Acetoin formation

Unfortunately diacetyl formation is still not well understood. Acetoin formation occurs either by nonspecific interaction of acetaldehyde with the a-hydroxyethyl thiamine pyrophosphate intermediate in pyruvate decarboxylation (209) or by decarboxylation of a-acetolactate (210), which in turn arises either from interaction of pyruvate with a-hydroxyethyl thiamine pyrophosphate (211) or as a specific intermediate in valine biosynthesis (212, 213). Diacetyl does not appear to be formed directly from acetoin (208, 214). It is formed from a-acetolactate, in absence of cells, by O2 oxidation (215), and even under N2 (216), although an oxidation must occur. It is also formed from acetyl CoA (217, 218), probably by interaction with a-hydroxyethyl thiamine pyrophosphate [cf. stimulation by acetyl CoA addition to a solution of pyruvate and pyruvate decarboxylase (2i5)]. It is not known whether this involves a specific enzyme or is a mere side reaction. 

The results plotted at Figure 1 seem to be useful for elucidation of the reaction pathway. In the course of butadione formation two hydrogen molecules of 2,3-butanediol must be eliminated. The elimination can proceed either simultaneously or step by step via consecutive elimination of hydrogen molecules and intermediate formation of acetoin. One can see that conversion of the latter into diacetyl proceeds faster and at lower temperature as compared to the conversion of butanediol. By increasing the temperature and conversion of butanediol the curve of acetoin formation passes maximum and the curve of diacetyl formation has an induction period. Thus, one can believe that the conversion proceeds mainly via consecutive elimination of hydrogen molecules and intermediate formation of acetoin ... 

The lipoic acids are believed to have a general function in the oxidative decarboxylation of a-keto acids.The outstanding example is pyruvic acid, but it also functions in the oxidation of a-ketoglutaric and a-ketobutyric acids. The latter acid is employed as a substrate to study the characteristics of the enzyme system to avoid the complicating effect of acetoin formation which occurs with pyruvate. ... 

Ketol condensation as in acetoin formation involves the linkage of two carbon atoms each bearing an aldehyde function ... 

Cocoa bean fermentation is a mixed-culture process, consisting initially of fermentations by yeast and lactic acid bacteria followed by oxidation of the fermentation products ethanol and lactic acid into acetic acid and acetoin by several Acetohacter strains, of which /I. pasteurianus is the prominent one (Moens et al. 2014). A C-based carbon flux analysis of Acetohacter during cocoa pulp fermentation-simulating conditions revealed a functionally separated metabolism during co-consumption of ethanol and lactate. Acetate was almost exclusively derived from ethanol, whereas lactate served for formation of acetoin and biomass building blocks. This switch was attributed to the lack of phosphoenolpyruvate carboxykinase and malic enzyme activities, which prevents conversion of oxalo-acetate and malate formed by acetate metabolism in the TCA cycle to PEP and pyruvate and subsequently to acetoin (Adler et al. 2014). Lactate, on the other hand, can be converted to pyruvate, which is then used for acetoin formation or, after conversion to PEP by pymvate phosphate dikinase, for gluconeogenesis. The inability of conversion of TCA cycle intermediates to PEP resembles the situation in G. oxydans, where in addition no enzyme for conversion of pyruvate to PEP is present. 

Microbial acetoin formation has been extensively studied due to a special industrial interest, since this is a key step for the fermatative production of 2,3-butanediol (31), an important starting material in polymer industries. 2,3-Butanediol has been produced by means of Serratia marcescens [53], Bacillus polymyxa [53,54], Klebsiella oxytoca [55,56], and Lactobacillus plantarum [57]. In these cases, however, the biosynthesis of acetoin mainly proceeds through another pathway [58,59]. The key enzyme is acetolactate synthase (EC 4.1.3.18), which catalyzes the nucleophilic attack of TPP-thiazolium intermediate on another molecule of pyruvate [Eq. (20)]. The pathway from acetolactate to acetoin via the... 

The formation of acyloins (a-hydroxyketones of the general formula RCH(OH)COR, where R is an aliphatic residue) proceeds best by reaction between finely-divided sodium (2 atoms) and esters of aliphatic acids (1 mol) in anhydrous ether or in anhydrous benzene with exclusion of oxygen salts of enediols are produced, which are converted by hydrolysis into acyloins. The yield of acetoin from ethyl acetate is low (ca. 23 per cent, in ether) owing to the accompanying acetoacetic ester condensation the latter reaction is favoured when the ester is used as the solvent. Ethyl propionate and ethyl ji-butyrate give yields of 52 per cent, of propionoin and 72 per cent, of butyroin respectively in ether. 

In general, pyruvate decarboxylase (EC 4.1.1.1) catalyzes the decarboxylation of a 2-oxocar-boxylic acid to give the corresponding aldehyde6. Using pyruvic acid, the intermediately formed enzyme-substrate complex can add an acetyl unit to acetaldehyde already present in the reaction mixture, to give optically active acetoin (l-hydroxy-2-butanone)4 26. Although the formation of... 

From analysis of the data in Figure 5 it is clear that the rate of PAC formation is fairly constant for the first 9 h to approx. 180 mM PAC and subsequently declines after this to reach a maximum value of 300 mM in 54 h. Unpublished studies by our group have shown no significant inhibition effects of PAC up to concentrations of 154 mM [13]. However, it is possible that appreciable inhibition could occur at higher PAC concentrations. Byproducts acetoin and acetaldehyde may also be inhibitory in the latter stages of the biotransformation although both concentrations were below 5 mM at 9 h. 

The B. licheniformis JF-2 strain produces a very effective surfactant under conditions typical of oil reservoirs. The partially purified biosurfactant from JF-2 was shown to be the most active microbial surfactant found, and it gave an interfacial tension against decane of 0.016 mN/m. An optimal production of the surfactant was obtained in cultures grown in the presence of 5% NaCl at a temperature of 45° C and pH of 7. TTie major endproducts of fermentation were lactic acid and acetic acid, with smaller amounts of formic acid and acetoin. The growth and biosurfactant formation were also observed in anaerobic cultures supplemented with a suitable electron acceptor, such as NaNO3[1106]. 

Several products were also detected in base-degraded D-fructose solution acetoin (3-hydroxy-2-butanone 62), l-hydroxy-2-butanone, and 4-hydroxy-2-butanone. Three benzoquinones were found in the product mixture after sucrose had been heated at 110° in 5% NaOH these were 2-methylbenzoquinone, 2,3,5-trimethylbenzoquinone, and 2,5-dimethyl-benzoquinone (2,5-dimethyl-2,5-cyclohexadiene-l,4-dione 61). Compound 62 is of considerable interest, as 62 and butanedione (biacetyl 60) are involved in the formation of 61 and 2,5-dimethyl-l,4-benzenediol (63) by a reduction-oxidation pathway. This mechanism, shown in Scheme 10, will be discussed in a following section, as it has been proposed from results obtained from cellulose. 

The goal of this contribution is to review the formation of at least some of these compounds in model systems (consisting of aldehydes, acetoin, and ammonium sulfide), their identification, and analytical characterization (mass spectra, Kov ts indices) accomplished by using the GC-MS-SPECMA data bank. 

Scheme 23.5 Metabolic pathways of lactic acid bacteria leading from pyruvate to a-acetolactate and acetoin and chemical diacetyl formation. ALS a-acetolactate synthase, ALDB a-acetolactate decarboxylase, DDH diacetyl dehydrogenase. (Adapted from [72])...

Thiamin itself (in the absence of enzyme) had previously been shown to catalyse the formation of acetoin from acetaldehyde, albeit in very poor yield (Ukai et al., 1943 Mizuhara et al., 1951 Mizuhara and Handler, 1954). The reaction parallels the formation of benzoin from benzaldehyde, catalysed by cyanide ion. The mechanism of the latter reaction had been suggested in 1903 by Arthur Lapworth, who had shown how an aldehyde, R—CHO, could be converted into the equivalent of the anion R—C=0- (Lapworth, 1903). It is this idea that Breslow carried over to thiamin pyrophosphate and used to... 

The highly flavorable compound diacetyl is an important by-product of lactic acid bacterial fermentation. The mechanism of its formation has recently been unraveled (35). Diacetyl (measured as diacetyl rather than as diacetyl plus acetoin) is present in higher concentrations in wines with malo-lactic fermentation (cf. Ref. 36). At approximately threshold levels, this compound might contribute favorably to the flavor of wine (7) since increased complexity has been shown to enhance the quality of wine (37). 

As a side activity, many decarboxylases catalyze the formation of C-C bonds. In the reaction of two pyruvate molecules, catalyzed by pyruvate decarboxylase (PDC, E.C. 4.1.1.1), a-acetolactate is formed, an important intermediate of valine biosynthesis. In turn, a-acetolactale can be oxidatively decarboxylated by oxygen to diacetyl or enzymatically decarboxylated by acetolactate decarboxylase (ADC, E.C. 4.1.1.5) to (] )-acetoin (Figure 7.29). 

A detailed investigation of the carboligase reaction mediated by PDC from yeast, wheat germ and Z. mobilis revealed that the stereo-control of this reaction is only strict with aromatic aldehydes as acylanion-acceptors, while the formation of acetoin (3-hydroxybutan-2-one) resulted in mixtures of the (R)- and (S)-enantiomer. 

These differences in the control of the product stereochemistry have recently been investigated by molecular modeling techniques [60,154], From these studies, the relevance of the side-chain of isoleucine 476 (PDCS.c.) (Table 2) for the stereo-control during the formation of aromatic a-hydroxy ketones became obvious, since this side-chain may protect one site of the ot-carbanion/enamine 6 (Scheme 3) against the bulky aromatic cosubstrate. Nevertheless, the smaller methyl group of acetaldehyde can bind to both sites of the a-carbanion/en-amine. The preference for one of the two acetoin enantiomers has been interpre-tated in terms of different Boltzmann distributions between the two binding modes of the bound acetaldehyde [155],... 

There was no difference observed between wt-PDCZ.m. and the mutant enzymes with respect to the formation of acetoin. However, the biotransformation of benzaldehyde to (R)-PAC 1 was best performed with the mutant enzymes PDCW392M and PDCW392I (Fig. 5). As a consequence of these studies, Trp392 in PDCZ.m. could be characterized as a further key residue which is responsible for the differences between PDCS.c. and PDCZ.m.. Besides the absence of a regulatory apparatus in PDCZ.m., which was shown to impair the catalytic power of PDCS.c. [117], the Trp-residue is partially responsible for the higher stability and also for the higher activity of the bacterial enzyme. 

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