Efficient Enzymatic Preparation of Flavor Esters in Water

Article abstract of DOI:10.1021/acs.jafc.9b01790

A straightforward biocatalytic method for the enzymatic preparation of different flavor esters starting from primary alcohols (e.g., isoamyl, n-hexyl, geranyl, cinnamyl, 2-phenethyl, and benzyl alcohols) and naturally available ethyl esters (e.g., formate, acetate, propionate, and butyrate) was developed. The biotransformations are catalyzed by an acyltransferase from Mycobacterium smegmatis (MsAcT) and proceeded with excellent yields (80-97%) and short reaction times (30-120 min), even when high substrate concentrations (up to 0.5 M) were used. This enzymatic strategy represents an efficient alternative to the application of lipases in organic solvents and a significant improvement compared with already known methods in terms of reduced use of organic solvents, paving the way to sustainable and efficient preparation of natural flavoring agents.

Full text of DOI:10.1021/acs.jafc.9b01790

Subscriber access provided by University of Rochester | River Campus & Miner Libraries
Biotechnology and Biological Transformations
Efficient enzymatic preparation of flavor esters in water
Igor Chiarelli Perdomo, Stefania Gianolio, Andrea Pinto, Diego Romano,
Martina Letizia Contente, Francesca Paradisi, and Francesco Molinari
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01790 • Publication Date (Web): 17 May 2019
Downloaded from http://pubs.acs.org on May 17, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the
course of their duties.

Page 1 of 15
Journal of Agricultural and Food Chemistry
1
Efficient enzymatic preparation of flavor esters in water
a
a
a
a
2
3
Igor Chiarelli Perdomo, Stefania Gianolio, Andrea Pinto, Diego Romano, Martina
Letizia Contente,a, b* Francesca Paradisi, Francesco Molinaria*
b
a
4
5
Department of Food, Environmental and Nutritional Sciences (DeFENS), University of Milan, Via
Mangiagalli 25, 20133 Milan (Italy)
b
6
7
School of Chemistry, University of Nottingham, University Park, Nottingham, NG7 2RD, UK
8
9
KEYWORDS: Flavor esters, enzymatic acylation, biocatalysis, acyltransferase, Mycobacterium smegmatis
(MsAcT)
10
11
12
13
14
15
16
17
18
ABSTRACT: A straightforward biocatalytic method for the enzymatic preparation of different flavor esters starting
from primary alcohols (e.g., isoamyl, n-hexyl, geranyl, cinnamyl, 2-phenethyl, and benzyl alcohols) and naturally
available ethyl esters (e.g., formate, acetate, propionate, and butyrate) was developed. The biotransformations
are catalyzed by an acyltransferase from Mycobacterium smegmatis (MsAcT) and proceeded with excellent yields
(80-97%) and short reaction times (30-120 minutes), even when high substrate concentrations (up to 0.5 M) were
used. This enzymatic strategy represents an efficient alternative to the application of lipases in organic solvents
and a significant improvement compared to already known methods in terms of reduced use of organic solvents,
paving the way to a sustainable and efficient preparation of natural flavoring agents.
19
20
21
INTRODUCTION
Many esters are components of natural flavors and are used as flavor and fragrance in food, drinks and
1
cosmetics. Their preparation starting from natural substrates and using bioprocesses (e.g., fermentation or
1
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 2 of 15
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
enzymatic reactions) is appealing, since the final product can be labelled and commercialized in EU and USA as
natural.2, 3 Therefore, new biotechnological approaches for obtaining flavors are highly demanded as long as they
are efficient and sustainable.4, 5 Many flavor/fragrance esters can be enzymatically obtained using lipases that
catalyze esterification, transesterification or interesterification reactions in media (e.g., organic solvents)
characterized by low water activity.6-8 The presence of water is critical for the equilibrium of lipase-catalyzed
reactions in organic media, strongly limiting the overall yields; in fact water may compete as a nucleophile with
the alcohol in the attack of the acyl-enzyme intermediate, thus favoring hydrolysis over transesterification, 9-11
and for this reason water must be avoided or removed during the reaction for achieving high yields. 12,13 Flavor
ester biosynthesis in an aqueous system composed of coconut cream and fusel oil was achieved with limited
conversion (14.25 mg/g, based on cream weight), after optimization of the process catalyzed by the lipase
Palatase.14 The potential of lipases for flavor ester production has been exploited also using immobilized
enzymes15 and whole microbial cells;16-18 these systems have been especially employed for setting up robust
processes and/or facilitating product recovery. Acetate esters are amongst the most valuable flavor esters and
few of them has been produced in hydrophobic solvents (e.g., n-pentane, n-hexane or n-heptane) using different
immobilized lipases;19 in a large scale production of flavor esters, Novozym 435 (35 g/L) was able to give yields
ranging between 91 and 95% after 24 h starting from an alcohol concentration of 1.0 M and an excess of acetic
acid.12 Alternatively, the use of Candida rugosa lipase, immobilized in calcium alginate gel, allowed for a
productivity of 1600-2200 mol/h/g of biocatalyst for the preparation of isoamyl and butyryl acetate.20
An interesting enzymatic alternative for preparing esters is the use of the acyltransferase from Mycobacterium
smegmatis (MsAcT).21 MsAcT is characterized by a hydrophobic tunnel leading to the active site, where water
access is disfavored;21,22 moreover, MsAcT is functionally active as a closely aggregated octamer in aqueous
21
solution. This overall architecture means that MsAcT is able to favor transesterification reactions with respect to
hydrolysis even in water, unlike lipases that catalyze transesterification only under conditions of low water
activity.22 For these structural reasons, MsAcT has been used for catalyzing transesterification reactions of primary
and secondary alcohols with ethyl acetate in aqueous buffers, often with good enantioselectivity.22-24 The ratio
between the catalytic rate constants of the hydrolysis and synthesis of benzyl acetate were compared, showing
that MsAcT is indeed an effective acetyltransferase rather than a hydrolytic enzyme.24 At reasonably high
concentrations of the ester, the reaction mixtures of these biotransformations are actually two-liquid phase
2
ACS Paragon Plus Environment

Page 3 of 15
Journal of Agricultural and Food Chemistry
50
51
52
53
54
55
56
57
systems composed of the hydrophobic ester and water, and the reaction occurs in the aqueous phase. The
peculiar features of this enzyme makes it also suitable for catalyzing amide synthesis in aqueous media.25, 26
In this work, we have explored the potential of MsAcT for flavor ester preparation; the enzyme was able to work
in two-liquid phase systems composed of sparingly water-soluble esters and water, accomplishing the reaction
in the aqueous phase. Several (24) different flavor esters derived from transesterification of primary alcohols (e.g.,
isoamyl, n-hexyl, geranyl, benzyl, 2-phenylethyl, and cinnamyl) with ethyl esters bearing short-chain acyl groups
(e.g., formyl, acetyl, propionyl, and butyryl) were prepared in excellent yields and short reaction times loading
high concentration of substrates (up to 500 mM), making the process interesting at industrial level.
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
MATERIALS AND METHODS
Chemicals. All reagents and solvents were obtained from commercial suppliers and were used without further
purification.
Preparation of the acetyltransferase from Mycobacterium smegmatis (MsAcT). E. coli BL21 star (DE3) was used as host
for the production of the recombinant protein; cloning was carried out as described before, starting from a synthetic
gene encoding for MsAcT (GenBank accession: ABK70783) from Mycobacterium smegmatis str. MC2 155.26 Recombinant
E. coli was firstly grown at 37 °C in LB (Luria-Bertani) liquid medium in the presence of 25 μg/mL kanamycin for 16 h;
this starting culture was used to inoculate the cultivation medium (Terrific Broth: 12 g/L bacto-tryptone, 24 g/L yeast
2 4 2 4
extract, 4 g/L glycerol, 17 mM KH PO , 72 mM K HPO , 25 μg/mL kanamycin), which was brought to an initial OD600nm
of 0.1. Cultivation was carried out at 37 °C in orbital shakers with an agitation speed 110 rpm; cells were grown until an
OD600nm of 0.5-0.6. The expression of MsAcT was induced by adding isopropyl--d-thiogalactopyranoside (IPTG) to a
final concentration of 0.1 mM. The culture was further incubated for 16 h at 25 °C. Wet cells (2.0 g) were then harvested
by centrifugation (20 min, 9000 rcf at 4 °C) and washed with 20 mM phosphate buffer pH 8.0, resuspended in 10 mL of
buffer (100 mM phosphate buffer pH 8.0, 6 mM imidazole, 100 mM NaCl) and sonicated (5 cycles of 1 min on and 1
min off at 4° C). Cell debris were recovered by centrifugation (45 min, 36000 rcf at 4 °C). The enzyme was purified by
affinity chromatography with HIS-Select® Nickel Affinity Gel. The fractions showing activity were pooled and dialyzed
against phosphate buffer (100 mM, pH 8.0) and stored at 4 °C; 96 mg of pure protein were obtained. Activity was
spectrophotometrically evaluated at 400 nm by determining the release of p-nitrophenol after hydrolysis of p-
nitrophenylacetate at 25 °C in a half-microcuvette (volume 1 mL) for 2 min. One unit (U) of activity is defined as the
amount of enzyme which catalyzes the consumption of 1 μmol of p-nitrophenylacetate per minute. Reference
3
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 4 of 15
7
7
8
8
8
8
8
8
8
8
8
8
9
9
9
9
9
9
9
9
9
9
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
conditions were: 0.1 mg/mL p-nitrophenylacetate, 0.1% v/v EtOH, different amounts of MsAcT in 100 mM phosphate
buffer, pH 8.0. The specific activity of the purified enzyme was 110 U/mg.
Preparation of flavor esters. Standard transesterification reactions were carried out in 10 mL screw cap tubes: alcohols
(250 mM) and ethyl esters (10% v/v) were added to 2.5 mL of phosphate buffer (100 mM, in the range of pH 8.0)
containing MsAcT as purified enzyme (0.1-1.5 mg/mL; the reaction was left under magnetic stirring at 25 °C. The
reactions were stopped after 24 h and extracted with EtOAc (2x8 mL); the organic phases were collected, dried over
Na
2
SO
4
and evaporated. Products were purified by flash chromatography (eluent: n-hexane/EtOAc) and their structures
1
and purity secured by H NMR analysis by comparison with commercially available samples. Optimization of geranyl
and cinnamyl acetate synthesis was carried out using 1 mL reaction mixture in phosphate buffer (100 mM, in the range
of pH between 5.0 and 8.0), containing geraniol or cinnamyl alcohol (25-500 mM), MsAcT (0.1-1.5 mg/mL), and fixed
amount of ethyl acetate (10% v/v) were left under magnetic stirring at different temperatures (20-40 °C). Aliquots (50
μL) were withdrawn at different reaction times, extracted twice with EtOAc (2x100 μL) and the collected organic phase
2 4
was dried over Na SO . Preparative biotransformations at multi-milligram scale aimed at synthesizing geranyl acetate
and cinnamyl acetate were carried out using geraniol (250 mM) and cinnamyl alcohol (250 mM) dissolved in 9 mL of
phosphate buffer (0.1 M, pH 8.0) in the presence of 10 mg of MsAcT and 1 mL of EtOAc at 25 °C. The reactions were
2 4
stopped after 24 h and extracted with EtOAc (2x8 mL); the organic phases were collected, dried over Na SO and
evaporated. Products were purified by flash chromatography; 442 mg (90% yield) of geranyl acetate (eluent: n-
hexane/EtOAc 98:2, chemical purity > 98%) and 410 mg (93% yield) of pure cinnamyl acetate (eluent: n-hexane/EtOAc
1
95:5, chemical purity > 98%) were obtained. Purity of the product was confirmed by H NMR and gas-chromatography
analysis.
Analysis of the biotransformations. Aliquots of the biotransformation medium (100 µL) were withdrawn at different
reaction times, quenched with 5 l of 0.5 M sulfuric acid and extracted with 100 µL of ethyl acetate. The amounts of
produced flavor esters and the residual quantity of alcohol substrates were determined by gas-chromatographic (GC)
analysis. Geraniol, cinnamyl alcohol, n-hexanol, benzyl alcohol, 2-phenylethanol and their esters were analysed by gas-
GC analysis on a Carlo Erba Fractovap GC equipped with a fused-silica capillary column MEGA-SE30 (100% methyl
polysiloxane; 25 m x 0.25 mm i.d.), with the injector temperature at 200 °C. Oven temperatures ranged from 45 to 180
°C. Retention times (temperature gradient: from 80 °C to 180 °C with 5 °C/min): geraniol 7.3 min; geranyl formate 10.2
min; geranyl acetate 10.2 min; geranyl propionate 12.5; geranyl butyrate 14.5 min; Retention times (temperature
gradient: at 80 °C for 2 min then from 80 °C to 180 °C with 5 °C/min gradient): cinnamyl alcohol 9.9 min; cinnamyl
formate 10.7 min; cinnamyl acetate 13.2 min; cinnamyl propionate 15.5 min; cinnamyl butyrate 17.8 min; Retention
1
1
1
1
1
1
1
1
00
01
02
03
04
05
06
07
4
ACS Paragon Plus Environment

Page 5 of 15
Journal of Agricultural and Food Chemistry
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
times (temperature gradient: at 50 °C for 10 min then from 50 °C to 150 °C with 10 °C/min gradient): n-hexanol 5.3 min;
n-hexyl formate 7.7 min; n-hexyl acetate 8.8 min; n-hexyl propionate 15.2 min; n-hexyl butyrate 16.8 min; Retention
times (temperature from 80 °C to 180 °C with 10 °C/min gradient): benzyl alcohol 3.0 min; benzyl formate 3.4 min;
benzyl acetate 4.2 min; benzyl propionate 5.0 min; benzyl butyrate 6.5 min; 2-phenylethanol 3.7 min; 2-phenylethyl
formate 4.3 min; 2-phenylethyl acetate 5.4 min; 2-phenylethyl propionate 6.6 min; 2-phenylethyl butyrate 7.8 min.
Conversions of isoamyl alcohol into its esters were determined by GC analysis on a Carlo Erba Fractovap GC equipped
with a fused-silica capillary column MEGA-DEX DMP-Beta (dimethyl pentyl--cyclodextrin; 25 m x 0.25 mm i.d.), with
the injector temperature at 200 °C. Retention times (temperature gradient: from 40 °C to 180 °C with 5 °C/min): isoamyl
alcohol 6.7 min; isoamyl formate 5.8 min; isoamyl acetate 7.1 min; isoamyl propionate 9.4 min; isoamyl butyrate 11.5
min. Initial rates were defined as the amount of mol of ester produced after 30 min per amount of enzyme.
RESULTS and DISCUSSION
The reaction between geraniol and ethyl acetate (EtOAc) using the acyltransferase from Mycobacterium smegmatis
(MsAcT) as biocatalyst was studied in a two-liquid phase system composed by water and EtOAc; the product (geranyl
acetate) presents a green type flavour and a floral type odour.1 Different parameters (pH, temperature and enzyme
concentration) were optimized, while keeping the amount of geraniol (0.065 mM) and the phase ratio between water
and EtOAc (9:1) fixed; a Multisimplex optimization design was employed,27 using initial rate and molar conversions after
30 min as response variables. The best results (corresponding to a conversion >98% after 30 minutes) were obtained
using 1.0 mg/mL of MsAcT at pH 8.0 and 25 °C; under these conditions the production of the ester was studied using
different initial geraniol concentrations (Table 1).
128
129
130
131
Table 1. Acetylation of geraniol at different initial concentrations using MsAcT (1.0 mg/mL, 110 U/mL) in the presence
of EtOAc (10% v/v) in phosphate buffer (0.1 M, pH 8.0) at 25 °C. As determined by gas chromatography. b Time refers
to maximum conversion observed.
a
Geraniol (mM) Initial rate (μmol/mg
Maximum conversion
(%)a
Maximum product yield
(mg/mL)
Time (h)b
min)
5
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 6 of 15
100
200
250
300
400
500
2.25
4.56
6.36
6.57
7.68
7.65
> 98
> 98
98
19.6
39.2
48.1
55.9
62.0
66.7
2
24
24
24
48
48
95
79
68
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
The highest rates of geranyl acetate synthesis were achieved between 400-500 mM geraniol concentration, while almost
quantitative yields were obtained only at alcohol concentrations lower than 300 mM. Possible product inhibition was
evaluated by assessing the activity of MsAcT in the presence of increasing concentrations of EtOH and geranyl acetate
(0-500 M); the activity of the enzyme was assay (with p-nitrophenyl acetate) in the presence of concentrations of EtOH
between 250 and 500 mM showed that the enzyme was 40-50% inhibited, while no significant effects were observed
with geranyl acetate.
The best compromise between rates and conversion was observed starting from 250 mM geraniol, allowing for 98%
conversion (48.1 mg/mL analytical yield) after 24 hours. Notably, good conversion (68%) after 48 hours was achieved
even when the biotransformation was carried out starting from 500 mM geraniol, allowing for the accumulation of 66.7
mg/mL of geranyl acetate.
A similar evaluation of the effect of substrate concentration was carried out in the case of the acetylation of cinnamyl
alcohol (Table 2). Cinnamyl acetate has a balsamic-floral odour and a pineapple flavor.1
Table 2. Acetylation of cinnamyl alcohol at different initial concentrations using MsAcT (1.0 mg/mL, 110 U/mL) in the
presence of EtOAc (10% v/v) in phosphate buffer (0.1 M, pH 8.0) at 25 °C. a As determined by gas chromatography. b
Time refers to maximum conversion observed.
6
ACS Paragon Plus Environment

Page 7 of 15
Journal of Agricultural and Food Chemistry
Initial rate (μmol/mg
min)
2.54
Maximum conversion
(%)a
Maximum product yield
Cinnamyl alcohol (mM)
Time (h)b
(mg/mL)
17.6
100
200
250
300
400
500
> 98
> 98
> 98
92
1
3
5.07
6.32
6.82
8.53
8.65
35.2
44.0
48.6
59.2
69.6
6
24
24
24
84
79
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
Enzymatic acetylation of cinnamyl alcohol occurred with generally better rates and yields than observed with geraniol,
allowing for almost complete conversion of the alcohol up to 250 mM initial concentration after 6 hours. In the case of
the reaction carried out with the highest initial concentration of alcohol (500 mM), 79% molar conversion was obtained
after 48 hours.
Acetylation of geraniol and cinnamyl alcohol was performed on semipreparative scale (10 mL) starting from 250 mM
alcohol concentration; the reactions were stopped and worked-up when conversions reached the maximum yield,
allowing for the recovery of 442 mg of geranyl acetate (90% recovered yield) and 410 mg of cinnamyl acetate (93%
yield), respectively.
Acylation of geraniol and cinnamyl alcohol were then studied with other acyl donors, which varied in acyl chain length;
the results of the biotransformations carried out under optimized conditions (alcohol 250 mM, acyl donor 10% v/v,
enzyme 1.0 mg/mL, buffer pH 8.0, 25 °C) are summarized in Table 3.
Table 3. Acylation of geraniol (250 mM) and cinnamyl alcohol (250 mM) using MsAcT (1.0 mg/mL, 110 U/mL) in the
presence of different ethyl esters (10% v/v) in phosphate buffer (0.1 M, pH 8.0) at 25 °C. a As determined by gas
chromatography. b Time refers to maximum conversion observed.
7
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 8 of 15
Alcohol
Ester
Ester main flavor property 1 Initial rate (μmol/mg min)
Conv. (%)a
> 98
Time (h)b
Geraniol
Ethyl formate
rose
fruity
7.69
1.51
1.15
1.42
0.88
7.72
2.13
1.87
1.92
0.91
3
Geraniol
Geraniol
Ethyl propionate
Ethyl butyrate
86
62
48
48
48
48
1
fruity/apricot
rose/apricot
rose/sweet apple
fruity/apple
fruity/woody
fruity/honey
fruity/apple-banana
rose/apple
Geraniol
Ethyl isobutyrate
Ethyl isovalerate
Ethyl formate
74
Geraniol
38
Cinnamyl alcohol
> 98
> 98
90
Cinnamyl alcohol Ethyl propionate
Cinnamyl alcohol Ethyl butyrate
Cinnamyl alcohol Ethyl isobutyrate
Cinnamyl alcohol Ethyl isovalerate
3
24
24
48
87
44
8
ACS Paragon Plus Environment

Page 9 of 15
Journal of Agricultural and Food Chemistry
166
167
168
169
170
171
172
The ability of acting as acyl donor decreased by increasing the acyl chain length; geranyl formate was obtained in
quantitative yield after 1-3 h, whereas, with geranyl propionate and butyrate, the maximum yields (ranging between
65-98%) were reached only after 1-2 days.
Enzymatic acylation was then studied with different primary alcohols and different acyl donors; all the reactions studied
were aimed at preparing esters used as flavor or fragrance components (Table 4). Biotransformations were carried out
starting from 100, 250, and 500 mM alcohol concentrations.
173
174
175
176
Table 4. Acylation of different primary alcohols (starting 100-500 mM concentrations) with short chain ethyl esters using
MsAcT (1.0 mg/mL, 110 U/mL) in the presence of different ethyl esters (10% v/v) in phosphate buffer (100 mM, pH 8.0)
a
b
at 25 °C. As determined by gas chromatography. Time refers to maximum conversion observed.
R
1
Initial alcohol (mM)
R
2
Ester main flavor property 1
black currant/currant
Conv. (%)a Time (h)b
(
(
(
(
(
(
(
(
(
(
(
(
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
3
3
3
3
3
3
3
3
3
3
3
3
)
)
)
)
)
)
)
)
)
)
)
)
2
CHCH
2
CHCH
2
CHCH
2
CHCH
2
CHCH
2
CHCH
2
CHCH
2
CHCH
2
CHCH
2
CHCH
2
CHCH
2
CHCH
2
2
2
2
2
2
2
2
2
2
2
2
-
-
-
-
-
-
-
-
-
-
-
-
100
250
500
100
250
500
100
250
500
100
250
500
100
H-
H-
H-
93
93
86
94
95
88
97
97
95
94
93
92
97
0.5
0.5
1
CH
CH
CH
3
3
3
-
-
-
0.5
0.5
0.5
0.5
1
banana/pear
CH
CH
CH
2
CH
2
CH
2
CH
3
3
3
-
-
-
pineapple-apricot/apricot
1
CH
CH
CH
2
2
2
CH
CH
CH
2
2
2
CH
CH
CH
3
-
3
-
3
-
24
24
24
0.5
fruity
apple
CH
3
(CH
2
)
4
-
H-
9
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 10 of 15
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
3
3
3
3
3
3
3
3
3
3
3
3
3
(CH
(CH
(CH
(CH
(CH
(CH
(CH
(CH
(CH
(CH
(CH
(CH
(CH
2
2
2
2
2
2
2
2
2
2
2
2
2
)
)
)
)
)
)
)
)
)
)
)
)
)
4
-
4
-
4
-
4
-
4
-
4
-
4
-
4
-
4
-
4
-
4
-
4
-
4
-
250
500
100
250
500
100
250
250
500
500
100
250
500
100
250
500
100
250
500
100
250
500
100
250
500
100
250
500
100
H-
H-
97
88
97
95
90
72
50
35
30
24
94
96
95
92
84
86
92
90
87
96
95
89
95
92
95
95
88
86
96
1
3
CH
CH
CH
3
3
3
-
-
-
0.5
1
fruity/pear
3
CH
CH
CH
CH
CH
2
CH
2
CH
2
CH
2
CH
2
CH
3
3
3
3
3
-
-
-
-
-
1
1
earthy/metallic-fruity
2
1
2
CH
CH
CH
2
2
2
CH
CH
CH
2
2
2
CH
CH
CH
3
3
3
-
-
-
2
apricot/pineapple
floral-fruity/apricot
jasmine
6
24
0.5
1
Ph-
Ph-
Ph-
Ph-
Ph-
Ph-
Ph-
Ph-
Ph-
Ph-
Ph-
Ph-
H-
H-
H-
3
CH
CH
CH
3
3
3
-
-
-
0.5
1
3
CH
CH
CH
2
CH
2
CH
2
CH
3
-
3
-
3
-
0.5
1
floral-fruity/peach
plum/pear
3
CH
CH
CH
2
2
2
CH
CH
CH
2
2
2
CH
CH
CH
3
-
3
-
3
-
8
8
24
0.5
2
PhCH
PhCH
PhCH
PhCH
2
2
2
2
-
-
-
-
H-
H-
H-
hyacinth
3
CH
3
-
rose/raspberry
0.5
10
ACS Paragon Plus Environment

Page 11 of 15
Journal of Agricultural and Food Chemistry
PhCH
PhCH
PhCH
PhCH
PhCH
PhCH
PhCH
PhCH
2
2
2
2
2
2
2
2
-
-
-
-
-
-
-
-
250
500
100
250
500
100
250
500
100
250
500
100
100
250
100
250
500
100
250
500
CH
CH
3
-
-
91
82
0.5
0.5
0.5
0.5
0.5
4
3
CH
CH
CH
2
CH
2
CH
2
CH
3
3
3
-
-
-
97
red rose/strawberry
rose/honey
97
96
CH
CH
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
3
3
3
-
-
-
97
97
8
90
24
0.5
4
PhCH=CHCH
PhCH=CHCH
PhCH=CHCH
PhCH=CHCH
PhCH=CHCH
PhCH=CHCH
PhCH=CHCH
PhCH=CHCH
PhCH=CHCH
PhCH=CHCH
PhCH=CHCH
PhCH=CHCH
2
-
2
-
2
-
2
-
2
-
2
-
2
-
2
-
2
-
2
-
2
-
2
-
H-
H-
H-
>98
>98
82
fruity/apple
24
1
CH
CH
CH
3
-
3
-
3
-
>98
>98
96
floral/pineapple
fruity/woody
4
24
1
CH
CH
CH
3
CH
3
CH
3
CH
2
-
2
-
2
-
>98
>98
97
5
24
16
24
48
CH
CH
CH
3
CH
3
CH
3
CH
2
CH
2
CH
2
CH
2
-
2
-
2
-
>98
92
fruity/honey
80
177
178
179
180
181
182
183
184
185
Formylation and acetylation of all the tested primary alcohols allowed for quantitative or almost quantitative
conversions with high rates ( 90% within 30-60 min) when the initial concentration of alcohols was kept between 100
and 250 mM. When the biotransformation was carried out with higher alcohol concentration (500 mM), still high molar
conversions of the flavor-esters were observed (from 80 up to >98%), albeit with longer reaction times. Slower reactions
were generally found with acyl donors of increased chain length (propionyl- and butyryl-). The only biotransformation
occurring with relatively low yields (30-70% depending on the initial concentration) was the propionylation of n-hexanol
to give n-hexyl propionate; interestingly, in this case hydrolysis of the formed ester was predominant after reaching its
maximum accumulation, as noticeable from an increase of the alcohol concentration (data not shown).
11
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 12 of 15
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
In conclusion, a highly efficient preparation of different flavor-esters was achieved using the acyltransferase from
Mycobacterium smegmatis (MsAcT), which catalyzes the acylation of different primary alcohols in aqueous systems. The
results collected so far indicate that the biotransformation can be applied not only to the production of acetate esters,
but can be expanded to a range of other acyl donors (e.g., ethyl formate, propionate, butyrate), useful for the formation
of different flavor/fragrance esters, making this process strongly versatile. Notably, all the primary alcohols tested were
accepted as substrates, even at higher concentrations (up to 500 mM); preparation of geranyl acetate and cinnamyl
acetate was carried out on semipreparative scale (10 mL) starting from 250 mM alcohol concentration, allowing for the
recovery of 44.2 mg/mL (geranyl acetate) and 41.0 mg/mL (cinnamyl acetate) of the products. Biocatalysis is sometimes
perceived to be inefficient compared with conventional chemical processes because of the low productivity, mostly due
to substrate/product inhibition of the enzymatic activity which often occurs at somewhat low concentrations. To
become an industrially attractive technology. a biocatalytic process must be engineered to improve space time yields:28
in this work we have described that the use of relatively low amount of MsAcT allowed the conversion of remarkably
high substrate concentrations with good yields. The proposed enzymatic method may pave the way for an efficient and
environmentally sustainable preparation of natural esters; the biotransformation may be further intensified by
immobilizing the enzyme and carry out continuous processes in suited reactors,29 thus making it appealing at industrial
level.
REFERENCES
1. Burdock, G.A. In Fenaroli’s handbook of flavor ingredients (6th ed.). Boca Raton, CRC Press. 2010.
2. Berger, R.G. Biotechnology of flavours - the next generation. Biotechnol. Lett. 2009, 31, 1651–1659.
3. Sabisch, M.; Smith D. The complex regulatory landscape for natural flavor ingredients.
https://www.sigmaaldrich.com/technical-documents/articles/white-papers/flavors-and-
fragrances/natural-flavor-ingredients-regulations.html. 2018.
4. Hofmann, T.; Krautwurst, D.; Schieberle, P. Current status and future perspectives in flavor research: highlights of
the 11th Wartburg symposium on flavor chemistry & biology. J. Agric. Food Chem. 2018, 66, 2197–2203.
5. Fraatz, M.A.; Goldmann, M.; Geissler, T.; Gross, E.; Backes, M.; Hilmer, J.M.; Ley, J.; Rost, J.; Francke, A.; Zorn, H.
Biotechnological production of methyl-branched aldehydes. J. Agric. Food Chem. 2018, 66, 2387–2392.
6. Dhake, K.P.; Dinesh, D.; Thakare, D.D.; Bhalchandra, M.; Bhanage, B.M. Lipase: A potential biocatalyst for the
synthesis of valuable flavour and fragrance ester compounds. Flavour Frag. J. 2013, 28, 71–83.
12
ACS Paragon Plus Environment

Page 13 of 15
Journal of Agricultural and Food Chemistry
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
7. Nikolaivits, E.; Makris, G.; Topakas, E. Immobilization of a cutinase from Fusarium oxysporum and application in
pineapple flavor synthesis. J. Agric. Food Chem. 2017, 65, 3505–3511.
8. Sà, A.G.A.; de Meneses, A.C.; de Araújo, P.H.H.; Oliveira, D. A review on enzymatic synthesis of aromatic esters
used as flavor ingredients for food, cosmetics and pharmaceuticals industries. Trends Food Sci. Technol. 2017, 69,
95–105.
9. Halling, P.J. Solvent selection for biocatalysis in mainly organic systems: Predictions of effects on equilibrium
position. Biotechnol. Bioeng. 1991, 35, 691–701.
10. Klibanov, A.M. Improving enzymes by using them in organic solvents. Nature. 2001, 409, 241–246.
11. Spizzo, P.; Basso, A.; Ebert, C.; Gardossi, L.; Ferrario, V.; Romano, D.; Molinari F. Resolution of (R,S)-flurbiprofen
catalysed by dry mycelia in organic solvent. Tetrahedron 2007, 63, 11005–11010.
12. Gubicza, L.; Kabiri-Badr, A.; Keoves, E.; Belafi-Bako, K. Large-scale enzymatic production of natural flavour esters
in organic solvent with continuous water removal. J. Biotechnol. 2000, 84, 193–196.
13. Tamborini L.; Romano D.; Pinto A.; Contente M.; Iannuzzi M.C.; Conti P.; Molinari F. Biotransformation with whole
microbial systems in a continuous flow reactor: resolution of (RS)-flurbiprofen using Aspergillus oryzae by direct
esterification with ethanol in organic solvent. Tetrahedron Lett. 2013, 54, 6090–6093.
14. Sun, J.; Yu, B.; Curran, P.; Liu, S.Q. Optimisation of flavour ester biosynthesis in an aqueous system of coconut
cream and fusel oil catalysed by lipase. Food Chem. 2010, 135, 2714–2720.
15. Rodriguez-Nogales, J.M.; Roura, E.; Contreras E. Biosynthesis of ethyl butyrate using immobilized lipase: a
statistical approach. Process Biochem. 2005, 40, 63–68.
16. Gandolfi, R.; Converti, A.; Pirozzi, D.; Molinari, F. Efficient and selective microbial esterification with dry mycelium
of Rhizopus oryzae. J. Biotechnol. 2001, 92, 21–26.
17. Converti, A.; Del Borghi, A.; Gandolfi, R.; Lodi, A.; Molinari, F.; Palazzi E. Reactivity and stability of mycelium‐bound
carboxylesterase from Aspergillus oryzae. Biotechnol. Bioeng. 2002, 77, 232–237.
18. Brault, G.; Shareck, F.; Hurtubise, Y.; Lepine, F.; Douce N. Short-chain flavor ester synthesis in organic media by an
E. coli whole-cell biocatalyst expressing a newly characterized heterologous lipase. Plos ONE. 2014, 9, e91872.
19. Ozyilmaz, G.;E. Gezer, E. J. Mol. Catal. B: Enzym. 2010, 64, 140–145.
20. Mathews I.; Soltis M.; Saldajeno M.; Ganshaw G.; Sala R.; Weyler W.; Cervin M.A.; Whited G.; Bott, R. Structure of a
novel enzyme that catalyzes acyl transfer to alcohols in aqueous conditions. Biochemistry. 2007, 46, 8969–8979.
21. Kazemi, M.; Sheng, X.; Wolfgang Kroutil, W.; Himo F. Computational study of Mycobacterium smegmatis acyl
transferase reaction mechanism and specificity. ACS Catal. 2018, 8, 10698–10706.
13
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 14 of 15
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
22. Wiermans, L.; Hofzumahaus, S.; Schotten, C.; Weigand, L.; Schallmey, M.; Schallmey, A.; Domínguez de María, P.
Transesterifications and peracid-assisted oxidations in aqueous media catalyzed by Mycobacterium smegmatis
acyl transferase. ChemCatChem 2013, 5, 3719−3724.
23. de Leeuw, N.; Torrelo, G.; Bisterfeld, C.; Resch, V.; Mestrom, L.; Straulino, E.; van der Weel, L.; Hanefeld, U. Ester
synthesis in water: Mycobacterium smegmatis acyl transferase for kinetic resolutions. Adv. Synth. Catal. 2018, 360,
242−249.
24. Mestrom, L.;. Claessen, J.G.R.; Hanefeld U. Enzyme-catalyzed synthesis of esters in water. ChemCatChem 2019,
11, 2004−2010.
25. Land, H.; Hendil-Forssell, P.; Martinelle, M.; Berglund, P. One- pot biocatalytic amine transaminase/acyl transferase
cascade for aqueous formation of amides from aldehydes or ketones. Catal. Sci. Technol. 2016, 6, 2897−2900.
26. Contente, M.L.; Pinto, A.; Molinari, F.; Paradisi F. Biocatalytic N-acylation of amines in water using an
acyltransferase from Mycobacterium smegmatis. Adv. Synth. Catal. 2018, 360, 4814−4819.
27. Romano, D.; Gandolfi, R.; Guglielmetti, S.; Molinari, F. Enzymatic hydrolysis of capsaicins for the production of
vanillylamine using ECB deacylase from Actinoplanes utahensis. Food Chem. 2011, 124, 1096−1098.
28. Sheldon, R. A.; Pereira, P. C. Biocatalysis engineering: the big picture. Chem. Soc. Rev., 2017, 46, 2678−2691.
29. Tamborini, L.; Fernandes, P.; Paradisi, F.; Molinari F. Flow bioreactors as complementary tools for biocatalytic
process ntensification.Trends Biotechnol. 2018, 36, 73−88.
14
ACS Paragon Plus Environment

Page 15 of 15
Journal of Agricultural and Food Chemistry
ACS Paragon Plus Environment