flavour production via enzyme catalysis
DESCRIPTION
Of recent Aroma compounds have gained momentum in its application but the challenge has been its production through a conventionally clean method. Biotechnology is a promising field to address this challenge.TRANSCRIPT
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ENZYME APPLICATION IN FOOD INDUSTRY
Enzyme catalysis for flavour production
By Kissa R. Alunga
Abstract
Of recent, biocatalytic production of aroma compounds has rapidly gained momentum.
Natural flavours belong to many different structural classes and their industrial production has been of
great challenge to academic and research scientists. Here, an overview of the potential offered by
biocatalysis for the synthesis of natural odorants, highlighting relevant biotransformations using enzymes.
The examples of industrial processes based on biocatalytic methods are discussed, their advantages over
classical chemical synthesis is also highlighted. Lastly the challenges facing the biocatalytic production
are expounded upon.
Key words; Enzyme catalysis; Flavour production; Bioreduction; Ehrlich pathway; Biotransformation;
Esterification.
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Contents
Abstract ................................................................................................................................................... 1
1. Introduction ......................................................................................................................................... 3
2. History of enzyme catalysis for flavour production .............................................................................. 3
3. Advantages of biocatalysis over conventional chemical synthesis......................................................... 4
4. Examples of enzyme catalysis for flavour production ........................................................................... 5
4.1 Ehrlich pathway: the route for 2-phenylethanol (2-pe) production .................................................. 5
4.2 Rose oxide biosynthesis using Chloroperoxidase (CPO) ................................................................. 8
4.3 Production of Flavours via Bioreduction ........................................................................................ 9
4.4 Esterification by lipase ................................................................................................................. 11
5. Challenges ......................................................................................................................................... 13
5.1 Low yield and high costs of production ........................................................................................ 13
5.2 Toxicity of the substrate and products .......................................................................................... 13
5.3 Enzymes deactivation .................................................................................................................. 13
5.4 Other challenges .......................................................................................................................... 14
6. Conclusion ........................................................................................................................................ 15
7. References ......................................................................................................................................... 16
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1. Introduction
Flavours and fragrances are more the same playing a similar role because of their volatile odor
characteristic. They are natural and vital ingredients of most essential oils which play an
important role in the food, beverage, perfume and pharmaceutical industries among others [3, 6].
Because natural flavours are obtained from natural raw materials using microorganism, are
regarded as safer over chemically synthesized ones [6]. The US and European laws have marked
them ‘natural flavours’ because they are obtained naturally using living cells and that makes
them have a market advantage over the non-natural flavours [6, 7].
The high demand for natural flavours and fragrances is the reason for the upsurge of the number
of research scientists currently studying and developing biocatalysts for producing these
molecules.
Thus, the microbial and enzymatic biotransformation of some substances such as
monoterpenoids, in particular a few ketones and aldehydes (e.g., carvone, menthol, citronellol,
myrtenal and geraniol) into highly valuable flavouring derivatives is becoming of increasing
interest because of their economic potential for the perfume, soap, food, and beverage
industries[6].
2. History of enzyme catalysis for flavour production
Enzymes have been used since the discovery of the fermentation process for beer, wine, and
other related products; they were of significant importance in the early stages of food aroma
industry till this very day. The small beginning of enzyme catalysis evolved into a major
technological process applicable in major industries today. It is believed that more than a century,
benzaldehyde was the pioneer flavour compound ever discovered.
The isolation, identification and production of vanillin signaled the start of modern flavour
industry. Starting in the early 1950s, the replacement of classical organic methods of analysis by
the modern analytical and separation methods such as gas chromatography facilitated the
separation and structural elucidation of volatile compounds.
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Back in the days, the microbiologists concentrated on screening microorganisms and the aroma
compounds generated. Contemporary microbiological techniques including genetic engineering
are now increasingly applied to enhance efficiency of the biocatalysts.
According to Perfumer & Flavourist magazine, the flavours and fragrance industry was worth
US$20.3 billion in 2009 (estimate may vary from other sources), of which the lion‘s share is
flavours. There is a significant amount of natural volatiles available but a few have been
manufactured on a scale greater than 1 ton per annum.
Bioprocesses for volatile compounds have emerged only recently. Technical scale processes are
operating for some aliphatic alkenols and carbonyls, carboxylic and benzoic esters including
lactones, vanillin and certain specialities.
3. Advantages of biocatalysis over conventional chemical synthesis.
Much of the production of the flavours has been via chemical synthesis. Of recent, most
customers do prefer food addiditives from natural compounds leaving behind chemical additives
from the chemical process; this is due to racemic mixtures associated with them.
Reactions catalyzed by biological systems frequently exhibit high selectivity [6]. Enzymes are
potent analytical tools because of its specificity and sensibility that allows them to quantify
substances at very low concentrations with minimal interference [enzyme biocatalysis, andress
illanes].
Biocatalysis reaction processes are considered environmentally friendly because they typically
occur under mild conditions [5, 6] where as chemical synthesis is environmentally unfriendly
(high temperature, high pressure, and strong acid or alkali) and are associated with the
production of unwanted byproducts, thus reducing efficiency and increasing downstream costs.
Biocatalysis transformation is associated with few byproducts, and is considered to be a
promising strategy for the production of high-valued compounds.
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4. Examples of enzyme catalysis for flavour production
4.1 Ehrlich pathway: the route for 2-phenylethanol (2-pe) production
2-Phenylethanol (2-PE) is a flavour alcohol, the main use of 2-PE in the world market is to
modify certain flavour compositions. Although the above compound can be synthesized
microbiologically, the final output is usually low; 2-PE is an intermediate in the microbial
transformation of L-phenylalanine (L-Phe), which is an essential amino acid in humans. It is
produced on a large-scale by enzymatic transformation with a low production cost, in a process
that can be considered a natural process.
Ehrlich pathway explanation of the transformation of L-Phe to 2-PE.
Several biotechnological processes are based on this pathway which has stimulated studies to
establish enzymes that are actively involved in this process. 2-PE Dehydrogenase was discovered
as the sole carbon source and has broad substrate specificity and catalyzes the reversible
oxidation of various primary alcohols to aldehydes.
Illustration of this pathway.
L-Phe is transaminated to phenylpyruvate by a transaminase, decarboxylated to
phenylacetaldehyde by phenylpyruvate decarboxylase, and then reduced to 2-PE by a
dehydrogenase. 2-PE also can be transformed to phenylaldehyde and phenylacetate in a reaction
catalyzed by a dehydrogenase as shown on figure 1 below.
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Fig.1 Ehrlich pathway for 2-PE production from L-Phe [D. Hua et al, 2011]
To address the challenge of low product yield, scientists have come up with techniques such as
the ISPR (in situ product-removal) techniques which are effective and promising methods. This
technique has been applied in the production of 2-PE production from L-Phe as may be
explained below.
ISPR techniques, which are the continuous in-situ removal of product from reaction system, are
widely used. These techniques include two-phase extraction, adsorption and solvent
immobilization these methods maintain the product concentration around cells below an
inhibitory level, and the strains are able to continue the production of target product.
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To illustrate one of the techniques applied; two phase extraction, using aqueous–organic two-
phase extraction in 2-PE production from L-Phe. Biotransformation of LPhe to 2-PE is carried
out in the aqueous phase. The produced 2-PE is continuously extracted into the organic phase as
may be illustrated in the figure 2
Fig 2 two phase extraction (D. Hua et al, 2011)
If successful high yield of 2-PE is achieved, them more valuable aromatic compounds can also
be achieved highly. 2-PE is used as a substrate for the synthesis of other aroma compounds such
as phenylethyl acetate (scheme 1).
Scheme 1, Biotransformation of 2-PE to other valuable chemicals (D. Hua et al, 2011).
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4.2 Rose oxide biosynthesis using Chloroperoxidase (CPO)
Chloroperoxidase (CPO) is a 42-kDa haem-thiolate enzyme that is secreted by the fungus
Caldariomyces fumago. CPO is an attractive catalyst for bio-oxidation reactions using low cost
oxidising agents like hydrogen peroxide.
Studies have shown that monoterpenoids are the major substrates for this enzyme [2]. The
challenge in here was the low yield and attempting to develop a straightforward and
environmentally friendly route from citronellol to rose oxide proved unsuccessful. Until recently
a novel biocatalytic approach for the synthesis of rose oxide was discovered by combining the
CPO catalysed oxyfunctionalisation of citronellol with a chemical two step synthesis with a high
yield.
To illustrate the synthetic usefulness of the CPO-catalysed bromohydroxylation of citronellol
(scheme 2), the generated bromohydrins of citronellol bromohydrins were converted into rose
oxide 6 via the diols 4 and 5 in two reaction steps. The reaction steps involved treatment of the
bromohydrins with potassium tert-butylate followed by acid treatment. This reaction sequence
yields a high percentage of cis-rose oxide which is the most valuable and appreciated
diastereomer in the flavour and fragrance industry [2].
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Scheme 2, Chloroperoxidase-catalyzed formation of the diastereomeric bromohydrins 2a/2b from
(R)-citronellol (R)-1 and conversion of 2a/2b to the corresponding epoxides 3a/3b or to rose oxide 6
via the diols 4 and 5; DMSO, dimethyl sulfoxide; t-BuOK: potassium tert-butylate [2].
4.3 Production of Flavours via Bioreduction
Carvone is an aldehyde belonging to one of the largest classes of flavouring compounds
monoterpenes. It is available in two forms which differ by their odor characteristics; they include
(4R)-(−)-carvone, present in spearmint oil and S-(+)-enantiomer commonly extracted from
caraway and dill seeds.
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Carvone is an important element; their dihydrocarveols are valuable ingredients currently
applicable in the flavour and fragrance industry.
The biotransformations of the α,β-unsaturated ketone (4R)-(−)-carvone (1) catalyzed by whole-
cells of NCYs in aqueous media were investigated. The possible reaction pathway is illustrated
in Scheme 3.
According to the proposed scheme, the biotransformation resulted in the reduction of the α,β-
unsaturated C=C bond of the cyclic ketone, catalyzed by ene-reductases (ERs) associated to the
yeast cells, to give two dihydrocarvones 2a,b. The ER-catalysed reduction was thus followed by
the subsequent reduction of the carbonyl group of both dihydrocarvone isomers, catalyzed by
carbonyl reductases (CRs), which determined the formation of a mixture of four dihydrocarveols
3a–d.
Scheme 3, Bioconversion pathway of (4R)-(-)-carvone by whole-cells of NCYs (non-conventiomal
yeasts) [6]
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4.4 Esterification by lipase
Flavour esters of short-chain carboxylic acid (e.g. isoamyl acetate, citronellyl acetate, geranyl
propionate, neryl acetate, etc) are among the most important flavour and fragrance compounds
used in the food, cosmetic and pharmaceutical industries.
A lipase enzyme has been considered as the most efficient mediator of esterification reactions [3,
4] in the production of various flavours and fragrances. The esterification reaction approach is
most favourable in non aqueous phase [3]; the organic solvents here include ionic liquids (ILs),
supercritical fluids among others [4].
Enzyme-catalyzed direct esterification
The biocatalytic synthesis of different flavour alkyl esters is by direct esterification of an alkyl
carboxylic acid (acetic, propionic, butyric or valeric) with a flavour alcohol (citronellol, geraniol,
nerol or isoamyl alcohol) in the IL N, -hexadecyltrimethylammonium
bis(trifluoromethylsulfonyl) imide ([C16tma][NTf2], see Fig. 3B) as a switchable ionic
liquid/solid phase, used for the reaction and subsequent product separation by centrifugation .
Fig. 3 (A) Flavour esters synthesized by lipase-catalyzed esterification. (B) The IL [C16tma][[NTf2], as
an example of switchable ionic liquid/solid phase.
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Fig 3B. Scheme of the cyclic protocol for the production of flavor esters by lipase-catalyzed direct
esterification in switchable ionic liquid/solid phases, and reusing the enzyme/IL system.
In summary, The ability of hydrophobic ILs based on long alkyl side chains in cations (e.g.
[C16tma][NTf2]) to melt at temperatures compatible with enzyme catalysis (e.g. lower than
80 °C) permitted development of a two-step protocol for flavour ester production: (i) lipase
catalyzed direct esterification between an aliphatic acid and a flavour alcohol with a product
yield close to 100%, and (ii) clean separation of the reaction product by a cooling/centrifugation
method [4].
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5. Challenges
5.1 Low yield and high costs of production
The problem facing the biocatalysis processes is the low yields of the products and high costs
associated to separation and purification of the isolated enzymes; this renders it truly
uncompetitive with the conventional chemical synthesis [6, 7].
An example to illustrate this challenge, synthesis of these aroma compounds has been restricted
in food, beverages, and cosmetics for instance natural 2-PE can be extracted from the essential
oils of certain flowers (e.g. rose flowers) [6]. However, the concentration of 2-PE in flowers is
very low, and the extraction process is therefore complicated and costly. The harvest of flowers
is also influenced by weather conditions; therefore, natural 2-PE from botanical sources cannot
meet the large market demands and is significantly more expensive than its chemically produced
counterpart.
5.2 Toxicity of the substrate and products
Secondly, toxicity of the substrate and products, this has been observed in the production of
nootkatone from nootkatol [7] which is a high value ingredient for the flavor industry because of
its grapefruit flavor/odor. The substrate toxicity is significant only at high concentration
(≥100mgL-1) and the trapping of β-nootkatol in the membranes and cell walls of
microorganisms. Toxicity due to product toxicity was as a result of product accumulation in the
endomembranes [7].
5.3 Enzymes deactivation
Most enzymes are deactivated under extreme conditions such as high pH and temperature as
illustrated in tables below. The optimum pH for enzyme activity ranges from 6 to 8, at extremely
highly temperatures, metabolic rate activity is slowed down.
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Effects of pH (table 1) and temperature (table 2) on enzyme activity.
Table 1
Table 2
5.4 Other challenges
Other challenges affecting application of enzymes industrially include;
i) Instability and often very short life times of biocatalysts in application.
ii) The enzymes in most cases require cofactors to assist them in their catalyzed reactions
such as NAD and ATP.
iii) Enzymes catalyze only a single step of a reaction which limits continuous production of
the target compound and hence resulting into low product yield.
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6. Conclusion
Because of the ever-growing demands for natural products in the world market, flavour
production by biocatalysis has become the focus of extensive research and the ever-increasing
reports on biochemical pathways, genetic modifications, and metabolic engineering will be
useful to further improve the yield of the target product.
Well-established biocatalytic processes have been described both to point out their actual
performance in the flavour production industry. The new outstanding production techniques
offered by biocatalysis have been illustrated by description of some methods of industrial and
academic interest with particular attention to the legal differentiation of flavours.
New strategies for natural flavour biogeneration will take advantage of the current studies on
biotechnology, biochemical pathways and microbiology and the preference of consumers for
natural compounds will support their production. The production of natural flavours using
biocatalysis will enhance the future prospects offered by chemical syntheses rather than compete
with them.
In this field, the most promising biocatalysts are certainly lipases because of their versatility and
selectivity. Lastly but not the least, future research should focus on process scale-up and product
recovery for industrialization. It is important to scale up the production process from flask to
industrial application.
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7. References
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origin:Terpenoids, steroids, phenolics and related compounds.
Bioresource Technology 115 (2012) 237–243
[2] U. Piantini, J. Schrader, A. Wawrzun, M. Wüst, A biocatalytic route towards rose oxide using
Chloroperoxidase. Food Chemistry 129 (2011) 1025–1029
[3] Zi Jin, J. Ntwali, Shuang-Yan Han, S. Zheng, Y. Lin, Production of flavor esters catalyzed by CALB-
displaying Pichia pastoris whole-cells in a batch reactor. J. Biotechnology 159 (2012) 108– 114
[4] P. Lozano, J. M. Bernal and A. Navarro, A clean enzymatic process for producing flavour esters by
direct esterification in switchable ionic liquid/solid phases. Green Chem., 2012, 14, 3026
[5] D. Hua, Ping Xu, Recent advances in biotechnological production of 2-phenylethanol
Biotechnol. Advances 29 (2011) 654–660.
[6] M. Goretti, B. Turchetti, M. R. Cramarossa, L. Forti and P. Buzzini, Production of Flavours and
Fragrances via Bioreduction of (4R)-(-)-Carvone and (1R)-(-)-Myrtenal by Non-Conventional Yeast
Whole-Cells. Molecules 2013, 18, 5736-5748
[7] C. Gavira, R. Höfer, Agnès Lesot , F. Lambert, J. Zucca, D. Werck-Reichhart,
Challenges and pitfalls of P450-dependent (þ)-valencene bioconversion by Saccharomyces cerevisiae.
Metabolic Engineering 18 (2013) 25–35.
[8] M. J. Fink, F. Rudroff, M. D. Mihovilovic, Baeyer–Villiger monooxygenases in aroma compound
synthesis. Bio-organic & Medicinal Chemistry Letters 21 (2011) 6135–6138.
[9] Carla C.C.R. de Carvalho, Enzymatic and whole cell catalysis: Finding new strategies for old processes.
Biotechnology Advances 29 (2011) 75–83
[10] O. Bortolini, P. P. Giovanninia, S. Maiettib, A. Massia, P. Pedrinib, G. Sacchettib, V. Venturib,
An enzymatic approach to the synthesis of optically pure (3R)- and (3S)-enantiomers of green tea
flavour compound 3-hydroxy-3-methylnonane-2,4-dione.
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