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.

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

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 flavo urs 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 lions 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.

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.

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.

To illustrate one of the techniques applied; two phase extraction, using aqueous organic twophase 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). 7

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].

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.

Carvone is an important element; their dihydrocarveols are valuable ingredients currently applicable in the flavour and fragrance industry. The biotransformations of the ,-unsaturated ketone (4R)-()-carvone (1) catalyzed by wholecells 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 ,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 3ad.

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 -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
[1] A-L Groussin, S. Antoniotti, Valuable chemicals by the enzymatic modification of molecules of natural origin:Terpenoids, steroids, phenolics and related compounds. Bioresource Technology 115 (2012) 237243 [2] U. Piantini, J. Schrader, A. Wawrzun, M. Wst, A biocatalytic route towards rose oxide using Chloroperoxidase. Food Chemistry 129 (2011) 10251029 [3] Zi Jin, J. Ntwali, Shuang-Yan Han, S. Zheng, Y. Lin, Production of flavor esters catalyzed by CALBdisplaying 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) 654660.

[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. Hfer, Agns Lesot , F. Lambert, J. Zucca, D. Werck-Reichhart,

Challenges and pitfalls of P450-dependent ()-valencene bioconversion by Saccharomyces cerevisiae. Metabolic Engineering 18 (2013) 2535. [8] M. J. Fink, F. Rudroff, M. D. Mihovilovic, Baeyer Villiger monooxygenases in aroma compound synthesis. Bio-organic & Medicinal Chemistry Letters 21 (2011) 61356138. [9] Carla C.C.R. de Carvalho, Enzymatic and whole cell catalysis: Finding new strategies for old processes. Biotechnology Advances 29 (2011) 7583 [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. J. Molecular Catalysis B: Enzymatic 85 86 (2013) 93 98

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