Acetoin reductase

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.

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%. 

Siemerink MAJ, Kuit W, Lopez Contreras AM, Eggink G, van der Oost J, Kengen SWM. (2011). D-2,3-Butanediol production due to heterologous expression of an acetoin reductase in Clostridium acetobutylicum. Appl Environ Microbiol, 11, 2582-2588. 

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... 

A newer model for K. pneumoniae was similar to the earlier one in that it included an acetoin racemase. However, in this newer model, D(-) acetoin is converted to meso-2,3-BD, and L(-l-) acetoin is converted to L(-l-) 2,3-BD. This model is based on the purification and separation of the two acetoin reductases and the determination of their stereospecificity (Voloch et al. 1983) (O Fig. 2.6). 

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). 

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

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. 

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. 

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. 

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