Metabolism Of Polyethylene Glycol Research Articles

Cloning and expression of an ether-bond-cleaving enzyme involved in the metabolism of polyethylene glycol

A gene encoding an ether-bond-cleaving enzyme, diglycolic acid dehydrogenase (DGADH) from polyethylene glycol-utilizing Pseudonocardia sp. strain K1, was cloned and expressed in Escherichia coli. The deduced amino acid sequence had a high homology with superoxide dismutases (SODs) from various bacteria. The recombinant protein showed the same activities as those of DGADH from Pseudonocardia sp. strain K1, namely, SOD activity and ether-bond-cleaving activity.

Cloning and Expression of an Ether-Bond-Cleaving Enzyme Involved in the Metabolism of Polyethylene Glycol

A gene encoding an ether-bond-cleaving enzyme, diglycolic acid dehydrogenase (DGADH) from polyethylene glycol-utilizing Pseudonocardia sp. strain K1, was cloned and expressed in Escherichia coli . The deduced amino acid sequence had a high homology with superoxide dismutases (SODs) from various bacteria. The recombinant protein showed the same activities as those of DGADH from Pseudonocardia sp. strain K1, namely, SOD activity and ether-bond-cleaving activity.

Sphingomonads involved in the biodegradation of xenobiotic polymers.

Sphingomonads involved in the microbial degradation of xenobiotic polymers are introduced. The metabolism of polyethylene glycol was the primary focus of the study. Several others, including polyvinyl alcohol, polyethylene and polyaspartate were also studied. It is suggested that these xenobiotic polymers are metabolized by intracellular enzymes located in the periplasmic space or bound to membranes, indicating that transport of these polymers through outer membranes is requisite for their metabolism. Involvement of specific membrane structures of sphingomonads such as unusual sphingolipids is suggested for membrane transport of xenobiotic compounds, especially hydrophobic materials.

Purification and characterization of an ether bond-cleaving enzyme involved in the metabolism of polyethylene glycol

An ether bond-cleaving enzyme was purified as diglycolic acid (DGA) dehydrogenase associated with 3-(4,5-dimethyl-2-thioazolyl)-2,5-diphenyl-2H tetrazolium bromide and phenazine methosulfate. DGA dehydrogenase was inductively formed in a polyethylene glycol (PEG)-utilizing symbiotic mixed culture, E-1, composed of Sphingomonas terrae and Rhizobium sp. which were grown on PEG 6,000. DGA dehydrogenase was solubilized with 0.5% n-dodecyl-β- d-maltoside from membrane fractions of the mixed culture E-1 and purified almost to homogeneity, as determined by SDS-polyacrylamide gel electrophoresis, by DEAE-Toyopearl column chromatography and Sepharose 6B gel filtration. The molecular weight of the enzyme was determined to be about 40,000–41,000 Da by SDS-polyacrylamide gel electrophoresis and 480,000 by gel filtration on Sepharose 6B in the presence of 0.5% n-dodecyl-β- d-maltoside. The absorption spectrum of the purified enzyme showed two peaks at 345 nm and 430–450 nm in addition to a major peak at 280 nm. The optimal pH and temperature were 8.9 and 40°C, respectively. The enzyme was stable in the pH range of 7.8 to 9.0 and at below 30°C. The enzyme was activated by neither metal ions nor ammonium ion, and strongly inhibited by 1,4-benzoquinone. The enzyme catalyzed the degradation of DGA, PEG dicarboxylic acids, glycolic acid and glyoxylic acid, but not primary alcohols, ethylene glycol (EG), EG oligomers, PEG 6,000 and aldehydes.

Role of novel dye-linked dehydrogenases in the metabolism of polyethylene glycol by pure cultures of Sphingomonas sp. N6

Polyethylene glycol and diglycolic acid dehydrogenase acitivities linked with3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2 H-tetrazolium bromide and phenazine methosulfate were found in the participate fraction of the sonic extract of a newly isolated polyethylene glycol 20 000-utilizing bacterium ( Sphingomonas sp. N6). The amount of glyoxylic acid formed from diglycolic acid increased proportionally with the increase in reaction time and enzyme concentration to show that diglycolic acid can be a model compound for an ether-cleaving enzyme. Both enzymes were formed inducibly when the organism was grown on polyethylene glycol 10000. Both enzymes transferred many more electrons to3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2 H-tetrazolium bromide plus phenazine methosulfate than to 2,6-dichlorophenolindophenol plus phenazine methosulfate. Also, ferricyanide, nitroblue tetrazolium and horse heart cytochrome c served as electron acceptors, among which3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2 H-tetrazolium bromide plus phenazine methosulfate was most active for polyethylene glycol dehydrogenase and ferricyanide was most active for diglycolic acid dehydrogenase irrespective of the presence of phenazine methosulfate. NAD and NADP did not act as electron acceptors. Polyethylene glycol dehydrogenase was completely inhibited by 1,4-benzoquinone and partially inhibited by quinine and glyoxylic acid. Diglycolic acid dehydrogenase was strongly inhibited by 1,4-benzoquinone and partially inhibited by α-benzoin oxime, quinacrine and glyoxylic acid. The enzymes appear to be different from each other and also from polyethylene glycol and diglycolic acid dehydrogenases of polyethylene glycol 20 000-utilizing symbiotic mixed culture E-1in which S. terrae was responsible for polyethylene glycol degradation in the coupling with electron acceptors and in the effect of inhibitors.

Role of novel dye-linked dehydrogenases in the metabolism of polyethylene glycol by pure cultures ofSphingomonassp. N6

Abstract Polyethylene glycol and diglycolic acid dehydrogenase acitivities linked with3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2 H-tetrazolium bromide and phenazine methosulfate were found in the participate fraction of the sonic extract of a newly isolated polyethylene glycol 20 000-utilizing bacterium ( Sphingomonas sp. N6). The amount of glyoxylic acid formed from diglycolic acid increased proportionally with the increase in reaction time and enzyme concentration to show that diglycolic acid can be a model compound for an ether-cleaving enzyme. Both enzymes were formed inducibly when the organism was grown on polyethylene glycol 10000. Both enzymes transferred many more electrons to3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2 H-tetrazolium bromide plus phenazine methosulfate than to 2,6-dichlorophenolindophenol plus phenazine methosulfate. Also, ferricyanide, nitroblue tetrazolium and horse heart cytochrome c served as electron acceptors, among which3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2 H-tetrazolium bromide plus phenazine methosulfate was most active for polyethylene glycol dehydrogenase and ferricyanide was most active for diglycolic acid dehydrogenase irrespective of the presence of phenazine methosulfate. NAD and NADP did not act as electron acceptors. Polyethylene glycol dehydrogenase was completely inhibited by 1,4-benzoquinone and partially inhibited by quinine and glyoxylic acid. Diglycolic acid dehydrogenase was strongly inhibited by 1,4-benzoquinone and partially inhibited by α-benzoin oxime, quinacrine and glyoxylic acid. The enzymes appear to be different from each other and also from polyethylene glycol and diglycolic acid dehydrogenases of polyethylene glycol 20 000-utilizing symbiotic mixed culture E-1in which S. terrae was responsible for polyethylene glycol degradation in the coupling with electron acceptors and in the effect of inhibitors.

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.

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.