipase
was clearly suppressed (Fig. 3B). We concluded that active Ser
is insignicant for Knoevenagel condensation, though the true
catalytic group(s) is situated within the active site. The same con-
clusion was reached by other authors when Ser105Ala mutant
of CALB exhibited enhanced activity in the reactions of aldol
and Michael addition [13,14,27]. Yet there was a difference in
some of the observed effects. For example, the reaction of aldol
addition with hexanal, performed by the immobilized wild type
CALB in non-aqueous medium (cyclohexane), was essentially ham-
pered via modication of Ser-residue by methyl p-nitrophenyl
n-hexylphosphonate [29]. On the contrary, the analogous modica-
tion of PPL, MJL and YlLip2 by THL showed no effect on Knoevenagel
condensation in the current work (Fig. 3B). The observed dis-
crepancy can be ascribed to different types of substrates or/and
conformational differences in the enzyme active sites induced
by the surrounding medium, e.g., the immobilized lipase sus-
pended in a highly hydrophobic cyclohexane [29] versus the free
enzymes in a relatively polar mixture of ethanol and water (this
study).
4.3. Specic condensation (lipases)
Comparison of the two condensation reactions (1 + 2 and 1 + 3)
was performed at different temperatures and lipase concentrations,
because the essentially different reaction velocities precluded
monitoring of the two processes under the identical conditions.
When analyzing the reaction kinetics 1 + 3, a noticeable velocity of
spontaneous condensation was observed (Figs. 2A and 4). This irrel-
evant component was subtracted from the velocity of enzymatic
assay. In the case of another reaction (1 + 2) spontaneous conden-
sation was regarded as insignicant. The protein-related kinetics
included inputs of both the active site of lipase (major compo-
nent) and the unspecic residues (minor component). We analyzed
a pooled catalytic rate of each enzyme, because accurate discrim-
ination between specic and unspecic elements of the reaction
was not possible.
First we found that the mechanism of enzymatic Knoevenagel
condensation does not involve the esterase activity of lipase,
though 2 has a hydrolyzable ester bond. For example, no hydrolysis
of compound 2 was detected using material balance of 2 (incu-
bated with lipase but without 1), or looking at pH records of the
reaction progress (Fig. 3A). We can speculate that polar nitrile
group within the tail of a short carboxylic acid precludes its
correct positioning inside the active site, thereby hindering hydrol-
ysis of ethyl cyanoacetate (2). On the other hand, ethyl butyrate
(the hydrophobic structural analog of 2) is hydrolyzed by lipases
[30]. The hydrolytic step was chemically impossible when using
the second substrate of our study (malononitrile, 3). Disregard-
ing this hindrance, 3 showed accelerated condensation with the
aldehyde 1. Another instance of the successful enzymatic con-
densation using a non-hydrolyzable substrate (acetylacetone) was
recently described in the literature [31]. Despite the mentioned
obstacles for hydrolysis, all reactions of condensation were suc-
cessfully accomplished. We can conclude that the lipases of this
study (together with the examples from literature) do perform
promiscuous catalysis via an unusual reaction mechanism.
His224 (CALB numbering) from the catalytic triad was suggested
as the major basic catalyst in aldol condensation [31]. Its role can
be, however, discussed. For example, Asp134 of CALB (situated in
the active site right in front of Ser105) is also a feasible candidate,
considering the observed catalytic activity of sodium acetate. At
pH > 5, the residue of Asp134 is expected to be dissociated providing
a basic group RCOO capable of proton abstraction.
The three lipases did not show very pronounced variation in
the kinetic constants of the reaction 1 + 2, roughly assessed by Eq.
(4). The highest turnover number (k+ according to Scheme 4) was
found for PPL (upper part of Table 2). Yet the catalytic efciency of
PPL becomes less impressive if normalizing it per mg of the pro-
tein. For example, considerably smaller MJL used 2.5 less protein
material to build the active site of comparable efciency (at least
in respect to Knoevenagel condensation). The lipases responded
differently to the changes in the substrate concentrations (1 + 2).
Velocity of YlLip2 steeply increased in the range of 00.5 M, where-
upon no acceleration was observed (Fig. 5C). On the contrary, both
PPL and MJL had gentle slopes, which continued to increase well
above 0.5 M if changing both ligands simultaneously (Fig. 5A and
B). The performed comparison of the three enzymes is not abso-
lutely unbiased. For example, the highly efcient reaction with PPL
was explored at low water content, which was interesting from
a technological point of view. On the contrary, activation of MJL
and YlLip2 (Fig. 2A) required presence of 45% water. Yet the activ-
ity of PPL was stable within a broad range of water concentrations
(Fig. 2A), which allows us to conjecture similar kinetic parameters
of PPL at both 5% and 45% of H2 O.
The reaction 1 + 2 can be also interpreted in terms of the ordered
steady state model (Scheme 5). This is one of several possible mech-
 W. Li et al. / Biochemical Engineering Journal 101 (2015) 99107
anisms, and we doubt that discrimination between them is possible
based on the current datasets. We consider Scheme 5 as a plausi-
ble hypothetical model, because it has a close resemblance to the
assumed mechanism of spontaneous solvent catalysis (Scheme
3). According to the conjectured model (second part of Table 2),
PPL had high values of the two critical forward rate constant (k+ea
and k+ b ) combined with a moderate afnity for ligand 2 (sub-
strate A in the Scheme 5). This provided response over a wide
range of substrate concentrations, where velocity tended to high
values at increasing 1 and 2. MJL had pretty similar characteris-
tics with somewhat decreased overall turnover at both steady state
steps. A different example was found in YlLip2, which exhibited a
strong binding of 2 combined with a low rate of the deprotonation
step (k+ea ). The hindered deprotonation might be a direct conse-
quence of excessively strong interactions with 2 where multiple
protein-ligand contacts can complicate approach of the deproto-
nating group.
Comparison of the two variants of reaction 1 + 2 and 1 + 3 (exam-
ined for YlLip2) shows that deprotonation of malononitrile 3 is
very fast, especially considering a much lower temperature of this
reaction (25 C instead of 50 C). In aqueous ethanol the deproto-
nation step was accelerated even further (last column in Table 2).
Frequency of efcient collision at the second steady state step
(between 1 and the complex EH+ 3 ) was also high if compared to
EH+ 2 , resulting in a steady increase of velocity along axis b (1), see
Fig. 5D. All these observations are in line with better dissociation
of proton in the free molecule of 3 if compared to 2.
5. Conclusions
We have established that enzymatic Knoevenagel condensa-
tion does not require hydrolysis of ester substrates (as might
have been hypothesized). The examined reaction examples can
be, therefore, classied as a true promiscuous catalysis. Enzymes
provided a signicant acceleration of conversion, if compared to
that of unspecic protein groups and simple catalysts. Ser residue
of lipase active site was not involved in catalysis because its
covalent modication did not hamper the condensation. On the
contrary, a competitive inhibitor (caffeic acid) suppressed the con-
version. Simple basic salts and polar solvents provided a detectable
level of activity, pointing to a mechanism without Schiff base for-
mation. Reaction velocities where characterized by saturation
kinetics for both simple and complex catalysts. The most plausi-
ble kinetic scheme (applicable in both cases) implies the following
major steps: (i) abstraction of the proton from active hydrogen
component and formation of a temporary ionic complex with
the catalyst; (ii) efcient collision of this complex with polarized
aldehyde and subsequent formation of the condensation prod-
uct.
Acknowledgments
This work was supported by the National Basic Research Pro-
gram of China (973 program: 2013CB733600, 2012CB725200)
the National Nature Science Foundation of China (21390202,
21436002), and National High-Tech R&D Program of China (863
program: 2014AA022100, 2014AA021904). The support provided
by China Scholarship Council (No. +2011688018) during a visit to
Aarhus is acknowledged.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bej.2015.04.021
107
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