PQQ-ADH

PQQ-ADH is classified into three groups type I (ADH I), type II (ADH II), and type III (ADH III) (Toyama et al. 2004). Type II and type III have heme C as well as PQQ in the catalytic site, and thus are called quinohemoprotein ADH, whereas type I has only PQQ in the catalytic subunit (quinoprotein ADH). The phylogeny of PQQ-ADH is divided into large two clades, ADH I and ADH n, both of which include functionally validated enzymes of Pseudomomas aeruginosa (ADH I), Pseudomonas fluorescence (ADH II), and Comamonas testosterord (ADH II)... 

A direct link has been established between acetic acid resistance and the capability to oxidize ethanol during the diauxic growth curve in AAB (Ohmori et al. 1982). The PQQ-ADH activity in Komagataeibacter strains reached higher values (at least twice as high) than those in Acetobacter, under the same growth conditions (Trcek et al. 2006, 2007). 

Experimental evidence caused us to suspect that Komagataeibacter strains share most of the resistance mechanisms shown by Acetobacter sp., although they also must develop specific strategies that enable tolerance to the drastic conditions of submerged vinegar production. We have already addressed the higher activity and stability of PQQ-ADH enzyme compared with that in Acetobacter sp., as well as their specialized lipid membrane composition (Trcek et al. 2006, 2007). 

Biosensors constructed for ethanol and D-glucose measurements in beverages were built using ferrocene compounds as electron transfer mediators between the coenzyme PQQ of immobilized enzymes glucose (GDH) and alcohol (ADH) dehydrogenases and a carbon electrode surface <2003JOM(668)83>. 

Trcek, J., Toyama, H., Czuba, J., Misiewicz, A., and Matsushita, K. 2006. Correlation between acetic acid resistance and characteristics of PQQ-dependent ADH in acetic acid bacteria. Applied Microbiology and Biotechnology 70 366-373. 

A direct electron transfer from entrapped quinohemoprotein alcohol dehydrogenase (QH-ADH) to a Pt electrode, via chains of the polypyrrole, acting as immobilization matrix, was demonstrated [152]. QH-ADH is able to translocate in a fast inner-enzymatic reaction, the electrons primarily accepted by PQQ to heme units located close to the outer protein shell, from where they can be transferred on the conducting-polymer chains (Fig. 13). A similarity between the electron-transfer pathway in multicofactor proteins and that of mediator-modified electroenzymes is apparent, if one considers that a multicofactor enzyme can be regarded as a combination of a primary redox site and protein-integrated electron-transfer relays. 

Evidence for the direct electron transfer from PPy to an entrapped quinohemoprotein alcohol dehydrogenase (QH-ADH) from Gluconobacter sp.33 prepared via an in situ polymerization of pyrrole in the presence of QH-ADH has been demonstrated by Ramanavicius et aL [70]. It was proposed that the cooperative action of the pyrroloquino-line-quinone (PQQ) and heme-containing enzymes permit electron transfer from the enzyme active site to the ICP. Ethanol is said to diffuse to the PQQ enzyme centre where it is oxidized to an aldehyde. The PQQ centre is subsequently regenerated by the heme sites in the enzyme. Resulting is an electron that can be readily transferred to the PPy at a viable kinetic rate. 

The acetic acid bacteria produce acetic acid from ethanol hy two enzyme-catalyzed reactions of memhrane-hound alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). In addition, some acetic acid bacteria can oxidize various sugars and sugar alcohols. These reactions are all incomplete oxidations because the oxidation products are usually accumulated in large amounts in the bacterial beers. Both ADH and ALDH have pyrroloquinohne quinone (PQQ) bound as a prosthetic group and are linked to the respiratory chain in the cytoplasmic membrane (O Fig. 1.3). A second set of NAD(-P) -dependent ADHs and ALDHs is found in the cytoplasm of acetic add bacteria. These latter two enzymes have much lower specific activities than those of the PQQ-enzymes and are not involved in acetic add production (seeO Fig. 1.3). 

The membrane-bound PQQ-alcohol dehydrogenase (ADH) is to be considered the key enzyme in the production of vinegar because of its essential place in the oxidation of ethanol to acetaldehyde, the intermediate that will be later oxidized by the MCD (molybdopterin cytosine dinucleotide)-aldehyde dehydrogenase (ALDH) to acetic acid. This idea is based on the finding that a defect in membrane-bound ADH has been associated with a reduction in acetic acid resistance (Chinnawirotpisan et al. 2003 Okumura et al. 1985 Takemura et al. 1991). Additionally, the elevation in membrane-bound ALDH activity by gene amplification has been reported to enhance the acetic acid concentration finally attained in Acetobacter (Fukaya et al. 1989). 

Trcek J, Barja F (2015) Updates on quick identification of acetic acid bacteria with a focus on the 16S-23S rRNA gene internal transcribed spacer and the analysis of cell proteins by MALDI-TOF mass spectrometry. Int J Food Microbiol 196 137-144 Trcek J, Raspor P, Teuber M (2000) Molecular identification of Acetobacter isolates fiom submerged vinegar production, sequence analysis of plasmid pJK2-l and application in development of a cloning vector. Appl Microbiol Biotechnol 35 1899-1901 Trcek J, Toyama H, Czuba J, Misiewicz A, Matsushita K (2006) Correlation between acetic acid resistance and characteristics of PQQ-dependent ADH in acetic acid bacteria. Appl Microbiol Biotechnol 70(3) 366-373... 

The growth of Acetobacter species on ethanol results in the accumulation of acetate, which is then completely oxidized by the phenomenon termed acetate overoxidation. The sequential oxidation of ethanol and acetate leads to a diauxic growth profile that is characterized by acetate accumulation in the first exponential growth phase and complete oxidation of the accumulated acetate in the secrnid exponential phase (Saeki et al. 1997). However, in some conditions, ethanol is oxidized to acetate both in the periplasm, by membrane-bound PQQ-dependent ADH and aldehyde dehydrogenase (ALDH), and in the cytoplasm, by soluble NAD (P) -dependent ADH and ALDH. These conditimis are unfavorable for vinegar production because acetate generated in the cytoplasm tends to be rapidly metabolized via the tricarboxylic acid (TCA) cycle. 

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