Reductases diacetyl reductase

Diacetyl, acetoin, and diketones form during fermentation. Diacetyl has a pronounced effect on flavor, with a threshold of perception of 0.1—0.2 ppm at 0.45 ppm it produces a cheesy flavor. U.S. lager beer has a very mild flavor and generally has lower concentrations of diacetyl than ale. Diacetyl probably forms from the decarboxylation of a-ethyl acetolactate to acetoin and consequent oxidation of acetoin to diacetyl. The yeast enzyme diacetyl reductase can kreversibly reduce diacetyl to acetoin. Aldehyde concentrations are usually 10—20 ppm. Thek effects on flavor must be minor, since the perception threshold is about 25 ppm. 

Figure 13.8 Pathway for metabolism of citrate by Leuconostoc spp. and S. lactis subsp. diacetylactis. (1) Citrate permease, (2) citrate lyase, (3) oxaloacetic acid decarboxylase, (4) pyruvate decarboxylase, (5) a-acetolactate synthetase, (6) a-acetolactate carboxylase, (7) diacetyl synthetase, (8) diacetyl reductase, and (9) acetoin reductase.

Diacetyl reductase (acetoin dehydrogenase) isolated from Lactobacillus kefir converts the prochiral diacetyl into optically pure (+)-acetoin (ee>94%) [145]. 

Diacetyl reductase (acetoin dehydrogenase, 1.1.1.5) is widespread in bacteria (207, 208, 219, 220, 221), including the species (Streptococcus diacetilactis, Lactobacillus casei) used to prepare cultured dairy products. Mutants lacking diacetyl reductase also fail to synthesize diacetyl (222). The enzyme has been purified 30-fold from L. casei (223). The activity was not fully separable from an NADH oxidase activity, and the enzyme appeared to be a flavoprotein. Maximum activity was at pH 4.5. The NADH oxidase activity is associated with diacetyl reductase in other sources. 

Figure 15-40. Reduction of a-diketones by diacetyl reductase from Bacillus stearothermophilus 233).

Figure 16.2-22. Kinetic resolution of racemic syn-diols by Bacillus stearothermophilus diacetyl reductase (BSDR). A reaction with LDH-catalyzed regeneration of NAD+ B selection of syn-diols applied.

Recently, diacetyl reductase (Acetoin reductase, E.C. 1.1.1.5) from Bacillus stearo-thermophilus (BSDR) was reported to be a powerful catalyst in the oxidative kinetic resolution of vic-diols (Fig. 16.2-22)1901. All syn-diols tested yielded the enantiopure (R,R) diols in almost maximum theoretical yields, a-hydroxy ketones were largely further oxidized to the corresponding diketones. Oxidation of vic-anti diols only gave ee values in the range of 62-76%. 

Recently isolated from baker s yeast are two diacetyl reductases. One enzyme shows a high S and the other a high R selectivity for the reductions of 2,3-pentanedione, 2,3-hexanedione and other mono- and dikctoncs218. 

Unfortunately diacetyl is not stable in most cultured food products. The microorganisms that synthesize diacetyl also contain diacetyl reductase that reduce diacetyl to acetoin and 2,3-butanediol (Figure 5.9). Thus fermented products such as buttermilk, which depend on diacetyl for flavor have an optimum or peak flavor. As the product is stored, the diacetyl is reduced and flavor strength and quality decrease. 

Fig. 10.33. Formation of diacetyl and butanediol from citrate by Streptococci. 1 citratase, 2 oxaloac-etate decarboxylase, 3 pyruvate decarboxylase, 4 a-acetolactate synthase, 5 diacetyl reductase, 6 a-acetolactate decarboxylase, 7 2,3-butanediol dehydrogenase...

A novel mechanism for stereoisomer formation was described for Bacillus polymyxa. The RR-acetoin formed from pyruvate is converted into RR-butanediol by diacetyl (acetoin) reductase. The same enzyme reduces diacetyl to RR-acetoin. An S-acetoin-forming diacetyl reductase converts diacetyl to SS-acetoin. The racemic acetoin molecules are acted upon by a butanediol dehydrogenase, which generates either RR-butanediol or meso-butanediol (Ui et al. 1986). 

Three enzymes are involved in the synthesis of 2,3-BD a-acetolactate synthase (EC 4.1.3.18), a-acetolactate decarboxylase (EC 4.1.1.5), and butanediol dehydrogenase (also known as diacetyl [acetoin] reductase Larsen and Stormer 1973 Johansen et al. 1975 Stormer 1975). Two different enzymes form acetolactate from pyruvate. The first, termed catabolic a-acetolactate synthase, has a pH optimum of 5.8 in acetate and is part of the butanediol pathway. The other enzyme, termed anabolic a-acetolactate synthase or acetohydroxyacid synthetase, has been well studied and characterized and will not be discussed here. This enzyme is part of the biosynthetic pathway for isoleucine, leucine, and valine and is coded for by the ilvBN, ilvGM, and ilvH genes in E. colt and Salmonella typhimurium (Bryn and Stormer 1976). 

The second enzyme in the butanediol pathway is acetolactate decarboxylase, which has a pH optimum of about 6.3 and which catalyzes the decarboxylation of acetolactate to acetoin. The third enzyme, diacetyl (acetoin) reductase, catalyzes a reversible reduction of acetoin to 2,3-BD and an irreversible... 

Larsen SH, Stormer FC (1973) Diacetyl (Acetoin) reductase from Aerobacter aerogenes. Eur J Biochem 34 100-106... 

Figure 1.2. Citrate metabolism in Lactococcus, Leuconostoc, and Weissella species. Key for the enzymes CL, citrate lyase OAD, oxaloacetate decarboxylase LDH, lactate dehydrogenase PDC, pyruvate decarboxylase ALS, a-acetolactate synthase ADC, a-acetolactate decarboxylase DAR, diacetyl acetoln reductase BDH, 2,3-butanediol dehydrogenase Tppi, thiamine pyrophosphate.

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