Hydroxycinnamate decarboxylase

Grapes contain several hydroxycinnamic acids, p-coumaric, caffeic, ferulic and sinapic acids, which exist as free acids and esterified with tartaric acid. Saccha-romyces species can take up free acids to produce the corresponding vinyl phenol catalysed by hydroxycinnamate decarboxylase (phenylacrylic acid decarboxylase Padlp) (Fig 8D.11) (Chatonnet et al. 1992b Chatonnet et al. 1993 Edlin et al. 1995). Vinyl phenols are unstable and highly reactive. Dekkera bruxellensis is one of few wine microorganisms that can further reduce vinyl phenols to highly stable ethyl phenols in wine. Vinyl phenols can also react with anthocyanins to form vinyl derivatives, a reaction that is favoured by fermentation yeast having hydroxycinnamate decarboxylase activity (Morata et al. 2006). 

Dekkera brwcellensis Spoilage yeast Active hydroxycinnamate decarboxylase and vinylphenol reductase producing ethylphenols in synthetic media, juices and wines Heresztyn (1986) Chatonnet et al. (1995) Shinohara et al. (2000) Rodrigues et al. (2001) Dias et al. (2003a, 2003b)... 

Pichia guilliermondi Contamination yeast Active hydroxycinnamate decarboxylase and vinylphenol reductase producing ethylphenols in synthetic media and grape juices Barata et al. (2006)... 

Volatile phenols originate from hydroxycinnamic acids (ferulic, p-coumaric, or caffeic acid) by the action of hydroxycinnamate decarboxylase enzyme, which turn the hydroxycinnamics acid into vinylphe-nols (Albagnac, 1975 Grando et al., 1993). Then, these compounds are reduced to ethyl derivatives by vinylphenol reductase enzymes characteristic of species, such as Dekkera bruxellensis, Dekkera anomala, Pichia guillermondii, Candida versatilis, Candida halophila, and Candida mannitofaciens (Edlin et al., 1995 1998 Dias et al., 2003 Chatonnet et al., 1992 1995 1997 Dias et al., 2003), apart from very small quantities produced by some yeasts and lactic acid bacteria under peculiar growth conditions (Chatonnet et al., 1995 Barata et al., 2006 ... 

Edlin, D.A.N., Narbad, A., Gasson, M. J., Dickinson, J. R., and Lloyd, D. (1998). Purification and characterization of hydroxycinnamate decarboxylase from Brettanomyces anomalus, Enzyme Microbiol Technol., 22, 232-239. 

Hydroxycinnamic Decarboxylase Vinyl/Ethyl phenols Lactobacilli, Pediococci Off flavors... 

The decarboxylation of 4-hydroxycinnamic acid to 4-hydroxystyrene, and of fernlic acid (3-methoxy-4-hydroxycinnamic acid) to 4-vinylgnaiacol by several strains of Haf-nia alvei and H. protea, and by single strains of Enterobacter cloacae and K. aerogenes (Fignre 2.7d) (Findsay and Priest 1975). The decarboxylase has been pnrihed from Bacillus pumilis (Degrassi et al. 1995). 

Grape compounds which can enter the yeast cell either by diffusion of the undissociated lipophilic molecule or by carrier-mediated transport of the charged molecule across the cell membrane are potentially subject to biochemical transformations by enzymatic functions. A variety of biotransformation reactions of grape compounds that have flavour significance are known. One of the earlier studied biotransformations in yeast relates to the formation of volatile phenols from phenolic acids (Thurston and Tubb 1981). Grapes contain hydroxycinnamic acids, which are non-oxidatively decarboxylated by phenyl acryl decarboxylase to the vinyl phenols (Chatonnet et al. 1993 Clausen et al. 1994). 

Precursors. Both hydroxycinnamic acids and 4-vinylphenols can lead to the formation of hydroxyphenyl-pyranoanthocyanins. The main hydroxycinnamic acids present in wines are p-coumaric, caffeic, ferulic and sinapic acids. 4-Vinylphenol and 4-vinylguaiacol are volatile phenols associated with off flavors in wine (Eti6vant 1981) and arise from the decarboxylation of p-coumaric and ferulic acid, respectively, via the yeast cinnamate decarboxylase (CD) (Chatonnet et al. 1993). 

The precursors of VPs are hydroxycinnamic acids which are enzymatically decar-boxylated by a cinnamate decarboxylase, leading to vinylphenol derivatives, and... 

After the decarboxylation step, vinylphenols may be reduced to ethylphenols but the sequential decarboxylase and reductase activities, regarding wine yeasts, have only been demonstrated in D. bruxellensis and in P. guilliermondii (Barata et al. 2006). The former species may also convert 4-VP into 4-EP in the absence of hydroxycinnamic acids (Dias et al. 2003b). 

The use of certain pectolytic enzyme preparations to facilitate the extraction or clarification of white must may lead to an increase in the vinyl-phenol content of white wines and a deterioration of their aromatic qualities (Chatonnet et al 1992a Dugelay etal., 1993 Barbe, 1995). Indeed, certain industrial pectinases, made from Aspergillus niger cultures, have a cinnamyl esterase (CE) activity. This enzyme catalyzes the hydrolysis of tartrate esters of hydroxycinnamic acids in must during the pre-fermentation phase (Figure 8.7). Feruhc and j9-coumaric acids are then converted into vinyl-phenols during alcohohc fermentation due to the cinnamate decarboxylase activity of Saccharomyces cerevisiae. 

It is believed that these compounds, characteristic of Brettanomyces and Dekkera, result from decarboxylation of hydroxycinnamic acids, yielding vinyl phenol intermediates and subsequent reduction to produce the ethyl analog (Steinke and Paulson, 1964). As seen in Fig. 3-3 initial decarboxylation is mediated by cinnamate decarboxylase, whereas the reduction step utilizes a vinyl phenol reductase. 

Biochemically, 4-ethyl guaiacol and 4-ethyl phenol originate from ferufic acid and /vcoumaric acid, respectively. The reaction is a two-step process with an initial decarboxylation of the hydroxycinnamic acids catalyzed by cinnamate decarboxylase and the reduction of the vinyl phenol intermediates by vinyl phenol reductase (Fig. 11.1). Although the specific coenzyme involved remains unknown, one possible metabolic benefit of the second reaction to Brettanomyces could be reoxidation of NADH. Under low oxygen conditions such as those found in wines, the availability of NAD can be limited so that carbohydrate metabolism is inhibited (Section 1.5.1). Reduction of the vinyl phenols to the ethyl phenols would allow the cell to increase the availability of NAD and thus maintain metabolic functions. 

Some phenolic acid decarboxylases [60, 62-65] which catalyze the conversion of hydroxycinnamic acids into styrenes are also of interest. Considering the structural data available to date, both enzymes work with different mechanisms ... 

Priest et al. (2000) isolated seven strains of Lactobacillus (Lactobacillus brevis, L. crispatus, L. fermentum, L. hilgardii, L. paracasei, L. pentosus and L. planta-rum) from malt whisky fermentation that contained genes for hydroxycinnamic acid (p-coumaric acid) decarboxylase (Scheme 3.25). 

With the exception of L. hilgardii, these bacteria decarboxylated p-coumaric acid and/or ferulic acid, with the production of 4-VP and/or 4-VG, respectively, although the relative activities on the two substrates varied between strains. The addition of p-coumaric acid or ferulic acid to cultures of L. pentosus in MRS broth induced hydroxycinnamic acid decarboxylase mRNA within 5 min, and the gene was also induced by the indigenous components of malt wort. 

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