Facile One-Pot Immobilization of a Novel Esterase and Its Application in Cinnamyl Acetate Synthesis

Article abstract of DOI:10.1007/s10562-020-03168-1

Abstract: Cinnamyl acetate has a wide application in food industries. This work focuses on cinnamyl acetate synthesis with a novel esterase. est_GUZ753, an esterase gene from Geobacillus uzenensis DSMZ 13551, was first cloned and expressed in Pichia pastoris KM71. The optimal activity of Est_GUZ753 towards p-NP caprylate was at pH 8.0 and 70?°C, and the half-life at 70?°C was 28?h. Furthermore, Est_GUZ753 showed marked tolerance in methanol. The activity of Est_GUZ753 increased up to 1.16-fold in 90% methanol after 72?h, and the half-life in it was 336?h. The crude fermentation broth of Est_GUZ753 was immobilized directly onto the epoxy resin (Lx-105s), and the immobilized Est_GUZ753 exhibited a 99% conversion for cinnamyl acetate synthesis at a high cinnamyl alcohol concentration in 6?h (cinnamyl alcohol: 1.0?M, enzyme dosage: 3%). These characteristics of Est_GUZ753 indicated its great potential for organic synthesis. Graphic Abstract: A novel esterase gene from Geobacillus uzenensis DSMZ 13551 was first cloned and expressed in Pichia pastoris KM71. Esterase Est_GUZ753 showed marked thermostability at high temperature and tolerance in methanol. Furthermore, the crude fermentation broth of Est_GUZ753 was immobilized directly onto the epoxy resin, and immobilized Est_GUZ753 exhibited a 99% conversion for cinnamyl acetate synthesis.[Figure not available: see fulltext.]

Full text of DOI:10.1007/s10562-020-03168-1

Catalysis Letters
https://doi.org/10.1007/s10562-020-03168-1
Facile One‑Pot Immobilization of a Novel Esterase and Its Application
in Cinnamyl Acetate Synthesis
Fengying Dong1 · Lin Lin · Wei Wei · Dongzhi Wei
2
1
1
Received: 9 February 2020 / Accepted: 1 March 2020
©
Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Cinnamyl acetate has a wide application in food industries. This work focuses on cinnamyl acetate synthesis with a novel
esterase. est , an esterase gene from Geobacillus uzenensis DSMZ 13551, was ꢀrst cloned and expressed in Pichia
GUZ753
pastoris KM71. The optimal activity of Est
towards p-NP caprylate was at pH 8.0 and 70 °C, and the half-life at 70 °C
GUZ753
was 28 h. Furthermore, Est
showed marked tolerance in methanol. The activity of Est
increased up to 1.16-fold
GUZ753
GUZ753
in 90% methanol after 72 h, and the half-life in it was 336 h. The crude fermentation broth of Est
was immobilized
GUZ753
directly onto the epoxy resin (Lx-105s), and the immobilized Est
exhibited a 99% conversion for cinnamyl acetate
GUZ753
synthesis at a high cinnamyl alcohol concentration in 6 h (cinnamyl alcohol: 1.0 M, enzyme dosage: 3%). These character-
istics of Est indicated its great potential for organic synthesis.
GUZ753
Graphic Abstract
A novel esterase gene from Geobacillus uzenensis DSMZ 13551 was ꢀrst cloned and expressed in Pichia pastoris KM71.
Esterase Est showed marked thermostability at high temperature and tolerance in methanol. Furthermore, the crude
GUZ753
fermentation broth of Est
was immobilized directly onto the epoxy resin, and immobilized Est
exhibited a 99%
GUZ753
GUZ753
conversion for cinnamyl acetate synthesis.
Keywords High thermostability · High organic solvents tolerance · Esterase · Transesteriꢀcation · Cinnamyl acetate
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s10562-020-03168-1) contains
supplementary material, which is available to authorized users.
Extended author information available on the last page of the article
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3

F. Dong et al.
1
Introduction
expressed P. pastoris. Furthermore, EstGUZ753 fermentation
broth was directly immobilized on epoxy-functionalized
supports via one-pot strategy. The immobilized EstGUZ753
exhibited a high conversion for cinnamyl acetate synthesis
towards cinnamyl alcohol with a high concentration. The
enzyme dosage, substrate concentrations, reaction time
and temperature were invested.
Cinnamyl acetate is one of the most important ꢁavor esters.
It has a wide application in the food, pharmaceuticals and
cosmetic industries due to its sweet, balsamic and ꢁoral
odor [1]. Moreover, cinnamyl acetate has many beneꢀcial
biological properties, such as antioxidant activity, which is
ascribed to its special structural features [2]. Traditionally,
cinnamyl acetate was synthesized by chemical synthesis of
an alcohol with an organic acid in the presence of strong
acids. But it was not low-cost input or environmentally
friendly approach. Chemical synthesis produced undesir-
able, non-speciꢀc, toxic byproducts and harsh conditions
to carry out the reactions [3, 4]. Thus, recently, enzyme
catalyzed transesteriꢀcation and esteriꢀcation are attrac-
tive alternatives to avoid these drawbacks, which are the
most economical approaches to reach the green products
with no harm to human health [5].
2
Materials and Methods
2.1 Materials, Strains and Plasmids
Epoxy resin Lx-105s was provided by Xian Lanxiao Tech-
nology New Materials Co., Ltd. (Xian, China). G. uzenen-
sis DSMZ13551 was preserved in our laboratory. P. pas-
toris strain KM71 was grown in YPD broth (2% dextrose,
2
% tryptone, 1% yeast extract) at 30 °C, or MD broth (2%
Current researchers are targeting lipase-mediated catal-
ysis reaction for ꢁavor esters [6–9]. Sharma et al. obtained
the maximum conversion was only 55% after 27 h with
porcine pancreatic lipase [10]. Yadav et al. used Novozym
−
5
dextrose, 4 × 10 % biotin, 1.34% YNB and 2% agar) or
−
5
BMMY broth (1% yeast extract, 1.34% YNB, 4 × 10 %
biotin, 2% dextrose, 100 mM potassium phosphate buꢂer,
0
.5% methanol) at 30 °C. The plasmid pPIC9K (Invitrogen,
4
35 to synthesize ethyl cinnamate with 60% conversion
Carlsbad, USA) was the vector used to construct the protein
expression. The substrate p-NP esters were purchased from
Sigma (St. Louis, MO, USA). DNA polymerase and restric-
tion enzymes were purchased from Takara (Kyoto, Japan).
Genomic DNA was isolated using genomic DNA isolation
kit (Vitagene, China).
in 2 h at 30 °C [11], Wolfson et al. obtained a high yield
of ethyl cinnamate at room temperature, whereas it took
9
6 h to obtain it [12]. The mostly studies are reported at a
low concentration or conversion of cinnamyl alcohol using
lipase. One reason may be that most enzymes exhibited an
undesired activity and stability in organic solvents, espe-
cially in polar organic solvents [13]. In order to meet the
industrial demands, many esterases have been engineered
to enhance their thermostability by site directed mutagen-
esis. However, site-directed mutations mostly leaded to a
decrease in activity. Thus the wild-type thermostable ester-
ase with high activity and organic solvents tolerance has
much commercial value. In addition, in order to increase
recyclability and economics of those excellent enzymes,
various esterases expressed in Escherichia coli (E. coli)
had been immobilized onto epoxy-functionalized supports,
which was very stable at neutral pH and wet conditions for
a long time [14–16]. However, the esterase expressed in
E. coli needed to be isolated from E. coli, which resulted
in an unsatisfactory decreased activity. To overcome this
major drawback, the enzyme expressed in Pichia pastoris
KM71 was fully released into the medium, which behaved
like a puriꢀed enzyme, thereby obviating the need for cost-
intensive isolation, puriꢀcation, and could be immobilized
directly on some supports via one-pot strategy.
2
.2 Construction and Expression of EstGUZ753 in P.
pastoris
Using the primers GUZ753-U/GUZ753-D (Table S1), the
PCR product was cloned into pPIC9K by digested with
EcoR I. To enable gene integration, the recombinant plasmid
was linearized with Sal I enzyme and puriꢀed. Then, it was
transformed into the expression strain P. pastoris KM71 by
the Gene Pulser Xcell™ Electroporation System (Bio-Rad,
Hercules, CA, USA). The conditions were 1.5 kV, 200 Ω,
2
5 μF, 5 ms followed by pulsing. The recombinant strains
were grown at 30 °C on MD agar plates until single yeast
colonies appeared. The single yeast colonies were identi-
ꢀ
ed with two pairs of primers, AOX sequencing primers,
and primers GUZ -U/GUZ -D (Table S1). Subsequently,
753
753
colonies with large hydrolyzing halos were selected and
grown in BMMY broth for 5–7 days, and the expression of
Est
was induced by methanol with 0.5% (v/v) every
GUZ753
In this study, a novel esterase gene, est
from
GUZ753
12 h.
Geobacillus uzenensis DSMZ 13551 was successfully
1
3

Facile One-Pot Immobilization of a Novel Esterase and Its Application in Cinnamyl Acetate…
2
.3 Biochemical Characterization of EstGUZ753
(https://swissmodel.expasy.org/SWISS-MODEL.html) was
adopted for predicting its three-dimensional structure. The
model was visualized by PDB Viewer and the ꢀgure was
constructed by Pymol.
Enzyme activities of Est
solution were assayed by
GUZ753
measuring the absorbance at 405 nm of liberated p-nitro-
phenol. One unit of enzymatic activity was deꢀned as
the amount of enzyme needed to release 1 μM p-nitro-
phenol per minute. We detected the substrate speciꢀcity
using the following substrates: pNP-acetate (C2), pNP-
butyrate (C4), pNP-caproate (C6), pNP-caprylate (C8),
pNP-decenoate (C10), pNP-laurate (C12), pNP-myristate
2.5 Immobilization of the EstGUZ753 on Epoxy Resin
(Lx‑105s) and Enzymatic Synthesis of Cinnamyl
Acetate
(
C14), and pNP-palmitate (C16). The enzyme assay was
The yeast cells were centrifuged at 7000×g for 10 min at
4 °C and the supernatants was passed through a 0.22 μm
ꢀlter to remove large particles of impurities. Then the solu-
tion was concentrated (5 folds) using a 50 mL Amicon Ultra
Centrifugal Filter Device with a molecular weight cut-oꢂ
of 10 kDa (Millipore, USA). The hydrolytic activity of
carried out under standard assay conditions.
The pH optimum for the enzyme activity was studied
over a range from pH 4–9 for 5 min (60 °C). The following
buꢂer systems were used: pH 4.0–5.0 with 100 mM cit-
ric acid-sodium citrate, pH 6.0–7.0 with 200 mM sodium
phosphate, pH 8.0 with 50 mM Tris–HCl, and 9.0 with
Est
was 410 U/mg according to the method of Wang
GUZ753
5
0 mM glycine–NaOH. The eꢂect of temperature was
assayed at 30–90 °C (pH 8.0). The thermostability of
Est was evaluated by assaying its residual activity
et al. [19]. Because a ꢀvefold concentrated supernatant of
Est still had a high purity, the supernatant of Est
GUZ753
GUZ753
was immobilized directly on supports and the procedure
was as follows: 10 g epoxy resin (Lx-105s) was equili-
brated with 100 mM phosphate buꢂer (pH 8.0) in a coni-
cal ꢁask (200 mL) and stirred at 30 °C for 1 h (200 rpm).
Subsequently, 1 g of wet supports and diꢂerent amounts of
GUZ753
after incubation at various temperatures (50–90 °C) for a
certain time. Aliquots were removed at various intervals
and the residual esterase activity was determined. Non-
incubated enzyme was determined as the 100% activity
value.
Est
suspension mixed with equal volume of phosphate
GUZ753
Half-life (t ) of Est in methanol (50%, 60%, 70%,
GUZ753
buꢂer were added to the 250 mL conical ꢁask, which was
stirred in a thermostatic air bath shaker at 30 °C for 7 h
(200 rpm). After immobilization, the suspension was ꢀltered
and the resins were washed with buꢂer until no protein was
1
/2
8
0% and 90%, v/v) was calculated by measuring the resid-
ual activities after diꢂerent incubation times as described
by Li et al. [17]. The Est
was mixed with diꢂerent
GUZ753
volumes of methanol solution to a ꢀnal volume of 1 mL
detected in the eluate. Finally, the immobilized Est
GUZ753
and incubated for periods at 4 °C (200 rpm). (For example,
was dried in a vacuum desiccator at room temperature for
a 60% methanol solution contained 100 μL Est
solu-
10 h and stored at 4 °C until use [20].
GUZ753
tion, 300 μL Est
buꢂer, and 600 μL methanol). Then
The cinnamyl acetate was synthesized through the trans-
GUZ753
2
0 μL aliquots was removed from the mixture and trans-
esteriꢀcation using the immobilized Est
as catalyst.
GUZ753
ferred into 960 μL esterase buꢂer with 20 μL substrates,
and residual activities were determined under standard
conditions. For other organic solvent tolerance of the
The reaction was carried out in a 50 mL ꢁat bottom glass
reactor. The entire reactor was immersed in an ultrasonic
thermostatic water bath, which was maintained at desired
temperature. The reaction parameters were as follows:
Est
, aliquots of the recombinant Est
solution
GUZ753
GUZ753
were respectively incubated in 20%, 50% and 90% (v/v)
organic solvents, such as toluene, cyclohexane, n-hexane,
n-heptane and isooctane, After 24 h treatment, the residual
activity was measured as described above.
2.5.1 Eꢀect of Reaction Solvent
2
+
2+
2+
2+
The eꢂects of metal ions (Mg , Ca , Mn , Zn ,
Diꢂerent organic solvents not only aꢂect the solubility of
hydrophobic substrates but also the activity of the enzyme.
Thus the medium toluene, cyclohexane, n-hexane, n-heptane
and isooctane were tested, respectively.
2
+
2+
2+
Co , Ni and Fe ) on the activity of Est
were
GUZ753
determined at ꢀnal concentrations of 1, 5 and 10 mM.
2.4 Sequence Analysis
2
.5.2 Eꢀect of Molar Ratio
The nucleotide sequences and predicted amino acid
sequences were analyzed by the BLAST program (NCBI).
ClustalX (version 1.83) was employed to analyze multi-
ple sequence alignments [18]. The SWISS MODEL server
The addition of cinnamyl alcohol (1 M) remained
unchanged. The molar ratio of alcohol and vinyl acetate was
1:1, 1:2, 1:3, 1:4 and 1:5.
1
3

F. Dong et al.
2
.5.3 Eꢀect of Reaction Temperature
The conversion of cinnamyl alcohol is represented
by the reduction rate of cinnamyl alcohol in the HPLC
chromatograms:
The eꢂect of reaction temperature was studied at 20 °C,
0 °C, 40 °C, 50 °C, 60 °C and 70 °C, and other conditions
stayed unchanged.
3
conversion ratio = 1 − A ∕A
t
(1)
0
where A is the area cinnamyl alcohol at time 0 and A is the
0
t
area cinnamyl alcohol at time t.
2.5.4 The Eꢀect of Water Content
Other conditions stayed unchanged. Diꢂerent water content
0.5%, 1%, 5%, 10%, 20% and 50% (w/w, with respect to
total substrates)) was analyzed.
3
Results and Discussion
(
3.1 Gene Cloning and Sequence Analysis
of EstGUZ753
2.5.5 Eꢀect of Reaction Time
The esterase gene est
from the G. uzenensis DSMZ
GUZ753
Other reaction conditions were under the optimum. The
eꢂect of reaction time was 2, 4, 5, 6 and 7 h.
1
3551 was ampliꢀed by primers GUZ753-U/GUZ753-D.
The nucleotide sequence had been deposited in the GenBank
under the accession number KY612321.
2.5.6 Eꢀect of Enzyme Dosag
Multiple sequence alignment (Fig. 1a) analysis indicated
Est
was a member of α/β hydrolase family. Est
GUZ753
GUZ753
Diꢂerent amounts of immobilized Est
was investi-
contained a single catalytic triad (Ser97, Asp196, and
GUZ753
gated under the optimal conditions (N-hexane as the solvent,
His226) and the consensus pentapeptide G-x-S-x-G, which
molar ratio = 1:4, temperature = 60 °C, water content was
further revealed that Est
was a typical member of
had not been previously
GUZ753
0
.5%). The enzyme dosage was as follows: 1%, 2%, 3%, 4%.
α/β hydrolase family. As Est
GUZ753
Fig. 1 Protein sequence and structure of EstGUZ753. a Sequence align-
3 (identity: 80%); Geobacillus sp. 12AMOR1 (identity: 67%); ꢀlled
triangle, the catalytic site (Ser97, Asp196, and His226); b The whole
three-dimensional structure. The three-dimensional structure of
ment of the conserved region of Est
and other alpha/beta fold
GUZ753
hydrolases from diꢂerent bacterial sources. The alignment strains and
identities were as follows: G. kaustophilus HTA426 (identity: 93%);
G. thermoleovorans (identity: 88%); Geobacillus sp. Y412MC61
Est
was predicted by the SWISS-MODEL server; c The cata-
GUZ753
lytic site of Est
structure. The catalytic triad: Ser97, Asp196,
GUZ753
(
identity: 86%); G. thermodenitrifcans (identity: 85%); G. genomosp.
and His226 residues are marked in yellow
1
3

Facile One-Pot Immobilization of a Novel Esterase and Its Application in Cinnamyl Acetate…
reported, the three-dimensional structure of EstGUZ753 was
generated with a speciꢀc template (PDB code: 4LHE (89.6%
identity)) (Fig. 1b), which was a lipase from Bacillus sp.
pH 9.0 (Fig. 3b). In addition, compared with free Est
,
GUZ753
immobilized Est
exhibited a wider pH rage (Fig. S1a).
GUZ753
The eꢂect of temperature on free EstGUZ753 activity was
(
strain H-257), and the protein structure was viewed by
shown in Fig. 3c. Free Est
displayed activity over a
GUZ753
PDB Viewer. The catalytic triad was observed in the regions
Fig. 1c).
broad range from 30 to 90 °C, especially between 60 and
80 °C, it retained at least 70% of its maximal activity.
(
Est
exhibited the maximal activity at 70 °C. In addi-
GUZ753
3.2 Heterologous Expression of EstGUZ753
tion, compared with free Est
, immobilized Est
GUZ753 GUZ753
exhibited a wider temperature rage (Fig. S1b).
The thermostability of free Est was shown in
The est
without signal peptide coding sequence was
GUZ753
GUZ753
inserted into the pPIC9K vector and then transformed into
the P. pastoris KM71. The single colony with a big and clear
hydrolysis halo in tributyrin plate was selected to grow in
BMMY broth (Fig. 2a). Upon the induction of methanol
after six days, the supernatant of the fermentation broth
was concentrated by ꢀvefold and analyzed by SDS-PAGE
Fig. 3d. The t of Est
at 70 °C was 28 h, further-
GUZ753
1
/2
more, over 50% of its maximal activity remained after
incubation at 80 °C for 2 h. Kumar et al. reported the best
mutant (LipR5) displayed the t was only 2 h at 65 °C.
1
/2
Acharya et al. obtained the mutants, t of TM with signiꢀ-
1
/2
cant increase in residual activity was only 1 h at 55 °C [21,
(
Fig. 2b). The results showed that the Est
had a high
GUZ753
22]. Compared with the above mutants, the results suggested
expression and the molecular mass was about 27 kDa, which
coincided with the actual value (27.48 kDa).
that wild-type Est
had a broad temperature range and
GUZ753
excellent thermostability in high temperature. In addition,
about the thermostability, a comprehensive comparison of
esterases from diꢂerent sources at various temperatures
was shown in Table 1. The comparison results indicated
3
.3 Enzymatic Properties of Free EstGUZ753
and Immobilized EstGUZ753
that recombinant Est
was more thermotolerant than
GUZ753
The esterase activities toward various p-NP esters were
examined at 60 °C and pH 8.0 using recombinant Est
that of most reported esterases. Furthermore, compared with
.
free Est
, the t of immobilized Est
at 70 °C
GUZ753
GUZ753
GUZ753
1/2
Results showed that C8 was the most favorable substrate,
increased up to 47 h, which revealed that immobilization
and the speciꢀc activity of Est
towards C8 was 205
strategy further increased the thermostability of Est
GUZ753
GUZ753
U/mL (410 U/mg) of protein (Fig. 3a).
(Fig. S2).
The eꢂect of pH on free Est
activity was deter-
To estimate comprehensively the potential use of
GUZ753
mined using C8 as substrate at various pH. The Est
Est
in organic synthesis reactions, both hydrolytic
GUZ753
GUZ753
exhibited higher activities over a pH range of 6.0–8.0,
and hydrophobic solvents were studied. Figure 4 showed
that hydrophilic solvents, methanol noticeably activated
among which the highest speciꢀc enzyme activity was at pH
8
.0. The activity of Est
decreased signiꢀcantly more
the activity of Est
. The highest activity of Est
GUZ753 GUZ753
GUZ753
than pH 8.0 and only about 10% of the maximal activity at
increased up to 1.55 and 1.32 folds than the initial activity
Fig. 2 Enzyme activity identiꢀcation and SDS-PAGE analysis of
protein expression. a Hydrolysis halos formed by diꢂerent clones in
tributyrin plates, which indicated that recombinant P. pastoris cells
were with enzyme activity; b The SDS-PAGE analysis of recombi-
nant protein Est
. Lanes: M, protein marker; S, supernatant of
GUZ753
the Est
fermentation broth
GUZ753
1
3

F. Dong et al.
Fig. 3 Enzymatic properties of free Est
. a Substrate speciꢀty of
bility of Est
at temperatures ranging from 50 to 90 °C. Thermal
GUZ753
GUZ753
Est
using pNP esters with diꢂerent acyl chain lengths (C2, C4,
denaturing half-lives of the recombinant Est
were determined
GUZ753
GUZ753
C6, C8, C10, C12, C14, and C16); b Eꢂect of pH on Est
activ-
by measuring residual activities after the enzyme was treated at 50,
60, 70, 80 and 90 °C for a period of time, each value represented the
mean and SD of triplicate experiments
GUZ753
ity in diꢂerent buꢂers using C8 as substrate; c Optimal temperature
of Est at temperatures ranging from 30 to 90 °C; d Thermosta-
GUZ753
Table 1 Comparison of the thermostability of esterases from diꢂerent sources at diꢂerent temperatures
Enzyme Source Topt (°C) Thermal stability at various temperatures
0 °C 60 °C 70 °C 80 °C
61%, 52 h 54%, 28 h 49%, 28 h 55%, 2 h
5
90 °C
EstGUZ753 G. uzenensis DSMZ 13551 (this study)
70
20%, 15 min
–
Unstable
–
–
ThLip2
Est7
Thermoanaerobacterium thermosaccharolyticum [23] 75
–
–
–
–
–
50%, 2 h
Unstable
–
–
Stenotrophomonas maltophilia OUC_Est10 [24]
60
50
80
65
45
60
45%, 48 h
64%, 48 h
Unstable
–
GthFAE Geobacillus thermoglucosidasius DSM 2542T [25]
Aaeo1
Aquiꢀex aeolicus VF5 [26]
Bacillus gelatini KACC 12197 [27]
Bacillus licheniꢀormis [28]
Bacillus circulans [29]
–
–
–
–
75%, 2 h
–
70%, 1 h
–
–
Est-gela
Esterase
Esterase
59%, 1 h
20%, 1 h
100%, 1 h 80%, 1 h
0%, 30 min Unstable
Unstable
Unstable
0%, 1 h
in 50% and 60% methanol after 96 h, and in 70%, 80% and
eꢂect on Est
activity, the t values of Est
in
GUZ753
GUZ753
1/2
9
0% methanol, the highest activity of Est
respec-
50%, 60%, 70%, 80% and 90% methanol were 34,560,
23,040, 21,600, 20,400 and 20,160 min, respectively. For
the high methanol-tolerant lipase, Dror et al. had the similar
studies on lipase T6, but t values of the best variants (T6:
GUZ753
tively increased up to 1.26, 1.23 and 1.16 folds than the
initial activity after 72 h. Increasing the incubation time, the
methanol with diꢂerent concentrations displayed an inhibitor
1
/2
1
3

Facile One-Pot Immobilization of a Novel Esterase and Its Application in Cinnamyl Acetate…
Table 3 Eꢂect of hydrophobic organic solvents on EstGUZ753 activity
Organic solvents
logPa
Residual activity (%)b
2
0%
50%
90%
Control
Toluene
Cyclohexane
N-hexane
N-heptane
Isooctane
–
100± 1.0
62± 1.8
75± 1.6
80.8± 3.8
71.7± 2.9
55.1± 1.7
100± 0.9
78± 1.4
82± 2.5
91.4± 2.6
88.8± 1.5
71.1± 2.7
100± 1.2
85± 1.8
90± 1.2
98.7± 2.7
95.5± 2.7
90.1± 1.5
2.5
3.2
3.5
4.0
4.5
a
logP value is the partition coeꢃcient of an organic solvent between
water and n-octanol phases
b
After incubating Est
for 24 h in diꢂerent organic solvents, the
GUZ753
residual enzymatic activity was measured in 50 mM Tris–HCl buꢂer
(
pH 8.0) at 70 °C using C8 as the test substrate. The enzyme sample
incubated in buꢂer only was set as 100%. The presented results were
the average of three repeated experiments with SD of± 5%
Fig. 4 The relative activity proꢀles of free EstGUZ753 after diꢂerent
incubation time in 50%, 60%, 70%, 80% and 90% methanol (v/v).
The activity of Est
under the standard activity assay conditions
GUZ753
(
50 mM Tris–HCl (pH 8) buꢂer, C8 as substrate) was measured after
N-hexane after 24 h. Generally, many transesteriꢀcation and
ester synthesis reactions are in hydrophobic organic solvents
medium, such as n-hexane, therefore, the remarkable stabil-
diꢂerent incubation times in diꢂerent alcohol concentrations. The
residual activity was calculated by comparing the activity of each
Est
before and after incubation in alcohol. The activity in the
GUZ753
Tris–HCl buꢂer without methanol at 70 °C was set as 100%. Each
value represented mean and SD of triplicate experiments
ity of Est
makes it an attractive candidate for transes-
GUZ753
teriꢀcation reactions [33, 34].
As shown in Table 4, the activity of Est
was stimu-
GUZ753
2
+
2+
H86Y/A269T) were respectively 2429, 1777 and 258 min
in 50%, 60% and 70% methanol (Table 2) [30]. There are no
reports of esterases showing so high tolerance in 80% and
lated with the addition of Mg and Ca at 1 mM, 5 mM
and10 mM, and the activity of Est
increased up to
GUZ753
2
+
1.25-fold by adding 1 mM Mg . Whereas the activity of
2+
2+
2+
9
0% methanol. For the activation phenomenon of metha-
Est
was inhibited by the addition of Mn , Zn , Co ,
GUZ753
2+
2+
2+
nol with suitable concentration for a period of time, similar
eꢂects of methanol on several lipases had been reported,
however, some previous studies were in a low concentration
of methanol, Cao et al. reported that lipaseTt from Thermus
thermophilus HB8 exhibited an activation phenomenon in
methanol, however, only in 45% methanol [31]. Yamashiro
et al. also reported that a cold-adapted and organic solvent-
tolerant lipase from Pseudomonas sp. Strain YY31 was very
active and stable in methanol, whereas it was only 1.02 and
Ni and Fe at diꢂerent concentrations. Furthermore, Zn
2
+
was an important inhibitor, the addition of Zn (10 mM),
Est nearly lost all the activity.
GUZ753
3.4 Immobilization of EstGUZ753 and Its Application
in the Synthesis of Cinnamyl Acetate
The immobilization process is outlined in Scheme 1a. The
eꢂects of the initial dosage of Est
, immobilization
GUZ753
1
.10 folds than the initial activity in only 25% and 50%
time on the loading capacity, and the relative esterase activ-
methanol [32]. There are no reports of activation in so high
ity were investigated (not shown). The ꢀnal results showed
concentration of methanol.
that the highest loading of Est
was 55.5 mg/g when
GUZ753
As shown in Table 3, Est
was also stable in hydro-
90 mg Est
and 1 g wet supports were initially added in
GUZ753
GUZ753
phobic organic solvents (log P ≥ 2.0) from 20 to 90%. In
0% hydrophobic organic solvents, the Est all retained
the system, and the immobilized yield was 61.7% (Table S2).
To obtain a higher conversion at a high substrate concen-
9
GUZ753
above 85% of its original activity after incubation for 24 h.
tration, the immobilized Est
-mediated transesteriꢀca-
GUZ753
Furthermore, Est retained over 98% of activity in
tion with cinnamyl alcohol and vinyl acetate was carried
GUZ753
Table 2 Comparison of the
Items
t1/2 (min)
50% methanol 60% methanol 70% methanol 80% methanol 90% methanol
half-life between T6 and
Est
GUZ753
EstGUZ753
lipase T6
T6: H86Y/A269T 2429
34,560
347
23,040
173
1777
21,600
4
258
20,400
–
–
20,160
–
–
1
3

F. Dong et al.
Table 4 Eꢂect of various metal ions on EstGUZ753
Ion
Residual activity (%)a
(1 mM)
(5 mM)
(10 mM)
None
100± 0
100± 0
100± 0
2+
Mg
125.3± 2.1
95.7± 1.8
89.7± 3.2
59.1± 3.7
97.5± 2.9
96.1± 2.3
86.5± 3.4
105.1± 1.9
105.7± 2.1
81.1± 3.6
23.5± 2.7
89.3± 3.5
92.9± 2.4
64.9± 2.6
103.3± 1.9
103.2± 2.2
48.8± 2.5
ND
80.4± 2.4
88.4± 2.0
41.4± 1.7
2
+
Ca
Mn
Zn
2
+
2
+
2
+
Co
2
+
Ni
Fe
2+
a
The esterase was pre-incubated with the metal ions for 1 h at 4 °C
before measuring the residual activity. The presented results were the
average of three repeated experiments with SD of± 5%. The activity
toward C8 without any metal ions was taken as 100%
ND not detectable
Fig. 5 Eꢂect of hydrophobic organic solvents on cinnamyl acetate
synthesis. The maximal activity was deꢀned as 100% and the relative
activity is shown as a percentage of maximal activity
out in n-hexane (Scheme 1b) and products were analyzed
by thin-layer chromatography (TLC) and High Perfor-
mance Liquid Chromatography (HPLC) (Scheme 1c). For
cinnamyl acetate synthesis, Fig. S3 showed that the low
concentration (< 1 M) of cinnamyl alcohol could be fully
converted to cinnamyl acetate in less than 5 h, however,
when the concentration increased up to 1 M, only 85% of
cinnamyl alcohol was converted to cinnamyl acetate, so 1 M
Scheme 1 Immobilized Est
-mediated transesteriꢀcation of
tion of cinnamyl acetate through transesteriꢀcation with immobilized
GUZ753
cinnamyl alcohol and vinyl acetate in n-hexane. a The immobiliza-
tion procedure of Est on Epoxy Resin (Lx-105s); b Prepara-
Est
; c TLC and HPLC analysis of cinnamyl alcohol and cin-
GUZ753
namyl acetate
GUZ753
1
3

Facile One-Pot Immobilization of a Novel Esterase and Its Application in Cinnamyl Acetate…
of cinnamyl alcohol in this experiment was used to be opti-
mized. Because reaction solvent not only aꢂected the con-
version but also aꢂected the enzyme activity by changing the
three-dimensional conformation of proteins [7]. The eꢂects
of toluene, cyclohexane, n-hexane, n-heptane and isooctane
were shown in Fig. 5, cinnamyl alcohol had a high conver-
sion in diꢂerent hydrophobic organic solvents, and the high-
est conversion was obtained in n-hexane. The reason may be
that hydrophobic organic solvents do not strip the critical
water from the enzyme surface, so esterase will retain a high
enzyme retention activity in n-hexane [35, 36].
The eꢂect of temperature was shown in Fig. 6b. The
results showed that the conversion of cinnamyl alcohol
increased from 30 to 95% in 6 h with the temperature from
20 to 60 °C, however, an obvious decrease in the conver-
sion (79%) occurred at 70 °C. This is possible that the
higher temperature had a negative impact on the three-
dimensional structure of the enzyme, which reduced
the enzyme activity. Therefore, 60 ºC was suitable for
transesteriꢀcation.
Water content in organic bio-synthesis was essential for
enzyme ꢁexibility and catalytic activity [37]. The eꢂect of
water content on conversion was examined from 0.5% to
50% (w/w). As shown in Fig. 6c, under the addition of 0.5%
water, the conversion of cinnamyl alcohol had an obvious
increase compared to without water (Fig. 6b) at 60 °C for
6 h. But when the water content increased up to 50%, only
The eꢂect of the molar ratio of the two substrates was
investigated by TLC and HPLC under the concentration of
cinnamyl alcohol at 1 M for 6 h (60 °C) (Fig. 6a). The con-
version of cinnamyl alcohol was increased with the molar
ratio from 1:1 to 1:4, and the highest conversion was at 1:4.
1
0% conversion was obtained. This result was consistent
Fig. 6 Optimization of cinnamyl acetate synthesis by immobilized
of water content on conversion of cinnamyl alcohol with immobilized
Est ; d Eꢂect of time on conversion of cinnamyl alcohol with
Est
in n-hexane system via TLC and HPLC. a Eꢂect of molar
GUZ753
GUZ753
ratio on conversion of cinnamyl alcohol with immobilized Est
;
immobilized Est
. In the TLC, the lower dots represented the
GUZ753
GUZ753
b Eꢂect of temperature on conversion of cinnamyl alcohol; c Eꢂect
substrate content, and the upper dots represented the product content
1
3

F. Dong et al.
Fig. 7 Optimization of biocatalyst dosage and reusability of the
immobilized Est a Time course of cinnamyl acetate synthesis
tions: vinyl acetate as acyl donor; n-hexane as solvent; molar ratio,
1:4; temperature, 60 °C; b Reusability of the immobilized Est
GUZ753.
GUZ753
with diꢂerent amounts of immobilized Est
. Reaction condi-
GUZ753
with some previous studies that synthetic activity of most
lipases was optimal at relatively low water content in organic
systems (typically below 1% (w/w)) [38]. This was due to a
larger amount of water in organic medium would reduce the
solubility of the substrates and products. Therefore, the syn-
thesis of cinnamyl acetate should be carried out in a micro-
water system [39–41].
limited exposed surface area of catalyst with reactants and
accordingly decreased mass transfer [42].
The reusability of the catalyst was another important
factor in industrial applications. As can be seen in Fig. 7b,
immobilized Est
had a good reusability, which could
GUZ753
retain a high activity (80%) after 10 recycles. The decline
in activity could be due to partial enzyme leakage from the
carrier. After the reaction, the catalyst can be separated
and reused in subsequent experiments [43].
The reaction time was another key factor for the conver-
sion. The eꢂect of reaction time was analyzed under optimal
conditions (n-hexane as solvent, molar ratio=1:4, tempera-
ture=60 °C). As shown in Fig. 6d, the conversion presented
an upward trend from 0 to 6 h, and a conversion of 99% was
obtained at 6 h. So 6 h was the optimal time. In addition,
a comparison of conversion at diꢂerent concentrations of
cinnamyl alcohols was shown in Table S3. The comparison
4 Conclusions
In this research, the novel esterase gene est
from G.
GUZ753
results showed that immobilized Est
could make about
uzenensis DSMZ 13551 had a highly expression in P. pas-
GUZ753
tenfold concentrations of cinnamyl alcohol fully converted
toris KM71 and retained a high activity. Est exhib-
GUZ753
to cinnamyl acetate in only 6 h at 60 °C. The result revealed
ited an excellent thermostability. The t at 70 °C was
1
/2
that immobilized Est
higher temperature.
had a high catalytic eꢃciency at
28 h. Est
also had good tolerance towards metha-
GUZ753
GUZ753
nol compared to other thermophilic esterase. The t of
1
/2
In the case of industrial production, the increase
of enzyme dosage will accelerate the reaction rate and
enhance the conversion, but the enzyme dosage deter-
mined the cost of the production process. Figure 7a
showed that the conversion of cinnamyl alcohol increased
with the enzyme dosage from 1 to 3%, and 99% conversion
was obtained when the enzyme dosage was 3%. Increas-
ing the enzyme dosage up to 4%, the reaction rate was
accelerated at the initial time, but the maximum conver-
sion (99%) almost as the same time as that of 3% enzyme
dosage reached. This phenomenon was due to excessive
enzymes would aggregate together, which resulted in
Est
in 90% methanol was 20,160 min. Compared
GUZ753
with free Est
, the immobilized Est
showed
GUZ753
GUZ753
a wider temperature, pH rage and better thermostability.
Furthermore, the immobilized Est
was an eꢃcient
GUZ753
catalyst for cinnamyl ester synthesis. A conversion of 99%
was achieved in 6 h, and the high concentration of cinna-
myl alcohol (1 M) is reported for the ꢀrst time. The immo-
bilized Est
also showed a good reusability. On the
GUZ753
basis of the excellent characteristics of Est
reported
GUZ753
in this study, Est
will be a promising catalyst for
GUZ753
other organic synthesis.
1
3

Facile One-Pot Immobilization of a Novel Esterase and Its Application in Cinnamyl Acetate…
Acknowledgements This research was ꢀnancially supported by the
National Natural Science Foundation of China (No. C31570795),
the Shanghai outstanding technical leaders plan 19XD1431800, the
National Natural Science Foundation of China (Grant Nos. 81830052,
immobilization on epoxy-functionalized supports: modulation
of stability and selectivity. J Mol Catal B: Enzym 115:128–134
16. Chen B, Hu J, Miller EM, Xie W, Cai M, Gross RA (2008)
Candida antarctica lipase B chemically immobilized on epoxy-
activated micro-and nanobeads: catalysts for polyester synthe-
sis. Biomacromol 9(2):463–471
8
1530053) and Shanghai Key Laboratory of Molecular Imaging
(
18DZ2260400).
1
7. Li C, Tan T, Zhang H, Feng W (2010) Analysis of the confor-
mational stability and activity of Candida antarctica lipase B in
organic solvents insight from molecular dynamics and quantum
mechanics/simulations. J Biol Chem 285(37):28434–28441
8. Gouet P, Courcelle E, Stuart DI, Métoz F (1999) ESPript: analy-
sis of multiple sequence alignments in PostScript. Bioinformat-
ics 15(4):305–308
Compliance with Ethical Standards
Conflict of interest The authors declare no conꢁict of interest.
1
1
2
9. Wang W-f, Li T, Qin X-l, Ning Z-x, Yang B, Wang Y-h (2012)
Production of lipase SMG1 and its application in synthesizing
diacylglyecrol. J Mol Catal B: Enzym 77:87–91
References
1
.
Schrader J, Etschmann M, Sell D, Hilmer J-M, Rabenhorst J
0. Fy D, Tang Xd, Yang Xh, Lin L, Dn He, Wei W, Dz W (2019)
Immobilization of a novel ESTBAS esterase from Bacillus alti-
tudinis onto an epoxy resin: characterization and regioselective
synthesis of chloramphenicol palmitate. Catalysts 9(7):620
1. Kumar R, Singh R, Kaur J (2014) Combinatorial reshaping of
a lipase structure for thermostability: additive role of surface
stabilizing single point mutations. Biochem Biophys Res Com-
mun 447(4):626–632
(
2004) Applied biocatalysis for the synthesis of natural ꢁavour
compounds–current industrial processes and future prospects.
Biotechnol Lett 26(6):463–472
2
3
4
.
.
.
Nenadis N, Wang L-F, Tsimidou M, Zhang H-Y (2004) Estima-
tion of scavenging activity of phenolic compounds using the
ABTS•+ assay. J Agric Food Chem 52(15):4669–4674
Devulapelli VG, Weng H-S (2009) Synthesis of cinnamyl ace-
tate by solid–liquid phase transfer catalysis: Kinetic study with
a batch reactor. Catal Commun 10(13):1638–1642
2
2
2
2. Acharya P, Rajakumara E, Sankaranarayanan R, Rao NM (2004)
Structural basis of selection and thermostability of laboratory
evolved Bacillus subtilis lipase. J Mol Biol 341(5):1271–1281
3. Li W, Shi H, Ding H, Wang L, Zhang Y, Li X, Wang F (2018)
Characterization of two novel thermostable esterases from Ther-
moanaerobacterium thermosaccharolyticum protein. Expression
Purif 152:64–70
Bokade VV, Yadav GD (2009) Transesteriꢀcation of edible
and nonedible vegetable oils with alcohols over heteropo-
lyacids supported on acid-treated clay. Ind Eng Chem Res
4
8(21):9408–9415
5
6
7
8
9
.
.
.
.
.
Wang Y, Zhang D-H, Chen N, Zhi G-Y (2015) Synthesis of
benzyl cinnamate by enzymatic esteriꢀcation of cinnamic acid.
Bioresour Technol 198:256–261
2
2
4. Gao X, Mao X, Lu P, Secundo F, Xue C, Sun J (2019) Cloning,
expression, and characterization of a novel thermostable and
alkaline-stable esterase from Stenotrophomonas maltophilia
OUC_Est10 catalytically active in organic solvents. Catalysts
Geng B, Wang M, Qi W, Su R, He Z (2012) Cinnamyl acetate
synthesis by lipase-catalyzed transesteriꢀcation in a solvent-free
system. Biotechnol Appl Biochem 59(4):270–275
9
(5):401
Yadav GD, Devendran S (2012) Lipase catalyzed synthesis of
cinnamyl acetate via transesteriꢀcation in non-aqueous medium.
Process Biochem 47(3):496–502
5. Sal FA, Colak DN, Guler HI, Canakci S, Belduz AO (2019)
Biochemical characterization of a novel thermostable feruloyl
esterase from Geobacillus thermoglucosidasius DSM 2542 T.
Mol Biol Rep 46(4):4385–4395
Wu Z, Qi W, Wang M, Su R, He Z (2014) Lipase immobilized
on novel ceramic supporter with Ni activation for eꢃcient cin-
namyl acetate synthesis. J Mol Catal B: Enzym 110:32–38
Kim Y-O, Park I-S, Kim H-K, Nam B-H, Kong HJ, Kim W-J,
Kim D-G, Kim B-S, Jee Y-J, Song J-H (2013) Shewanella sp.
Ke75 esterase with speciꢀcity for p-nitorphenyl butyrate: gene
cloning and characterization. J Korean Soc Appl Biol Chem
2
2
2
6. Guo Y-Y, Yu X-W, Xu Y (2016) Cloning, expression and char-
acterization of two thermostable esterases from Aquiꢀex aeolicus
VF5. J Mol Catal B: Enzym 133:S220–S229
7. Kim J, Deng L, Hong E, Ryu Y (2015) Cloning and characteri-
zation of a novel thermostable esterase from Bacillus gelatini
KACC 12197. Protein Expr Purif 116:90–97
5
6(1):55–62
8. Alvarez-Macarie E, Augier-Magro V, Baratti J (1999) Charac-
terization of a thermostable esterase activity from the moderate
thermophile Bacillus licheniꢀormis. Biosci Biotechnol Biochem
1
0. Sharma CK, Chauhan GS, Kanwar SS (2011) Synthesis of
medically important ethyl cinnamate ester by porcine pancre-
atic lipase immobilized on poly (AAc-co-HPMA-cl-EGDMA)
hydrogel. J Appl Polym Sci 121(5):2674–2679
6
3(11):1865–1870
2
3
3
3
9. Kademi A, Aı̈t-Abdelkader N, Fakhreddine L, Baratti JC (2000)
1
1
1
1. Yadav GD, Dhoot SB (2009) Immobilized lipase-catalysed syn-
thesis of cinnamyl laurate in non-aqueous media. J Mol Catal
B: Enzym 57(1–4):34–39
Characterization of a new thermostable esterase from the mod-
erate thermophilic bacterium Bacillus circulans. J Mol Catal B:
Enzym 10(4):395–401
2. Wolfson A, Dlugy C, Karanet A, Tavor D (2012) A sustainable
one-pot synthesis of cinnamyl acetate in triacetin. Tetrahedron
Lett 53(34):4565–4567
0. Dror A, Shemesh E, Dayan N, Fishman A (2014) Protein engi-
neering by random mutagenesis and structure-guided consensus
of Geobacillus stearothermophilus lipase T6 for enhanced sta-
bility in methanol. Appl Environ Microbiol 80(4):1515–1527
1. Cao H, Nie K, Xu H, Xiong X, Krastev R, Wang F, Tan T, Liu
L (2016) Insight into the mechanism behind the activation phe-
nomenon of lipase from Thermus thermophilus HB8 in polar
organic solvents. J Mol Catal B: Enzym 133:S400–S409
2. Yamashiro Y, Sakatoku A, Tanaka D, Nakamura S (2013) A
cold-adapted and organic solvent-tolerant lipase from a psychro-
trophic bacterium Pseudomonas sp. strain YY31: identiꢀcation,
3. Siddiqui KS, Cavicchioli R (2005) Improved thermal stability
and activity in the cold-adapted lipase B from Candida antarc-
tica following chemical modiꢀcation with oxidized polysac-
charides. Extremophiles 9(6):471–476
1
1
4. Trytek M, Jędrzejewski K, Fiedurek J (2015) Bioconversion of
α-pinene by a novel cold-adapted fungus Chrysosporium pan-
norum. J Ind Microbiol Biotechnol 42(2):181–188
5. Ashjari M, Mohammadi M, Badri R (2015) Chemical ami-
nation of Rhizopus oryzae lipase for multipoint covalent
1
3

F. Dong et al.
cloning, and characterization. Appl Biochem Biotechnol
71(4):989–1000
3. Gao W, Wu K, Chen L, Fan H, Zhao Z, Gao B, Wang H, Wei D
39. Cai X-h, Wang W, Lin L, He D-n, Shen Y, Wei W, Wei D-z (2017)
Cinnamyl esters synthesis by lipase-catalyzed transesteriꢀcation
in a non-aqueous system. Catal Lett 147(4):946–952
40. Lv DS, Xue XT, Wang N, Wu Q, Lin XF (2004) Enzyme cata-
lyzed synthesis of some vinyl drug esters in organic medium. Prep
Biochem Biotechnol 34(1):97–107
1
3
3
3
3
(
2016) A novel esterase from a marine mud metagenomic library
for biocatalytic synthesis of short-chain ꢁavor esters. Microb Cell
Fact 15(1):41
4. Jin P, Pei X, Du P, Yin X, Xiong X, Wu H, Zhou X, Wang Q
41. Cao X, Mangas-Sánchez J, Feng F, Adlercreutz P (2016) Acyl
migration in enzymatic interesteriꢀcation of triacylglycerols:
eꢂects of lipases from Thermomyces lanuginosus and Rhizopus
oryzae, support material, and water activity. Eur J Lipid Sci Tech-
nol 118(10):1579–1587
(
2012) Overexpression and characterization of a new organic
solvent-tolerant esterase derived from soil metagenomic DNA.
Bioresour Technol 116:234–240
5. Duarte SH, del Peso Hernández GL, Canet A, Benaiges MD,
Maugeri F, Valero F (2015) Enzymatic biodiesel synthesis from
yeast oil using immobilized recombinant Rhizopus oryzae lipase.
Bioresour Technol 183:175–180
42. Puskas JE, Chiang CK, Sen MY (2011) Green cationic polym-
erizations and polymer functionalization for biotechnology.
Green Polym Methods. https://doi.org/10.1002/9783527636
167#page=316
6. Nasaruddin RR, Alam MZ, Jami MS (2013) Enzymatic biodiesel
production from sludge palm oil (SPO) using locally produced
Candida cylindracea lipase. Afr J Biotechnol 12(31):4966–4974
7. Klibanov AM (2001) Improving enzymes by using them in
organic solvents. Nature 409(6817):241–246
43. Hou C, Zhu H, Li Y, Li Y, Wang X, Zhu W, Zhou R (2015) Facile
synthesis of oxidic PEG-modiꢀed magnetic polydopamine nano-
spheres for Candida rugosa lipase immobilization. Appl Micro-
biol Biotechnol 99(3):1249–1259
3
3
8. Brault G, Shareck F, Hurtubise Y, Lépine F, Doucet N (2014)
Short-chain ꢁavor ester synthesis in organic media by an E. coli
whole-cell biocatalyst expressing a newly characterized het-
erologous lipase. PLoS ONE. https://doi.org/10.1371/journ
al.pone.0091872
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jurisdictional claims in published maps and institutional aꢃliations.
Aꢀliations
Fengying Dong1 · Lin Lin · Wei Wei · Dongzhi Wei
2
1
1
2
*
*
Wei Wei
Research Laboratory for Functional Nanomaterial,
weiwei@ecust.edu.cn
National Engineering Research Center for Nanotechnology,
Shanghai 200241, People’s Republic of China
Dongzhi Wei
dzhwei@ecust.edu.cn
1
State Key Laboratory of Bioreactor Engineering, Newworld
Institute of Biotechnology, East China University of Science
and Technology, 130 Meilong Road, Shanghai 200237,
People’s Republic of China
1
3