Generation mechanism and purification of an inactive form convertible in vivo to the active form of quinoprotein alcohol dehydrogenase in Gluconobacter suboxydans

Abstract

Alcohol dehydrogenase (ADH) of acetic acid bacteria is a membrane-bound quinohemoprotein-cytochrome c complex involved in vinegar production. In Gluconobacter suboxydans grown under acidic growth conditions, it was found that ADH content in the membranes was largely increased but the activity was not much changed, suggesting that such a condition produces an inactive form of ADH (inactive ADH). A similar phenomenon could be also observed in Acetobacter aceti, another genus of acetic acid bacteria. Furthermore, aeration conditions were also shown to affect ADH production; the ADH level was increased and was present as an active form under low-aeration conditions, while the ADH level was decreased and was present mainly as an inactive form under high-aeration conditions. Inactive ADH was solubilized from the membranes of G. suboxydans grown in acidic and high-aeration conditions and was purified separately from the normal, active form of ADH (active ADH). In spite of having 10 times less enzyme activity than active ADH, inactive ADH could not be distinguished from active ADH with respect to their subunit compositions, molecular sizes, and prosthetic groups. Inactive ADH, however, had a relatively loose conformation with a partially oxidized state, while active ADH had a tight conformation with a completely reduced state, suggesting that inactive ADH may lack a right subunit's interaction and that one of the heme c components may be inactivated. Reactivation from such an inactive ADH occurred either by shifting of the pH of the culture medium up during the cultivation or by incubation of the resting cells at the neutral pH region in the presence of an energy source such as D-sorbitol. Such an activation of ADH was repressed by the addition of a proton uncoupler and could not occur in the spheroplasts. Thus, the results suggest that inactive ADH could be generated abundantly under acidic growth conditions and converted to the active form at a neutral culture pH. The data also suggest that some periplasmic component may be involved in the conversion of inactive ADH into the active form by consuming some forms of energy.

Development of Thermotolerant Acetic Acid Bacteria Useful for Vinegar Fermentation at Higher Temperatures

“…It is interesting to see that inactive ADH is formed in G. suboxydans when the organism is grown under vigorous aeration in acidic pH. 10) It is said that a similar phenomenon also occurs in the genus Acetobacter. The highest concentration of acetic acid (about 5.5%) was detected after 10 days of incubation when the pH of the culture medium dropped from 3.5 to 2.9.…”

“…9 ) Furthermore, an inactive ADH convertible in vivo to the active form is formed in Gluconobacter suboxydans and the generation mechanism and physiological function are under investigation. 10) Hot summer in these couple of years has brought indoor temperature increases beyond 30°C even in night time in many countries. That is a serious challenge to not only vinegar fermentation but also other fermentation industries, since they need a huge amount of cooling water to maintain the optimum fermentation temperature.…”

Microbiological Aspects of Acetate Oxidation by Acetic Acid Bacteria, Unfavorable Phenomena in Vinegar Fermentation

“…pasteurianus SKU 1102, SKU 1106, and SKU 1111 (G. Theeragool et at., unpublished observations). Recently, Matsushita et al 30) reported the formation of inactive ADH when G. suboxydans was grown in acidic and high-aeration conditions. It is interesting to see that a similar phenomenon can be observed in A. aceti.…”

Molecular and Catalytic Properties of the Aldehyde Dehydrogenase of Gluconacetobacter diazotrophicus , a Quinoheme Protein Containing Pyrroloquinoline Quinone, Cytochrome b , and Cytochrome c

“…The oxidoreductase activity of ALDH was measured spectrophotometrically in a Shimadzu UV-2401 PC UV-visible light (UVVis) spectrophotometer (Shimadzu, Kyoto, Japan) at pH 3.5 using potassium ferricyanide as an electron acceptor as described by Matsushita et al (22) or at pH 7.0 using PMS plus 2,6-dichlorophenolindophenol (DCPIP) as electron acceptors, as described previously (8). One unit of the enzyme activity is the amount of enzyme that catalyzes the oxidation of 1.0 mol of substrate per min under the indicated conditions.…”