Diacetyl (Acetoin) Reductase from Aerobacter aerogenes

Abstract

The kinetics of the reversible reduction of acetoin and NADH to 2,3-butanediol andNAD by diacetyl (acetoin) reductase from Aerobacter aerogenes, have been investigated at pH 7.0, 5.8, and 4.8. The reaction follows an ordered bi-bi mechanism.Acetate acts as an inhibitor of the enzyme with respect to the substrate 2,3-butanediol. This inhibition increases for decreasing pH, indicating that it is acetic acid that is the effector.These results, in combination with earlier observations, show that high acetate concentration allows maximal 2,3-butanediol and NAD production at 5.8 from pyruvate and NADH. The kinetics of the pH 6 acetolactate-forming enzyme has been investigated [3,4]. It has a sharp pH-optimum at pH 5.8 in acetate, which also acts as an activator for the enzyme. Acetate also acts as an inducer for the three enzymes leading from pyruvate to 2,3-butanediol [2,5]. Acetolactate decarboxylase has a pH-optimum a t about 6.3 in phosphate, and gives about 75O/, of optimal activity a t yH 5.8. Acetate has no effect upon the enzyme activity [6]. This indicates that the formation of acetoin is maximally favored a t pH 5.8 in the presence of acetate.Diacetyl (acetoin) reductase has recently been purified into homogeneity [2]. The enzyme is a tetramer, consisting of four equal-sized subunits [7]. The native enzyme consists of at least 12 isoelectric species, all possessing enzyme activity [8].To gain an insight into the mechanism of diacetyl (acetoin) reductase reaction, a kinetic study was carried out on reaction 3 in Fig. 1 a t pH 5.8 in acetate, phosphate and 2-(N-morpholino)-ethane-sulfonate, pH 7.0 in phosphate and pH 4.8 in acetate and morpholinoethane sulfonate. EXPERIMENTAL PROCEDURE ChemicalsThe following chemicals were purchased from Sigma Chemical Company: NAD, NADH, NADP, and NADPH. Acetoin was obtained from KochLight, 2,3-butanediol from Fluka AG, and 24N-morpho1ino)-ethane-sulfonic acid from Calbiochem. Preparation and Assay of Diacetyl (Acetoin) ReductaseThe enzyme was purified from crude extracts of Aerobacter aerogenes [2]. For spectrophotometric determinations of enzyme activity, the Zeiss model PMQII recording spectrophotometer with the thermostated cell compartment a t 25 "C was used.Initial reaction rates were measured by observing the rate of change in the absorbance at 340nm.

Process energy efficiency in pervaporative and vacuum membrane distillation separation of 2,3‐butanediol

“…As a fermentation product, 2,3‐butanediol has long been produced by distillation (Larson and Stormer, 1973; Magee and Kosaric, 1987; Garg and Jain, 1995; Lee, 1999; Jansen and Tsao, 2007). Because of the low concentration of 2,3‐butanediol in the fermentation broth, and the stronger hydrogen bonding interaction between water molecules, recovery of 2,3‐butanediol by distillation is a very energy‐intensive process (Neel and Huang, 1991; Shao and Huang, 2007).…”

Enhanced production of 2,3‐butanediol by overexpressing acetolactate synthase and acetoin reductase in Klebsiella pneumoniae

“…Lactate is the most abundant by-product that consumes carbon flux and NADH in K. pneumoniae and competes with 2,3-BD biosynthesis [9]. Reducing the lactate and acetate yield decreased the acidification rate of the media, which is not favorable to cell growth [9,27]. Previous studies have reported that lactate and acetate can effectively inhibit Klebsiella cell growth, thereby affecting 2,3-BD production [28][29][30].…”

A 2,3‐butanediol dehydrogenase from Paenibacillus polymyxaZJ‐9 for mainly producing R,R‐2,3‐butanediol: Purification, characterization and cloning

“…The enzymatic characteristics of BDH purified from P. polymyxa ZJ-9 were compared with those of BDH from K. pneumoniae IAM 1063 [8], Klebsiella terrigena VTT-E-74023 [17], Aerobacter aerogenes 1033 [18], and Saccharomyces cerevisiae [19] ( Table 2). The BDH enzyme of P. polymyxa ZJ-9 differed from those of the other strains in terms of its molecular weight, subunits, and K m values for some substrates.…”