Functional investigation and applications of the acetylesterase activity of the Citrus sinensis (L.) Osbeck peel

Article abstract of DOI:10.1080/14786419.2020.1737055

The hydrolysis of acetyl moieties on a set of commercially relevant substrates was performed by employing the whole tissue of Citrus sinensis (L.) Osbeck peel as an efficient biocatalyst in mild reaction conditions with high degree of regioselectivity. The reaction is done in aqueous media and the product is easily recovered. Optimal reaction conditions were deduced and two practical applications were investigated: the elaboration of acetylglicerols and the preparation of vitamin K₁ precursor. Peel waste (flavedo and albedo) from orange juice manufacturing was successfully employed as a biocatalyst.

Full text of DOI:10.1080/14786419.2020.1737055

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Functional investigation and applications of the
acetylesterase activity of the Citrus sinensis (L.)
Osbeck peel
Gianfranco Fontana, Maurizio Bruno, Antonella Maggio & Sergio Rosselli
To cite this article: Gianfranco Fontana, Maurizio Bruno, Antonella Maggio & Sergio Rosselli
(2020): Functional investigation and applications of the acetylesterase activity of the Citrusꢀsinensis
(L.) Osbeck peel, Natural Product Research, DOI: 10.1080/14786419.2020.1737055
To link to this article: https://doi.org/10.1080/14786419.2020.1737055
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2020.1737055
Functional investigation and applications of the
acetylesterase activity of the Citrus sinensis (L.)
Osbeck peel
Gianfranco Fontanaa , Maurizio Brunoa , Antonella Maggioa
Sergio Rossellib
and
a
ꢀ
Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche (STEBICEF), Universita
b
degli Studi di Palermo, Palermo, Italy; Dipartimento di Scienze Agrarie, Alimentari e Forestali
(SAAF), Universita degli Studi di Palermo, Palermo, Italy
ꢀ
ABSTRACT
ARTICLE HISTORY
Received 29 November 2019
Accepted 22 February 2020
The hydrolysis of acetyl moieties on a set of commercially rele-
vant substrates was performed by employing the whole tissue of
Citrus sinensis (L.) Osbeck peel as an efficient biocatalyst in mild
reaction conditions with high degree of regioselectivity. The
reaction is done in aqueous media and the product is easily
recovered. Optimal reaction conditions were deduced and two
practical applications were investigated: the elaboration of acetyl-
glicerols and the preparation of vitamin K1 precursor. Peel waste
(flavedo and albedo) from orange juice manufacturing was suc-
cessfully employed as a biocatalyst.
KEYWORDS
Citrus sinensis; acetate
hydrolysis; enzymatic
biocatalysis; acetins;
vitamin K1
1. Introduction
Biocatalytic transformations are a wide class of processes in which a chemical modifi-
cation of a suitable organic substrate takes place, generally with a high degree of
regio- and stereoselectivity, in mild reaction conditions. The catalysts employed can
include pure isolated enzymes and intact tissues, as well as cell cultures suspended
CONTACT Gianfranco Fontana
gianfranco.fontana@unipa.it
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2020.1737055.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
G. FONTANA ET AL.
into a suitable growth broth (Hamada et al. 1996). The use of vegetables in this con-
text is attractive in consideration of the easy availability of a wide variety of possible
biocatalysts. From the pioneering work of Stohs and Staba (1965) on the chemical
modification of digitoxigenin with tissue cultures of Digitalis, a lot of work had been
made to develop protocols for performing many chemical transformations by vegeta-
bles (Cordell et al. 2007) including reduction of alkenes, carbonyl and nitro com-
pounds, oxidation of alcohols, ester hydrolysis. Among these enzymatic activities, the
esterase-like one is particularly attractive both for its potential application in organic
synthesis (Bracher and Krauss 1998) and from the economic point of view, in consider-
ation of the many important applications of esterases in food, perfume and pharma-
ceutical industries (Panda and Gowrishankar 2005). The commercial purified enzyme
acetylesterase from the flavedo of orange peels was employed, for example, by
Waldmann and Heuser (1994) as the catalyst in the removal of acetyl protecting
groups from monosaccharides.
The utilization of the whole plant preparation in order to perform the hydrolysis of
the ester bond was deeply investigated by Mironowicz and Siewinski; they disclosed
the possibility to hydrolyze various aromatic acetates by intact plants (Mironowicz
et al. 2014) of Spirodela punctata, Nephrolepis exaltata, Cyrtomium falcatum,
Nephrolepis cardifolia and the suspended coltures of Helianthus tuberosus. Further, ace-
tates and propionates of arylic, benzylic and alyphatic (terpenoidic) alcohols were suc-
cessfully hydrolyzed with the pulp of Solanum tuberosum and Malus silvestris
(Mironowicz et al. 2014). The same group performed the enantioselective hydrolysis of
a number of racemic chiral esters with Spirodella oligorrhiza (Pawlowicz and Siewinski
1987) Malus silvestris (Mironowicz 2014); Solanum tuberosum and Helianthus tuberosus
(Mironowicz 1998); Daucus carota, Apium graveolens and Armoracia lapatifolia (Maczka
and Mironowicz 2002). A set of 28 vegetables were tested by Vanderberghe et al.
(2013) as biocatalyst in the hydrolysis/kinetic resolution of the racemic 1-phenylethyl
acetate and more recently the peel of Citrus aurantium was employed for the same
purpose (Da Silva et al. 2016).
Citrus sinensis (L.) Osbeck is an endemic plant in the Italian region of Sicily, known
for its high nutraceutical value (Celano et al. 2019) and with several recently recog-
nized bioactivities, such as antioxidant (Tomasello et al. 2019) and mosquitocidal
(Badawy et al. 2018).
The aim of this work was to investigate the use of C. sinensis peel (CSP) as the bio-
catalyst for performing potentially useful chemical transformations, also in light of the
benefits connected with the potential application of recycled waste, such as fruit peel
derived from the industrial production of fruit juices and canned fruits.
Although the presence of an acetylesterase EC 3.1.1.6 enzyme in the orange peel is
known since a long time (Jansen et al. 1947), the use of the fresh peel and the raw
material, obtained from C. sinensis, as biocatalyst for the chemo- and regioselective
removal of acetyl groups was not deeply elucidated and the number of applications
already reported is still limited. For this reason, we decided to investigate the use of
easily obtained, low cost and eco-friendly catalyst in the deacetylation reaction of
some compounds, also useful in the preparation of valuable commercial products.
Geranyl acetate (1, Figure 1) is a natural product occurring in many plants essential

NATURAL PRODUCT RESEARCH
3
Figure 1. Biocatalytic transformations by Citrus sinensis peel.
Figure 2. Synthesis of K1 vitamin.
oils and its deacetylated derivative, the alcohol geranyol, is the constituent of many
natural essences used in perfume industry and a key starting material for other ter-
penes. Furthermore, it possesses many biological activities (Chen and Viljoen 2010).
1-Acetoxy-4-hydroxy-2-methylnaphthalene 4 is a key intermediate involved in the
preparation of the pharmacological relevant molecule vitamin K1 (Hirshmann et al.
1954), as depicted in Figure 2. The transformation of compound 3 to 4 reported in lit-
erature involve traditional chemicals such as tBuNH2 or MeONa in the highly toxic
methanol as solvent (Tomkuljak et al. 1980; Huang et al., 2006).
Finally, triacetin (TA) 5, diacetins (DA) 6,7 and monoacetins (MA) 8,9 are important
acetyl-derivatives of glycerol 10 with many commercially relevant applications (Kong
et al. 2016). MAs are used as plasticizers for vinyl polymers and cellulose-like material,
DAs are used as raw material in the production of polyesters and finally, TA is used as
solvent and excipient in pharmacy as well as additive in fuel mixture to modulate vis-
cosity and knocking properties. Further, as the functional profile of Citrus sinensis ace-
tylesterase was not yet fully investigated and the data available (Pasta et al. 2004,
Brenda 2019) at present for this enzyme are incomplete, the initial part of this work
was devoted to the identification of the most suitable reaction conditions (pH, tem-
perature, reaction time).
2. Results and discussion
The efficiency of the CSP as a biocatalyst in the deacetylation reaction was evaluated
on substrate 1 in order to establish the optimal reaction conditions. A range of pH

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G. FONTANA ET AL.
from 4.4 to 12.0 was explored using citric acid/phosphate buffers at a constant sub-
strate/catalyst ratio (0.0075 w/w) and temperature (30 ꢀC). The reaction mixture was
analyzed by GC/MS after 72 h. The higher rate of conversion is achieved at pH
comprised between 8.0 and 9.0 with a conversion quite complete (97%). The same
pH range was evaluated without the biocatalyst; as it can be seen from Figure S1a
(supplementary material), the basic, non-enzymatic, ester hydrolysis occurred over pH
9.7 and became predominant above pH 11.5. This point can also explain the appear-
ance of a relative minimum at pH 9.7 that is due to the decrease in the enzymatic
activity after pH 8 accompanied by the increase of the chemical hydrolysis rate after
pH 10.
The influence of temperature was investigated (Figure S1b). The higher conversion
was obtained at 40 ꢀC. However, the blank experiments gave a certain degree of ther-
mal hydrolysis taking place above 35 ꢀC.
The reaction is more sensitive to the pH variation than to the changes in tempera-
ture (45% at 30 ꢀC and 85% at 40 ꢀC without buffer, 97% at 30 ꢀC and 97% at 40 ꢀC
with pH 8.5 buffer). In order to follow the time course of the geranyl acetate conver-
sion, the substrate 1 was left to react with the usual substrate/catalyst ratio under the
optimal reaction conditions previously identified (pH 8.5, 40 ꢀC, Figure S1c). The
amount of produced geranyol reaches a plateau after about 60 h.
Finally, experiments were conducted for the evaluation of the efficacy of the deace-
tylation reaction performed with differently prepared biomass batches, i.e., chopped
fresh fruit peel, freeze-dried peel powder, residual material after industrial squeezing
of the fruit for juice production. No difference was evidenced between the fresh peel
and the orange industrial waste recovered directly from an industrial squeeze appar-
atus, dried and stored for one week at 4 ꢀC (fresh peel vs. waste conversion efficiency:
78,0/75.5 6.5%, r substrate/biocatalyst 0.0075 w/w, 40 ꢀC, pH 8.5, 50 h) that demon-
strating the possibility to employ this kind of waste as biocatalyst.
The optimized reaction conditions obtained for the compound 1 were employed
for substrates 3 and 5. After 72 h at 30 ꢀC and buffered pH of 8.5, the CSP catalyst
transforms partially (55% of yield) the 1,4-diacetyl-2-methylnaphtalene 3 in a single
product (TLC analysis) identified, after CC isolation and spectroscopic comparison with
a commercial sample, as the 1-monoacetylderivative 4 showing an interesting regio-
specificity of the enzymatic system towards this substrate. The method showed herein
can be considered as a possible valid eco-friendly, mild and safer alternative for
obtaining compound 4, as the methods already reported in literature involve the use
of toxic reactants (Huang et al. 2006).
TAG 5 was incubated in a suspension of the CSP biocatalyst at the usual reaction
conditions and the results are summarized in Table S1 (supplementary material).
It is relevant to note how it is possible to modulate the degree of acetylation of
the acetins mixture (TAG ! DAs ! MAs ! glycerol) by simply modulating the reac-
tion time. Indeed, the GC chromatogram showed two isomeric peaks for both the
diacetins and monoacetins due to the presence of regioisomeric mixtures, very prob-
ably arising from an acetyl shift occurred after the first removal of an acetyl group. We
did not collect direct evidences on the structure of the main component in the mono-
acetins mixture.

NATURAL PRODUCT RESEARCH
5
In conclusion, the catalytic activity of the Citrus sinensis peel in the hydrolysis of
acetates was investigated in relation to the pH of the medium, reaction temperature
and time. The method works in very mild conditions and in aqueous solvent. A signifi-
cant point of interest lies in the opportunity to exploit the use of the readily and
affordably available citrus canned food industry wastes as a green biocatalyst in rele-
vant industrial processes such as the production of fragrance ingredients as geraniol,
vitamin K1, mono- and diacetins.
3. Experimental
See supplementary material.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
Financial supports from “Fondo di Finanziamento della Ricerca di Ateneo (FFR2018)” and PON
a3_00053-PLASS are gratefully acknowledged. 1H (400 MHz) and 13C (100 MHz) NMR data were
ꢀ
provided by Centro Grandi Apparecchiature – UNINetLab - Universita di Palermo funded by
P.O.R. Sicilia 2000-2006, Misura 3.15 Quota Regionale.
ORCID
Gianfranco Fontana
Maurizio Bruno
Antonella Maggio
http://orcid.org/0000-0001-7329-9902
http://orcid.org/0000-0003-0583-0487
http://orcid.org/0000-0002-0861-626X
Sergio Rosselli
http://orcid.org/0000-0002-2448-9192
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