Purification and characterization of polyethylene glycol dehydrogenase involved in the bacterial metabolism of polyethylene glycol

Polyethylene glycol (PEG) dehydrogenase in crude extracts of a PEG 20,000-utilizing mixed culture was purified 24 times by precipitation with ammonium sulfate, solubilization with laurylbetaine, and chromatography with diethylamino-ethyl-cellulose, hydroxylapatite, and Sephadex G-200. The purified enzyme was confirmed to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme, which appeared to consist of four identical subunits, was 2.4 X 10(5). The enzyme was stable below 35 degrees C and in the pH range of 7.5 to 9.0. The optimum pH and temperature of the activity were around 8.0 and 60 degrees C, respectively. The enzyme did not require any metal ions for activity and oxidized various kinds of PEGs, among which PEG 6,000 was the most active substrate. The apparent Km values for tetraethylene glycol and PEG 6,000 were about 10.0 and 3.0 mM, respectively.     

Purification and characterization of polyethylene glycol dehydrogenase involved in the bacterial metabolism of polyethylene glycol.

Polyethylene glycol (PEG) dehydrogenase in crude extracts of a PEG 20,000-utilizing mixed culture was purified 24 times by precipitation with ammonium sulfate, solubilization with laurylbetaine, and chromatography with diethylamino-ethyl-cellulose, hydroxylapatite, and Sephadex G-200. The purified enzyme was confirmed to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme, which appeared to consist of four identical subunits, was 2.4 X 10(5). The enzyme was stable below 35 degrees C and in the pH range of 7.5 to 9.0. The optimum pH and temperature of the activity were around 8.0 and 60 degrees C, respectively. The enzyme did not require any metal ions for activity and oxidized various kinds of PEGs, among which PEG 6,000 was the most active substrate. The apparent Km values for tetraethylene glycol and PEG 6,000 were about 10.0 and 3.0 mM, respectively.     

Purification and characterization of constitutive polyethylene glycol (PEG) dehydrogenase of a PEG 4000-utilizing Flavobacterium sp. no. 203

Polyethylene glycol (PEG) 4000-utilizing bacterium no. 203 was identified as a Flavobacterium species. 2, 6-Dichlorophenol-indophenol (DCIP)-dependent PEG dehydrogenase was constitutively formed in nutrient broth, glucose and PEG media. However, the enzyme formation was repressed in the presence of an excess amount (over 0.25%) of PEG 400 or 1000. PEG dehydrogenase was purified approximately 34 fold by precipitation with ammonium sulfate, solubilization with benzalkonium chloride, chromatography with DEAE-Toyopearl 650 M and hydroxylapatite and gel filtration on Toyopearl HW-55. The molecular weight of the purified PEG dehydrogenase was calculated to be approximately 2.20 × 10⁵, a value which seemed to consist of four subunits with the same molecular weight of 5.70 × 10⁴. The enzyme was stable below 40°C and in the pH range of 7.0 and 8.0. The optimum pH and temperature of the activity were around 8.0 and 40°C, respectively. The enzyme reduced DCIP and coenzyme Q₁ and Q₂. PEG dehydrogenase showed activity toward various PEG molecules (dimer-PEG 20,000). The apparent K_m values for PEG 400, 1000, 4000 and 6000 were about 1.0, 1.7, 2.8 and 5.9 mM, respectively. The enzyme oxidized primary aliphatic alcohols of C₃–C₁₂, the corresponding aldehydes of C₃–C₇, aromatic alcohols and aldehydes, diols, etc. The enzyme was inactive on ethylene glycol, glycerol, secondary alcohols and sugar alcohols. The enzyme activity was strongly inhibited by sulfhydryl agents or heavy metals and 1, 4-benzoquinone. The purified enzyme showed absorption apectrum similar to that of PEG 6000 dehydrogenase which has already been reported to be a quinoprotein. The prosthetic group of the enzyme was extracted with methanol and identified as PQQ from its prosthetic group capability for glucose dehydrogenase and the fluorescence spectrum.     

Bacterial oxidation of polyethylene glycol.

The metabolism of polyethylene glycol (PEG) was investigated with a synergistic, mixed culture of Flavobacterium and Pseudomonas species, which are individually unable to utilize PEGs. The PEG dehydrogenase linked with 2,6-dichlorophenolindophenol was found in the particulate fraction of sonic extracts and catalyzed the formation of a 2,4-dinitrophenylhydrazine-positive compound, possibly an an aldehyde. The enzyme has a wide substrate specificity towards PEGs: from diethylene glycol to PEG 20,000 Km values for tetraethylene glycol (TEG), PEG 400, and PEG 6,000 were 11, 1.7, and 15 mM, respectively. The metabolic products formed from TEG by intact cells were isolated and identified by combined gas chromatography-mass spectrometry as triethylene glycol and TEG-monocarboxylic acid plus small amounts of TEG-dicarboxylic acid, diethylene glycol, and ethylene glycol. From these enzymatic and analytical data, the following metabolic pathway was proposed for PEG: HO(CH2CH2O)nCH2CH2OH leads to HO(CH2CH2O)nCH2CHO leads to HO(CH2CH2O)nCH2COOH leads to HO(CH2CH2O)n-1CH2CH2OH.     

Biodegradation of polyethylene glycol by symbiotic mixed culture (obligate mutualism)

Neither Flavobacterium sp. nor Pseudomonas sp. grew on a polyethylene glycol (PEG) 6000 medium containing the culture filtrate of their mixed culture on PEG 6000. The two bacteria did not grow with a dialysis culture on a PEG 6000 medium. Flavobacterium sp. grew well on a dialysis culture containing a tetraethylene glycol medium supplemented with a small amount of PEG 6000 as an inducer, while poor growth of Pseudomonas sp. was observed. Three enzymes involved in the metabolism of PEG, PEG dehydrogenase, PEG-aldehyde dehydrogenase and PEG-carboxylate dehydrogenase (ether-cleaving) were present in the cells of Flavobacterium sp. The first two enzymes were not found in the cells of Pseudomonas sp. PEG 6000 was degraded neither by intact cells of Flavobacterium sp. nor by those of Pseudomonas sp., but it was degraded by their mixture. Glyoxylate, a metabolite liberated by the ether-cleaving enzyme, inhibited the growth of the mixed culture. The ether-cleaving enzyme was remarkably inhibited by glyoxylate. Glyoxylate was metabolized faster by Pseudomonas sp. than by Flavobacterium sp., and seemed to be a key material for the symbiosis.     

Microbial degradation of polyethers

This paper summarizes studies on microbial degradation of polyethers. Polyethers are aerobically metabolized through common mechanisms (oxidation of terminal alcohol groups followed by terminal ether cleavage), well-characterized examples being found with polyethylene glycol (PEG). First the polymer is oxidized to carboxylated PEG by alcohol and aldehyde dehydrogenases and then the terminal ether bond is cleaved to yield the depolymerized PEG by one glycol unit. Most probably PEG is anaerobically metabolized through one step which is catalyzed by PEG acetaldehyde lyase, analogous to diol dehydratase. Whether aerobically or anaerobically, the free OH group is necessary for metabolization of PEG. PEG with a molecular weight of up to 20,000 was metabolized either in the periplasmic space (Pseudomonas stutzeri and sphingomonads) or in the cytoplasm (anaerobic bacteria), which suggests the transport of large PEG through the outer and inner membranes of Gram-negative bacterial cells. Membrane-bound PEG dehydrogenase (PEG-DH) with high activity towards PEG 6,000 and 20,000 was purified from PEG-utilizing sphingomonads. Sequencing of PEG-DH revealed that the enzyme belongs to the group of GMC flavoproteins, FAD being the cofactor for the enzyme. On the other hand, alcohol dehydrogenases purified from other bacteria that cannot grow on PEG oxidized PEG. Cytoplasmic NAD-dependent alcohol dehydrogenases with high specificity towards ether-alcohol compound, either crude or purified, showed appreciable activity towards PEG 400 or 600. Liver alcohol dehydrogenase (equine) also oxidized PEG homologs, which might cause fatal toxic syndrome in vivo by carboxylating PEG together with aldehyde dehydrogenase when PEG was absorbed. An ether bond-cleaving enzyme was detected in PEG-utilizing bacteria and purified as diglycolic acid (DGA) dehydrogenase from a PEG-utilizing consortium. The enzyme oxidized glycolic acid, glyoxylic acid, as well as PEG-carboxylic acid and DGA. Similarly, dehydrogenation on polypropylene glycol (PPG) and polytetramethylene glycol (PTMG) was suggested with cell-free extracts of PPG and PTMG-utilizing bacteria, respectively. PPG commercially available is atactic and includes many structural (primary and secondary alcohol groups) and optical (derived from pendant methyl groups on the carbon backbone) isomers. Whether PPG dehydrogenase (PPG-DH) has wide stereo- and enantioselective substrate specificity towards PPG isomers or not must await further purification. Preliminary research on PPG-DH revealed that the enzyme was inducibly formed by PPG in the periplasmic, membrane and cytoplasm fractions of a PPG-utilizing bacterium Stenotrophomonas maltophilia. This finding indicated the intracellular metabolism of PPG is the same as that of PEG. Besides metabolization of polyethers, a biological Fenton mechanism was proposed for degradation of PEG, which was caused by extracellular oxidants produced by a brown-rot fungus in the presence of a reductant and Fe3+, although the metabolism of fragmented PEG has not yet been well elucidated.     

Metabolism of polyethylene glycol by two anaerobic bacteria, Desulfovibrio desulfuricans and a Bacteroides sp

Two anaerobic bacteria were isolated from polyethylene glycol (PEG)-degrading, methanogenic, enrichment cultures obtained from a municipal sludge digester. One isolate, identified as Desulfovibrio desulfuricans (strain DG2), metabolized oligomers ranging from ethylene glycol (EG) to tetraethylene glycol. The other isolate, identified as a Bacteroides sp. (strain PG1), metabolized diethylene glycol and polymers of PEG up to an average molecular mass of 20,000 g/mol [PEG 20000; HO-(CH2-CH2-O-)nH]. Both strains produced acetaldehyde as an intermediate, with acetate, ethanol, and hydrogen as end products. In coculture with a Methanobacterium sp., the end products were acetate and methane. Polypropylene glycol [HO-(CH2-CH2-CH2-O-)nH] was not metabolized by either bacterium, and methanogenic enrichments could not be obtained on this substrate. Cell extracts of both bacteria dehydrogenated EG, PEGs up to PEG 400 in size, acetaldehyde, and other mono- and dihydroxylated compounds. Extracts of Bacteroides strain PG1 could not dehydrogenate long polymers of PEG (greater than or equal to 1,000 g/mol), but the bacterium grew with PEG 1000 or PEG 20000 as a substrate and therefore possesses a mechanism for PEG depolymerization not present in cell extracts. In contrast, extracts of D. desulfuricans DG2 dehydrogenated long polymers of PEG, but whole cells did not grow with these polymers as substrates. This indicated that the bacterium could not convert PEG to a product suitable for uptake.     

Metabolism of polyethylene glycol by two anaerobic bacteria, Desulfovibrio desulfuricans and a Bacteroides sp.

Two anaerobic bacteria were isolated from polyethylene glycol (PEG)-degrading, methanogenic, enrichment cultures obtained from a municipal sludge digester. One isolate, identified as Desulfovibrio desulfuricans (strain DG2), metabolized oligomers ranging from ethylene glycol (EG) to tetraethylene glycol. The other isolate, identified as a Bacteroides sp. (strain PG1), metabolized diethylene glycol and polymers of PEG up to an average molecular mass of 20,000 g/mol [PEG 20000; HO-(CH2-CH2-O-)nH]. Both strains produced acetaldehyde as an intermediate, with acetate, ethanol, and hydrogen as end products. In coculture with a Methanobacterium sp., the end products were acetate and methane. Polypropylene glycol [HO-(CH2-CH2-CH2-O-)nH] was not metabolized by either bacterium, and methanogenic enrichments could not be obtained on this substrate. Cell extracts of both bacteria dehydrogenated EG, PEGs up to PEG 400 in size, acetaldehyde, and other mono- and dihydroxylated compounds. Extracts of Bacteroides strain PG1 could not dehydrogenate long polymers of PEG (greater than or equal to 1,000 g/mol), but the bacterium grew with PEG 1000 or PEG 20000 as a substrate and therefore possesses a mechanism for PEG depolymerization not present in cell extracts. In contrast, extracts of D. desulfuricans DG2 dehydrogenated long polymers of PEG, but whole cells did not grow with these polymers as substrates. This indicated that the bacterium could not convert PEG to a product suitable for uptake.     

Isolation of bacteria able to grow on both polyethylene glycol (PEG) and polypropylene glycol (PPG) and their PEG/PPG dehydrogenases

Two bacterial consortia growing on a random copolymer of ethylene glycol and propylene glycol units were obtained by enrichment cultures from various microbial samples. Six major strains included in both consortia were purified and identified as Sphingomonads, Pseudomonas sp. and Stenotrophomonas maltophilia. Three of them (Sphingobium sp. strain EK-1, Sphingopyxis macrogoltabida strain EY-1, and Pseudomonas sp. strain PE-2) utilized both PEG and polypropylene glycol (PPG) as a sole carbon source. Four PEG-utilizing bacteria had PEG dehydrogenase (PEG-DH) activity, which was induced by PEG. PCR products from DNA of these bacteria generated with primers designed from a PEG-DH gene (AB196775 for S. macrogoltabida strain 103) indicated the presence of a sequence that is the homologous to the PEG-DH gene (99% identity). On the other hand, five PPG-utilizing bacteria had PPG dehydrogenase (PPG-DH) activity, but the activity was constitutive. PCR of a PPG-DH gene was performed using primers designed from a polyvinyl alcohol dehydrogenase (PVA-DH) gene (AB190288 for Sphingomonas sp. strain 113P3) because a PPG-DH gene has not been cloned yet, but both PPG-DH and PVA-DH were active toward PPG and PVA (Mamoto et al. 2006). PCR products of the five strains did not have similarity to each other or to oxidoreductases including PVA-DH. The paper was edited by a native speaker through American Journal Experts (http://www.journalexperts.com).     

Microbial Degradation of Polyethylene Glycols

Mono-, di-, tri-, and tetraethylene glycols and polyethylene glycols (PEG) with molecular weight up to 20,000 were degraded by soil microorganisms. A strain of Pseudomonas aeruginosa able to use a PEG of average molecular weight 20,000 was isolated from soil. Washed cells oxidized mono and tetraethylene glycols, but O₂ consumption was not detectable when such cells were incubated for short periods with PEG 20,000. However, the bacteria excreted an enzyme which converted low- and high-molecular-weight PEG to a product utilized by washed P. aeruginosa cells. Gas chromatography of the supernatant of a culture grown on PEG 20,000 revealed the presence of a compound co-chromatographing with diethylene glycol. A metabolite formed from PEG 20,000 by the extracellular enzyme preparation was identified as ethylene glycol by combined gas chromatography-mass spectrometry.     

Effects of polyethylene glycol on stability of alpha-chymotrypsin in aqueous ethanol solvent

The effects of polyethylene glycol (PEG) of different molecular weights (400, 2000, 6000, 12,000, 20,000, and 35,000) on the conformational stability and catalytic activity of alpha-chymotrypsin in 60% ethanol were studied. The inactivation caused by the organic solvent was not influenced by PEG 400. However, the PEGs with higher molecular weights up to 35,000 increased the stability of the enzyme, but this alpha-chymotrypsin stabilizing effect was molecular weight-independent. With increase of the molecular weight of PEG, a more stable tertiary structure of the enzyme was observed.     

A new ether bond-splitting enzyme found in Gram-positive polyethylene glycol 6000-utilizing bacterium, <Emphasis Type="Italic">Pseudonocardia</Emphasis> sp. strain K1

Pseudonocardia sp. strain K1 is the only Gram-positive bacterium among the bacteria aerobically metabolizing polyethylene glycol (PEG). Generally, PEG is metabolized by an oxidative pathway in which a terminal alcohol group of PEG is oxidized to aldehyde and to carboxylic acid and then an ether bond is oxidatively cleaved. As the cell-free extract of Pseudonocardia sp. strain K1 has PEG dehydrogenase, PEG aldehyde dehydrogenase and diglycolic acid (DGA) dehydrogenase (DGADH) activities, all of which are constitutively formed, the strain has a metabolic pathway similar to that so far known. We purified an ether bond-splitting enzyme as DGADH. The molecular mass of the enzyme was estimated to be 55 kDa; and it consisted of two identical subunits. The enzyme oxidatively cleaved both an ether bond of PEG 3000 dicarboxylic acid and DGA. The N-terminal amino acid sequence of the purified enzyme had high homology with various superoxide dismutases and the enzyme had also superoxide dismutase activity. The atomic absorption spectrum showed that approximately one atom of Fe was included in each subunit of the enzyme. DGADH activity increased in the cells grown in a PEG medium supplemented with FeCl₃. Thus, we concluded that the enzyme purified from Pseudonocardia sp. strain K1 is a new ether bond-splitting enzyme.     

Fermentation of polyethylene glycol via acetaldehyde in Pelobacter venetianus

Summary Pelobacter venetianus, a strictly anaerobic bacterium recently isolated with polyethylene glycol (PEG) as substrate, ferments PEG's with molecular masses of 106–40000, as well as acetoin, ethanolamine, choline, and ethoxyethanol, to acetate and ethanol. Ethylene glycol (EG) and acetaldehyde were fermented in the same manner at limiting concentrations in continuous culture. Growth with glycolaldehyde led to acetate as sole fermentation product. Acetaldehyde appeared as byproduct of PEG fermentation, and accumulated to high concentrations during degradation of PEG 4000 and PEG 6000. Utilization of PEG's was constitutive, whereas acetoin degradation was inducible. Acetaldehyde was shown to be the primary product of EG degradation, and inhibited utilization of other substrates. Enzymes involved in the fermentation of PEG, EG, acetoin, and glycolaldehyde were demonstrated in cell-free extracts, except for the PEG degrading enzyme and EG dehydrase. These results demonstrate that acetaldehyde plays a central role in the metabolism of Pelobacter venetianus. A scheme of intermediary metabolism and PEG degradation is discussed.Abbreviations EG ethylene glycol - Di-EG diethylene glycol - PEG (20 000) polyethylene glycol (molecular weight 20 000)     

α-Amylase production by Bacillus subtilis and B. amyloliquefaciens in different PEG solutions

-Amylase production by Bacillus subtilis and Bacillus amyloliquefaciens was investigated in polyethyleneglycol (PEG)-containing growth medium. Five different molecular weight PEGs (600, 3000, 4000, 8000 and 20,000) were used. Enzyme production with B. subtilis increased 21% in medium containing 5% PEG 3000, but enzyme production with B. amyloliquefaciens increased 31% in medium containing 5% PEG 600 and 21% in medium containing 2% PEG 8000.     

A novel nicotinoprotein aldehyde dehydrogenase involved in polyethylene glycol degradation

A gene (pegC) encoding aldehyde dehydrogenase (ALDH) was located 3.4 kb upstream of a gene encoding polyethylene glycol (PEG) dehydrogenase (pegA) in Sphingomonas macrogoltabidus strain 103. ALDH was expressed in Escherichia coli and purified on a Ni-nitrilotriacetic acid agarose column. The recombinant enzyme was a homotetramer consisting of four 46.1-kDa subunits. The alignment of the putative amino acid sequence of the cloned enzyme showed high similarity with a group of NAD(P)-dependent ALDHs (identity 36–52%); NAD-binding domains (Rossmann fold and four glycine residues) and catalytic residues (Glu225 and Cys259) were well conserved. The cofactor, which was extracted from the purified enzyme, was tightly bound to the enzyme and identified as NADP. The enzyme contained 0.94 mol NADP per subunit. The enzyme was activated by Ca²⁺, but by no other metals; no metal (Zn, Fe, Mg, or Mn) was detected in the purified recombinant enzyme. Activity was inhibited by p-chloromercuric benzoate, and heavy metals such as Hg, Cu, Pb and Cd, indicating that a cysteine residue is involved in the activity. Enzyme activity was independent of N,N-dimethyl-p-nitrosoaniline as an electron acceptor. Trans-4-(N,N-dimethylamino)-cinnamaldehyde was not oxidized as a substrate, but the compound worked as an inhibitor for the enzyme, as did pyrazole. The enzyme acted on n-aldehydes C₂–C₁₄) and PEG-aldehydes. Thus the enzyme was concluded to be a novel Ca²⁺-activating nicotinoprotein (NADP-containing) PEG-aldehyde dehydrogenase involved in the degradation of PEG in S. macrogoltabidus strain 103.     

Phosphorus contamination in polyethylene glycol

Concentrations of Fe, Mn, Cu, Zn, Ca, Mg, K, and P were examined in untreated and ion exchange resin-treated solutions of polyethylene glycol, molecular weight 3000 to 3700, polyethylene glycol (PEG 4000). Relatively high levels of P were found in untreated PEF-4000 solutions. The concentration of contaminating P in solutions prepared from untreated PEG 4000, even at high water potentials (−1 to −3 bars), was greater than what is usually found in soil solution. Occurrence of significant amounts of P in untreated PEG could introduce problems in experiments where ³²P and PEG are used together and where phosphate interactions may occur.     

Fermentative degradation of polyethylene glycol by a strictly anaerobic, gram-negative, nonsporeforming bacterium, Pelobacter venetianus sp. nov.

The synthetic polyether polyethylene glycol (PEG) with a molecular weight of 20,000 was anaerobically degraded in enrichment cultures inoculated with mud of limnic and marine origins. Three strains (Gra PEG 1, Gra PEG 2, and Ko PEG 2) of rod-shaped, gram-negative, nonsporeforming, strictly anaerobic bacteria were isolated in mineral medium with PEG as the sole source of carbon and energy. All strains degraded dimers, oligomers, and polymers of PEG up to a molecular weight of 20,000 completely by fermentation to nearly equal amounts of acetate and ethanol. The monomer ethylene glycol was not degraded. An ethylene glycol-fermenting anaerobe (strain Gra EG 12) isolated from the same enrichments was identified as Acetobacterium woodii. The PEG-fermenting strains did not excrete extracellular depolymerizing enzymes and were inhibited by ethylene glycol, probably owing to a blocking of the cellular uptake system. PEG, some PEG-containing nonionic detergents, 1,2-propanediol, 1,2-butanediol, glycerol, and acetoin were the only growth substrates utilized of a broad variety of sugars, organic acids, and alcohols. The isolates did not reduce sulfate, sulfur, thiosulfate, or nitrate and were independent of growth factors. In coculture with A. woodii or Methanospirillum hungatei, PEGs and ethanol were completely fermented to acetate (and methane). A marine isolate is described as the type strain of a new species, Pelobacter venetianus sp. nov. Its physiology and ecological significance, as well as the importance and possible mechanism of anaerobic polyether degradation, are discussed.     

Enhancement of enzymatic production of cyclodextrins by adding polyethylene glycol or polypropylene glycol

Enzymatic production of cyclodextrins (CDs) from soluble starch was studied using either Bacillus macerans or Bacillus ohbensis cyclomaltodextrin glucanotransferase (CGTase). The production yield of CDs was found to be increased up to 1.5–2 times by the addition of low molecular weight polyethylene glycol (PEG 400) or polypropylene glycol (PPG 425) to the reaction medium. Such results were interpreted as being due to a conformational change of the substrate as well as reduction of hydrolytic activity of the enzyme in the presence of these additives.     

Enhancement of ribonucleotide reductase activity at low enzyme concentrations by polyethylene glycol

The estimation of ribonucleotide reductase in cell extracts has been problematical on account of abnormally low activities at low enzyme concentrations, presumably due to subunit dissociation. This problem can be alleviated by assaying the enzyme in the presence of polyethylene glycol. The presence of 15% polyethylene glycol during the assay greatly stimulated ribonucleotide reductase activity at low enzyme concentrations and allowed measurement of enzyme activity in as little as 10(5) mouse L929 cells, a 30-fold enhancement of assay sensitivity. Enzyme activity measured in the presence of 15% polyethylene glycol was proportional to enzyme concentration, thus making possible the accurate measurement of very low levels of ribonucleotide reductase.     

(+)-Abscisic Acid and Two Compounds Showing Chlorophyll Degradation Activity in Cuscuta pentagona Engelm

The purified polyethylene glycol (PEG) dehydrogenase from cells of a synergistic mixed culture of Flavobacterium and Pseudomonas species showed a similar absorption spectrum to those of other quinoproteins reported so far. The prosthetic group of the PEG dehydrogenase after extraction with cold methanol and purification by DEAE-Sephadex A-25 column chromatography and Sephadex G-25 gel filtration showed the same elution profiles as those of authentic pyrrolo-quinoline quinone (PQQ). Absorption and fluorescence spectra of the purified prosthetic group and its prosthetic group capability for glucose dehydrogenase indicated that it was identical with authentic PQQ.

The enzyme was induced during bacterial cell growth on a medium containing PEG 6000 as a sole source of carbon. The purified enzyme oxidized primary alcohols of C₂-C₁₆ and the corresponding aldehydes of C₄-C₇. The enzyme also reacted with nonionic surfactants containing PEG residues. The enzyme reduced 2,6-dichlorophenolindophenol (DCIP) and the Km value for DCIP was calculated to be 1.4 × 10^?4m. The DCIP reductase activity was inhibited by carbonyl reagents like semicarbazide, hydrazine, hydroxylamine and 1,4-benzoquinone. 1,4-Benzoquinone inhibited the DCIP reductase activity competitively as to DCIP.