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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


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

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
Fig. 1. The lipase-catalyzed Knoeveagel reaction of our study. Substrate B benzalde- residue was not involved into Knoevenagel condensation. The
hyde (1) undergoes condensation with substrate A (active hydrogen component),
represented by either ethyl cyanoacetate (2) or malononitrile (3), giving the respec-
kinetic mechanism was suggested providing quantitative interpre-
tive products of 4 or 5. tation of velocity and its unusual depart from linearity in the case
of simple catalysts.

intermediate reacts with the carbonyl group, and the produced 2. Materials and methods
aldol undergoes subsequent elimination. Currently, there is no clear
answer, which mechanism takes place in reality (though the accu- 2.1. Materials
mulating data point to higher probability of the second scheme).
It was shown that some enzymes can mediate Knoevenagel con- Lipase from porcine pancreas (Type II) and Amano lipase M
densation, for example lipase from porcine pancreas [4], acylase from M. javanicus were purchased from SigmaAldrich Co. Crude
[5], papain [6] and alkaline protease from Bacillus licheniformis [7]. lipase Y. lipolytica (YlLip2) was prepared in our laboratory accord-
Lipases (triacylglycerol acylhydrolases, E.C. 3.1.1.3) are a subclass ing to previously published procedures [16]. Commercial reagents
of esterases (enzymes acting on ester bonds, E.C. 3.1). They usually benzealdehyde, ethyl cyanoacetate, malononitrile, caffeic acid,
catalyze hydrolysis of natural lipids, as well as a variety of syn- tetrahydrolipstatin (as well as salts and buffers) were obtained from
thetic compounds. All lipases investigated so far exhibit a surprising SigmaAldrich Co. Lipase suicide inhibitor methyl 4-nitrophenyl
degree of structural and functional similarity [8]. The catalytic triad hexylphosphonate (or ELSIMC) was purchased from EUCODIS
of the active site is composed of SerHis and Asp/Glu residues, Bioscience (Austria). Pierce BCA Protein Assay Kit (biuret with bicin-
which are also found in the respective triads of proteases, esterases choninic acid) was obtained from Thermo Scientic.
and thioesterases [9]. During catalysis, the negatively charged car-
boxylic group of Asp or Glu interacts with His residue and helps it 2.2. GC method
to deprotonate Ser [9]. The highly nucleophilic SerO attacks the
carbonyl group of the acylglycerol forming an acylenzyme inter- The yield was calculated by GC as described previously [17]. In
mediate, stabilized within so called oxyanion hole adjacent to short, the sample (1 L) was injected into a GC system (Thermo
the active Ser. The deacylation step is controlled by nucleophilic- scientic Trace GC Ultra system) equipped with an Omegawax TM
ity of the second substrate (e.g., H2 O, methanol etc.), which attacks 250 fusedsilica capillary column (30 m 0.25 mm 0.25 mm lm
the acylated enzyme. The reaction cycle ends by release of a prod- thickness) using hydrogen used as the mobile phase. The injector
uct (free fatty acid, its methyl ester etc.) and regeneration of the temperature was set to 250 C.
catalytic groups [8,9].
Apart from the normal catalytic mechanism, several exam- 2.3. Reaction conditions
ples of unusual lipase activities are mentioned in the literature
describing formation of carbon carbon, carbon heteroatom and Standard condensation of 1 + 2 was performed at 0.25 M of
heteroatom heteroatom bonds, as well as oxidative processes substrate 1, 0.375 M of substrate 2, 10 mg/mL of lipase prepara-
[1014]. For example, Li et al. [11] reported asymmetric aldol tion (total mass including proteins, carbohydrates and salts), all
reaction between acetone and different aromatic aldehydes cat- dissolved in 4 mL of ethanol with 3.23% (v/v) water added, 50 C,
alyzed by porcine pancreatic lipase (PPL) under aqueous reaction 500 rpm. Reactions progress was recorded over 30 min for PPL and
conditions. Later on, the same group of authors described decar- 90 min for MJL and YlLip2 (6080% of conversion was covered).
boxylative aldol reaction and Knoevenagel condensation between Standard condensation of 1 + 3 was performed at 0.17 M of
derivatives of benzaldehyde and -ketoesters [12]. A mutant substrate 1, 0.25 M of substrate 3, 2.5 mg/mL of lipase (total mass),
(Ser105Ala) of Candida antarctica lipase B (CALB) exhibited catalytic all dissolved in 6 mL of anhydrous ethanol, 25 C, 200 rpm. Reac-
activity under aldol addition [13,15] or Michael Addition [14]. The tions progress was recorded over 10 min (6080% of conversion
authors suggested His residue of active site to be the basic catalyst was covered).
in the absence of mutated Ser [13]. Hydrolysis of substrate 2 was performed at 0 M or 0.25 M of
Despite a number of well-documented examples of catalytic substrate 1 and 0.375 M of substrate 2, standard conditions.
promiscuity of lipases, some controversy still exists. For example, Reaction conditions with simple catalysts included standard
condensation of derivatives of benzaldehyde and -ketoesters was substrate concentrations, no catalyst, or 0.041 M of sodium acetate,
interpreted as the true promiscuous catalysis by Feng et al. [12]. or 0.062 M of sodium methylate, or BSA 2.5 mg/mL added, all
This interpretation was questioned by Evitt and Bornscheuer [15], dissolved in 6 mL of anhydrous ethanol, 25 C, 200 rpm, 5 min incu-
who pointed out that the ester substrates can be hydrolyzed by bation.
lipases in a conventional way. The generated products (most specif- Reaction conditions of kinetic study. Condensation of 1 + 2
ically carboxylic acid as stated in Ref. [15]) mediate a spontaneous involved 10 mg/mL of lipase (total mass) dissolved in 4 mL of the
condensation outside the active site of enzyme. A non-enzymatic 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


A+Y AY + BP + Y;
The total protein content in lipase preparations was measured
by PierceTM BCA Protein Assay Kit [19] as recommended by the ka
(3)
manufacturer (Thermo Scientic). k+a a k+b y0 k+a k+b y0
v= =
k+a a + k+b b + ka k+b k+a ka
+ +
2.5. Inhibition of lipase activity a b ab
here y0 is the total concentration of the catalyst (y0 = y + ay, y0 < a0
The lipase activity was suppressed using a competitive inhibitor and b0 ) and k+a , ka and k+b are the rate constants of the correspond-
caffeic acid [20], taken at the concentration of 0.1 M and added ing steps (ka might be relatively small). The opposite sequence of
to the standard reaction mixture of 1 + 2. Reactions were recorded collisions does not affect the general appearance of the rate equa-
over 1540 min. tion in Eq. (3) but changes k a to k b . More details about similar
In the alternative setup we used an irreversible inhibitor mechanisms can be found elsewhere [23].
tetrahydrolipstatin (THL) [21]. This compound covalently modies The reaction velocity depends on the concentration of each indi-
Ser residue of the active site [22], which is responsible for esterase vidual substrate either linearly (Eq. (1)) or shows a clear depart from
activity. Here a lipase preparation of 80 mg was dissolved in 1 mL linearity called saturation by the substrates (Eqs. (2) and (3)).
of the standard reaction medium (without substrates 1 and 2)
and mixed with THL (1.8 mg in 20 L DMSO) or DMSO (control). 2.7. Theory of enzymatic bisubstrate reactions
Each sample was incubated overnight, whereupon the enzyme was
mixed with the substrates to get the standard nal concentrations A bisubstrate reaction catalyzed by an enzyme E can follow vari-
of all compounds (see Section 2.3). The remaining enzymatic activ- ous mechanisms differing in the order of ligand binding (substrates
ity was tested in the condensation of 1 + 2 or in the hydrolysis of A and B) and the number of enzymesubstrate complexes [24].
p-nitrophenyl butyrate (pNPB), see next paragraph. (i) General model of such reaction (in the absence of products)
Esterase activity was measured via hydrolysis of 0.14 mM can be presented as shown in Scheme 4, where the sequence of sub-
pNPB added from a stock solution (50 mM pNPB in DMSO) to strate binding and presence of a ternary complex are not specied.
3 mL of 25 mM TrisHCl pH 7.5, 44 mM NaCl. The reaction was The model is described by the overall equation Eq. (4) [24]:
started immediately after mixing of pNPB with aqueous sol-
k+
vent by adding 20 L of 80 mg/mL lipase solution (either native E + A. . . + B. . . E (A) (B) P + E;
or inactivated sample). The reaction progress was recorded at
(4)
400 nm over 5 min. Concentration of the accumulated colored prod- V+
uct pNP was calculated from its coefcient of molar absorbance v= ; V+ = K+ e0
KmA KmB KsA KmB
400 = 10538 M1 cm1 , determined for the standard compound 1+ + +
a b a b
pNP (0.0080.1 mM) at pH 7.5. Comparison of the initial veloci-
Notation in Eq. (4) is as follows: v is the initial velocity of for-
ties (native versus inactivated lipase) was done for the same initial
ward reaction; V+ is the maximal velocity of forward reaction; a
pNPB concentrations.
and b correspond to the concentrations of free substrates A and B,
respectively; KmA and KmB are the Michaelis constants of ligands A
2.6. Theory of simple bisubstrate reactions and B, respectively; KsA is the substrate constant of A (often iden-
tical to the dissociation constant of E + A EA); k+ corresponds to
Reactions between two substrates A and B (taken at the con- the turnover number, i.e., maximal number of catalytic events per
centrations of a and b) can follow the mechanisms of different time per active site; e0 is the total concentration of active site E. The
complexity, often involving a simple catalyst Y (a solvent molecule, product of two constants KsB KmA might substitute for KsA KmB
acid, base etc.). Several relevant examples of irreversible reactions in Eq. (4) if required/allowed by the specic mechanism. The exact
with the corresponding velocity equations are presented. meaning of the Michaelis and substrate constants varies depending
(i) Mechanism of a simple collision (Eq. (1)) is shown below: on the model. The general form of Eq. (4) can be used to discriminate
k+
between different mechanisms depending on the set of none-zero
A + B (+Y) P (+Y) ; v = k+ ( y) a b (1) 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 and MJL was apparently dextrin, based on the product specica-
TheorellChance model) has one equilibrium step and two steady tion sheets. The main non-protein component of our preparation
state stages: of YlLip2 [26] was maltodextrin [27].
k+ea
Ka
k+b
3.2. Effect of water on the velocity of enzymatic reaction
E + AE A EA + B P + E;

kea (5) Water is the obligatory component of any hydrolytic reaction
k+ea k+b e0 (suspected to be crucial under condensation of 1 + 2 according to
v=
Ka (k+ea + kea ) /k+b Ka kea /k+b Ref. [15]). It is also an important effector of 3D structure of macro-
1+ + +
a b ab molecules in different organic solvents. Therefore, we have tested
the inuence of increasing water concentration on the velocity of
In Scheme 5, the substrate A binds to the enzyme in a fast equi-
Knoevenagel condensation for both 1 + 2 (2 is hydrolyzable) and
librium manner producing a temporary complex E A. Then, a
1 + 3 (3 is resistant to hydrolysis) using the ethanol medium in the
signicantly slower transition to a tighter complex takes place. This
presence and absence of lipases. In all cases, the time curves of prod-
process might be essentially shifted to the right. Finally, an efcient
uct accumulation tended to 90100% (Supplementary material, Fig.
collision of the low-afnity substrate B with the activated complex
S1). In other words, the condensation process could be regarded as
EA gives the product P without (or very little) formation of EAB
nearly irreversible.
complexes.
We observed a relatively weak spontaneous condensation in the
The equation Eq. (5) is identical in its form to Eq. (4) except for
mixture of 1 + 2 (the bottom curve in Fig. 2A). On the contrary, this
the meaning of the constants. If the rate constant of backward tran-
process was very noticeable in the mixture of 1 + 3 (the bottom
sition (kea ) is relatively small in comparison to two other steady
curve in Fig. 2B). The difference was attributed to the number of
state rate constants (k+ea and k+b ), the last element of denominator
strong electron withdrawing CN-groups: one in substrate 2, two in
in Eq. (5) can be canceled.
substrate 3 (Fig. 1). Kinetics of the spontaneous condensation was
examined in further details in Section 3.6.
2.8. Fitting and statistics Addition of lipases (PPL, MJL or YlLip2) noticeably accelerated
the condensation, especially in the medium with optimal aqueous
The data tting was performs by computer software KyPlot 5 ethanol (standard conditions). The maximal specic velocity was
(Keyens Inc., Japan). The error in parameters of best approximation reached at 1050% of water (Fig. 2A, 1 + 2) or 1025% of water
is given as the standard error of mean. Goodness of t was evaluated (Fig. 2B, 1 + 3). Application of purely aqueous solutions deceler-
by coefcient of determination R2 adjusted for the number of tting ated conversion, probably, because of a diminished solubility of
parameters. the substrates. The overall catalytic efciency of lipases under con-
densation of 1 + 2 (Fig. 2A) was signicantly lower if compared to
3. Results 1 + 3 (Fig. 2B), where shorter incubation times, lower enzyme con-
centrations and lower temperatures were used to bring the data
3.1. Lipase preparations of both panels within a similar scale (see standard conditions in
Section 2.3).
The concentration of active site in 10 mg/mL solution of each
catalyst was assessed according to the specic titration method, 3.3. Test of hydrolytic activity under Knoevenagel condensation
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- Potential hydrolytic cleavage of the ester compound 2 was
sponded to the lipase protein concentrations of 2.2 mg/mL (PPL), examined rst without substrate 1 but in the presence of lipases.
0.57 mg/mL (MJL) and 0.41 mg/mL (YlLip2) based on mass esti- The GC proles indicated that the content of substrate 2 did not
mates of their protein cores: Mw = 50 kDa for PPL (UniProtKB, change over 7 h of incubation (slope of +1.9 2.2% per hour based
P00591); Mw 20.5 kDa for MJL excluding 0.5 kDa of carbo- on linear tting). Incubation of 2 in a basic medium (0.1 M NaOH in
hydrates [25]; and Mw 34 kDa for YlLip2 excluding 5 kDa of ethanol +45% v/v water) also did not cause any changes over 20 h
carbohydrates [16]. The total protein content in 10 mg/mL solution (+0.4% 0.4% per h).
of the catalyst was equal to 3.29 mg/mL (PPL), 2.39 mg/mL (MJL) Additional tests were conducted by measurement of pH, which
and 2.30 mg/mL (YlLip2). The main non-protein component in PPL is expected to decrease if hydrolysis of esters takes place in a non-

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.
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


Condensation reaction catalyzed by simple basic catalysts.
the mixtures 2 + lipase or 1 + 2 + lipase (standard conditions). No
reliable change of pH was observed in any of the experiments (mean Reaction Sodium acetate Sodium methoxide No catalyst
initial value of pH 5.7 0.1, mean slope of +0.23 0.1 pH unit 0.041 M 0.062 M (control)
per hour), see Fig. 3A. At the same time, addition of acetic acid to 1+2 68.4% 28.4% 8.4%
the mixture 1 + 2 + enzyme caused a very noticeable decrease of pH 1+3 26.6% 74.0% 18.7%
(Fig. 3A). In other words, even a 0.1% hydrolysis of 2 (0.375 M) would Conditions: anhydrous ethanol, 25 C, 5 min of incubation
have caused a drop of pH by one unit. While pH measurements were
conducted, the reaction proceeded normally and reached 7590% of
BSA, indicating importance of the lipase active site. The competitive
conversion after 40 min of incubation (in the absence of acetic acid).
inhibitor CA signicantly decreased activity of all enzymes, where
We concluded that hydrolysis of ester-substrate 2 did not occur
the highest effect was observed for MJL and YlLip2. The Ser-targeted
under the experimental conditions and was not required for the
reagent THL had nearly no effect on the velocity of enzymatic Kno-
efcient Knoevenagel condensation 1 + 2 in the presence of lipases.
evenagel condensation. Yet the esterase activity of pNPB hydrolysis
was essentially reduced indicating successful modication of Ser
3.4. Test of reaction specicity under Knoevenagel condensation residue (see the bar labeled as X expected in Fig. 3B).
A visible rate of Knoevenagel condensation was discovered for
Specicity of enzymes in regard to Knoevenagel reaction was the unspecic catalyst BSA, as well as in pure polar solvents with-
tested by comparison to bovine serum albumin (BSA), as well as out amine compounds (Fig. 2). This attracted our attention, and we
using inhibitors targeted at the lipase active site. The inhibitors tested two organic salts not related to amines.
in question were caffeic acid (CA) and tetrahydrolipstatin (THL).
The rst one represents a competitive ligand, which occupies the 3.5. Reaction performed by simple basic catalysts
active site of lipase and hinders access of the substrates [20]. The
second one is a specic covalent modier of the active Ser-residue Sodium acetate and sodium methoxide were used as basic cat-
of lipase [21]. The results of specicity test are shown in Fig. 3B, alysts incapable to from a Schiff bases. Both of them facilitated
Here the rate of spontaneous conversion (initiated by solvent) was Knoevenagel conversion (Table 1), though the nature of substrates
subtracted, and the remaining protein-related velocity was nor- affected the efciency of reaction. Sodium acetate was a more
malized to 1 mg/mL of the total protein. The reaction 1 + 2 in the potent catalyst in the mixture of substrates 1 + 2 if compared to
presence of BSA was slow and remained unaffected by inhibitors. 1 + 3. The situation was opposite with sodium methoxide. Efcient
The lipase-mediated catalysis was noticeably faster than that of 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 Kinetic analysis of the reaction 1 + 2 was performed for all three
solutions of ethanol/water, especially in the case of benzaldehyde lipases in ethanol containing some water (0% or 5% for PPL; 45%
1 and malononitrile 3, see Fig. 2B. Therefore, we decided to exam- for MJL and 45% for YlLip2). We used the optimal water con-
ine kinetics of this process in further details to better understand centrations, to guarantee the highest enzymatic activities (Fig. 2A).
its nature and subtract the unspecic conversion from the specic PPL had the lowest demand for water and allowed exploration of
enzymatic reactions. the reaction in anhydrous ethanol or 5% H2 O, which contrasted
The 3D charts in Fig. 4 show the reaction velocities mea- this enzyme with two other lipases. All reaction velocities lin-
sured at different substrate concentrations in the absence of water early depended on the concentrations of enzymes (Supplementary
(Fig. 4A) or with 45% v/v water added. Quite surprisingly, both pan- material, Fig. S2) implying absence of any complicating effects, like
els showed a variant of saturation kinetics inconsistent with a enzyme dimerization and mass transfer limitations. Kinetic analy-
simple collision mechanism (Section 2.6, Scheme 1 and Eq. (1)). sis of the conversion for another substrate combination 1 + 3 was
Such deviation from linearity is usually interpreted in terms of a done only for YlLip2 in either anhydrous ethanol or the aqueous
complex-formation (e.g., Eq. (2)) or a steady state scheme with mixture ethanol + 45 % water. Velocities of spontaneous conden-
two-steps (e.g., Eq. (3)). Either of the models could be used for sation 1 + 3 determined earlier in Fig. 4 were subtracted from the
approximation of the data in Fig. 4. Yet formation of a temporary corresponding enzymatic measurements.
complex between A and B (according to Scheme 2) lacked physical Reaction rates in question were plotted versus substrate concen-
basis, and this mechanism was abandoned. trations as 3D charts, and the full list of examined reactions is shown
We therefore, examined the steady state Scheme 3, where the in Table 2. The most relevant data are depicted in Fig. 5 including the
catalytic events can be interpreted as follows. At the rst step, condensations of 1 + 2 at optimal water for all three lipases (panels
the active hydrogen compound 3 (written here as 3H) produces a A, B, C) and the alternative reaction 1 + 3 at optimal water for YlLip2
temporary complex with the activated solvent molecule (Y) char- (panel D). The experimental points were approximated by bivari-
acterized by an increased dipole moment. This causes transition of ate functions (Eqs. (4) and (5)). It should be mentioned that these
proton from 3H to Y and formation of an ionic complex YH+ . . 3 . equations do not consider presence of the products at the start of
The Scheme 3 treats these two events as a simultaneous process reaction. Yet water (both the product and the structural effector)
(though other variants are also feasible). At the second step, the was, in fact, added in a number of setups. This means that water is
aldehyde compound 1 (substrate B) collides with the complex YH+ . a hidden element of rate coefcients, which might change if the
. 3 . The rate constant (k+b ) reects frequency of the productive conditions are noticeably altered. The effect can be marginal (see
encounters between the negatively charged 3 and the positively Table 2, columns PPL, 1 + 2, H2 O and +H2 O) or very signicant
charged part of the aldehyde leading to formation of their conjugate (YlLip2, 1 + 3, H2 O and +H2 O).
and elimination of water. All sets of tting parameters are shown in Table 2. The upper part
Fitting of the reaction velocity in ethanol (Fig. 4A) by of this table presents the coefcients of unconstraint tting using
Eq. (3) gave the following parameters of best approx- Eq. (4) (general equation of a bisubstrate reaction). The major pur-
imation: y0 = 0.0064 0.002 M, k+a = 3.1 0.4 M1 min1 , pose was to assess the turnover numbers by extrapolating velocities
ka 0.01 0.3 min1 , k+b = 3.0 0.4 M1 min1 , R2 = 0.930. The to innite substrate concentrations. The approximating procedure
best approximation of the reaction in ethanol + water medium was, however, unstable and occasionally gave negative or innitely
(Fig. 4B) gave y0 = 0.16 0.05 M, k+a = 6.6 0.7 (M)1 min1 , large half-saturation coefcients (which make no sense in terms of
ka 0.46 0.45 min1 , k+b = 5.0 0.4 M1 min1 , R2 = 0.942. These chemistry). Such result means that the strayed parameter has
results indicate that a more polar mixture of ethanol + water low impact on the goodness of t and any value below or above
contained a higher concentration of catalytically active solvent 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- 4.1. Spontaneous condensation in ethanolwater
sible mechanisms, where a plausible constraint of parameters could
be introduced based on a general reasoning. This alternative model It is generally believed that Knoevenagel condensation requires
resembles Scheme 3 (used for pure solvents in Fig. 4). To make a catalyst of amine (imine) nature [1,2]. Yet the data from literature
Eqs. (4) and (5) identical in their layout, we added to Scheme 5 an [28] and of the current work indicate that polar solvents alone can
additional substrate binding step described as a fast equilibrium promote this reaction if the active hydrogen compound contains
(e.g., E + 2 E . . 2). We constrained the ratio of constants kea /k+ea strong electron withdrawing group(s). For example, spontaneous
at the next reaction step (interpreted as deprotonation, e.g., E . . condensation of 1 and Meldrums acid (with a highly dissociative
2H EH+ 2 ). This ratio is expected to be relatively low, based on proton of methylene bridge) was observed in pure aqueous solution
the results of the unconstraint tting trials (Table 2, upper part). The at room temperature [28].
values of kea /k+ea were assumed to be similar for the same enzyme In our assay, pure ethanol and ethanol + water mixture also
in all reactions. In other words, the value of 0.1 in one setup pre- enhanced the spontaneous condensation (Figs. 2 and 4). Reac-
cluded acceptance of 1000 in another. The trade-off variants were tion velocity was much higher for the substrate combination 1 + 3,
found in course of several ts to make a best possible approach to because of higher mobility of H+ in 3 (pK 11 for 3 versus pK 13
coefcient of determination in the unconstraint ts (Table 2, upper for 2). Presence of water at an optimal concentration accelerated
part). The results of the best alternative ts are shown in the lower condensation 1 + 3 if compared to both anhydrous ethanol and pure
part of Table 2. They should be regarded as plausible speculations water (lowest curve in Fig. 2B). Approximate content of the catalyti-
about the reaction kinetics if considering the mechanism of Eq. (5). cally active anhydrous ethanol was assessed as 0.0064 M (or 0.04%),
Additional interpretations are presented in the Section 4. 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
4. Discussion increased the apparent concentration of active solvent, as well
as provided elevated rate constants of conversion (see Fig. 4B and
Analysis of enzymatic Knoevenagel condensations 1 + 2 and 1 + 3 Section 3.6). Yet any direct interpretation is difcult when working
(Fig. 1) indicated presence of the following components of the reac- with liquid capable of various patterns of intermolecular hydrogen
tion kinetics: (i) spontaneous condensation in a polar solvent; (ii) bonding. The deduced concentration of catalytically active solvent
unspecic reaction associated with amino acid residues outside the (0.16 M) can be, therefore, ascribed to both individual molecules
active site; (iii) specic reaction performed within the active site of and their mixed combinations.
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].
106 W. Li et al. / Biochemical Engineering Journal 101 (2015) 99107

4.2. Unspecic condensation (simple catalysts and BSA) 4.3. Specic condensation (lipases)

Amines are not the only simple catalysts capable to promote Comparison of the two condensation reactions (1 + 2 and 1 + 3)
Knoevenagel condensation. One can nd in the literature exam- was performed at different temperatures and lipase concentrations,
ples with application of ionic liquids, salt of metals under solid because the essentially different reaction velocities precluded
catalysis ([2] and references thereof) and an organometallic cage monitoring of the two processes under the identical conditions.
in water [28]. A peculiar example concerns condensation of 4- When analyzing the reaction kinetics 1 + 3, a noticeable velocity of
nitrobenzaldehyde and lithium acetoacetate in aqueous solution spontaneous condensation was observed (Figs. 2A and 4). This irrel-
of MeCN + cyclen [15]. The authors interpreted this reaction as an evant component was subtracted from the velocity of enzymatic
example of acid catalysis because trace quantities of acetoacetic assay. In the case of another reaction (1 + 2) spontaneous conden-
acid (conjugated inactive acid according to BrnstedLowry) are sation was regarded as insignicant. The protein-related kinetics
formed in the presence of water. Yet their medium was obviously included inputs of both the active site of lipase (major compo-
basic as expected for any salt of a weak acid in an aqueous solu- nent) and the unspecic residues (minor component). We analyzed
tion (e.g. A + Li+ + H2 O AH + Li+ + OH ). In the produced mixture a pooled catalytic rate of each enzyme, because accurate discrim-
both the high excess of RCOO and/or a small amount of OH ination between specic and unspecic elements of the reaction
could act as basic acceptors of proton from the methylene bridge was not possible.
of acetoacetate. First we found that the mechanism of enzymatic Knoevenagel
In this regard, we have tested two simple basic catalysts (sodium condensation does not involve the esterase activity of lipase,
acetate and sodium methoxide). Both accelerated Knoevenagel though 2 has a hydrolyzable ester bond. For example, no hydrolysis
condensation (1 + 2 and 1 + 3) though to a different degree (Table 1). of compound 2 was detected using material balance of 2 (incu-
Formation of Schiff base was obviously impossible in both cases. bated with lipase but without 1), or looking at pH records of the
This experiment adds Asp and Glu residues (RCOO at neutral reaction progress (Fig. 3A). We can speculate that polar nitrile
pH), as well as highly nucleophilic Ser of lipase active site (RO group within the tail of a short carboxylic acid precludes its
at neutral pH under catalytic transition), to the list of amino acid correct positioning inside the active site, thereby hindering hydrol-
residues capable to initiate Knoevenagel condensation. If salts ysis of ethyl cyanoacetate (2). On the other hand, ethyl butyrate
alone could catalyze the reaction, the non-specic amino acid (the hydrophobic structural analog of 2) is hydrolyzed by lipases
residues were also fair candidates to do this job, as was tested on [30]. The hydrolytic step was chemically impossible when using
BSA. the second substrate of our study (malononitrile, 3). Disregard-
Some unspecic activity was indeed recorded for a non-catalytic ing this hindrance, 3 showed accelerated condensation with the
protein BSA (Fig. 3B). We can ascribe this effect mostly to basic aldehyde 1. Another instance of the successful enzymatic con-
amino acid residues (ArgLys and His) situated on the sur- densation using a non-hydrolyzable substrate (acetylacetone) was
face of BSA. We have earlier demonstrated that separate amino recently described in the literature [31]. Despite the mentioned
acids of basic nature promote Knoevenagel reaction at neu- obstacles for hydrolysis, all reactions of condensation were suc-
tral pH, though the catalytic efciency is not very high [17]. cessfully accomplished. We can conclude that the lipases of this
Existence of a background protein catalysis (associated with study (together with the examples from literature) do perform
unspecic surface residues) immediately raised a question about promiscuous catalysis via an unusual reaction mechanism.
the relation between specic and unspecic components of the His224 (CALB numbering) from the catalytic triad was suggested
enzymatic Knoevenagel condensation. The enzymes clearly out- as the major basic catalyst in aldol condensation [31]. Its role can
performed BSA if making comparison of activity per 1 mg of be, however, discussed. For example, Asp134 of CALB (situated in
each protein (Fig. 3B). We ascribe the acceleration of conver- the active site right in front of Ser105) is also a feasible candidate,
sion to presence of the catalytic site in each lipase molecule. considering the observed catalytic activity of sodium acetate. At
The specic nature of enzymatic condensation was addition- pH > 5, the residue of Asp134 is expected to be dissociated providing
ally demonstrated, when the competitive inhibitor CA noticeably a basic group RCOO capable of proton abstraction.
suppressed lipase activity (which was decreased nearly to the The three lipases did not show very pronounced variation in
level of unspecic protein catalysis). On the other hand, the Ser- the kinetic constants of the reaction 1 + 2, roughly assessed by Eq.
targeted specic covalent modier THL had practically no effect (4). The highest turnover number (k+ according to Scheme 4) was
on Knoevenagel condensation, though esterase activity of lipase found for PPL (upper part of Table 2). Yet the catalytic efciency of
was clearly suppressed (Fig. 3B). We concluded that active Ser PPL becomes less impressive if normalizing it per mg of the pro-
is insignicant for Knoevenagel condensation, though the true tein. For example, considerably smaller MJL used 2.5 less protein
catalytic group(s) is situated within the active site. The same con- material to build the active site of comparable efciency (at least
clusion was reached by other authors when Ser105Ala mutant in respect to Knoevenagel condensation). The lipases responded
of CALB exhibited enhanced activity in the reactions of aldol differently to the changes in the substrate concentrations (1 + 2).
and Michael addition [13,14,27]. Yet there was a difference in Velocity of YlLip2 steeply increased in the range of 00.5 M, where-
some of the observed effects. For example, the reaction of aldol upon no acceleration was observed (Fig. 5C). On the contrary, both
addition with hexanal, performed by the immobilized wild type PPL and MJL had gentle slopes, which continued to increase well
CALB in non-aqueous medium (cyclohexane), was essentially ham- above 0.5 M if changing both ligands simultaneously (Fig. 5A and
pered via modication of Ser-residue by methyl p-nitrophenyl B). The performed comparison of the three enzymes is not abso-
n-hexylphosphonate [29]. On the contrary, the analogous modica- lutely unbiased. For example, the highly efcient reaction with PPL
tion of PPL, MJL and YlLip2 by THL showed no effect on Knoevenagel was explored at low water content, which was interesting from
condensation in the current work (Fig. 3B). The observed dis- a technological point of view. On the contrary, activation of MJL
crepancy can be ascribed to different types of substrates or/and and YlLip2 (Fig. 2A) required presence of 45% water. Yet the activ-
conformational differences in the enzyme active sites induced ity of PPL was stable within a broad range of water concentrations
by the surrounding medium, e.g., the immobilized lipase sus- (Fig. 2A), which allows us to conjecture similar kinetic parameters
pended in a highly hydrophobic cyclohexane [29] versus the free of PPL at both 5% and 45% of H2 O.
enzymes in a relatively polar mixture of ethanol and water (this The reaction 1 + 2 can be also interpreted in terms of the ordered
study). steady state model (Scheme 5). This is one of several possible mech-
W. Li et al. / Biochemical Engineering Journal 101 (2015) 99107 107

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the online version, at http://dx.doi.org/10.1016/j.bej.2015.04.021 lipoprotein lipase, Catal. Commun. 64 (2015) 101104.