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PubMed1994

Description of the kinetic mechanism and the enantioselectivity of quinohaemoprotein ethanol dehydrogenase from Comamonas testosteroni in the oxidation of alcohols and aldehydes.

Initial rate studies were performed on the oxidation of (racemic) alcohols as well as aldehydes by quinohaemoprotein ethanol dehydrogenase, type 1, from Comamonas testosteroni with potassium ferricyanide as electron acceptor. The data could be fitted with an equation derived for a mechanism (hexa-uni ping-pong) in which alcohols are oxidized to the corresponding carboxylic acids and the intermediate aldehyde becomes released from the enzyme. However, for some substrates it was necessary to assume that they exert uncompetitive inhibition. The same model was used to fit the data of conversion processes. Reversible inactivation of the enzyme takes place during the conversion, the extent being inversely proportional to the concentration of ferricyanide present at the start. From the values of the kinetic parameters obtained for (R)- and (S)-solketal [2,2-dimethyl-4-(hydroxymethyl)-1,3-dioxolane] and their corresponding aldehydes, it appeared that the second step in (S)-solketal conversion is much faster than the first one and that opposite enantiomeric preferences exist for the alcohol and the aldehyde substrates. Since the initial rate measurements as well as the progress curve analysis gave similar kinetic parameter values and product analysis revealed intermediates in the amounts predicted, it is concluded that the kinetic and enantioselective behaviour of the enzyme is adequately described by the model presented here. Finally, the results indicate that both kinetic approaches should be used in conversions with consecutive reactions since they provide complementary information.

1-Propanol↗

PubMed1995

Quinohaemoprotein ethanol dehydrogenase from Comamonas testosteroni. Purification, characterization, and reconstitution of the apoenzyme with pyrroloquinoline quinone analogues.

Pyrroloquinoline-quinone(PQQ)-free quinohaemoprotein ethanol dehydrogenase (QH-EDH) apoenzyme was isolated from ethanol-grown Comamonas testosteroni. The purified apoenzyme, showing a single band of 71 kDa on native gel electrophoresis, could be only partially converted into active holoenzyme by addition of PQQ in the presence of calcium ions. In addition to a band with a molecular mass of 71 kDa, additional bands of 51 kDa and 25 kDa were observed with SDS/PAGE. Analysis of the N-terminal sequences of the bands and comparison with the DNA sequence of the gene, suggested that the latter two originate from the former one, due to scission occurring at a specific site between two vicinal residues in the protein chain. The extent of scission appeared to increase during growth of the organism. After addition of PQQ to apoenzyme, holoenzyme and nicked, inactive enzyme could be separated. Holoenzyme prepared in this way was found to contain equimolar amounts of PQQ, Ca2+ and covalently bound haem. EPR spectra of fully oxidized apo-QH-EDH and holo-QH-EDH showed g values typical for low-spin haem c proteins. In partially oxidized holo-QH-EDH an organic radical signal attributed to the semiquinone form of PQQ was observed. Binding of PQQ leads to conformational changes, as reflected by changes of spectral and chromatographic properties. Reconstitution of apoenzyme with PQQ analogues resulted in a decreased activity and enantioselectivity for the oxidation of chiral alcohols. Compared with PQQ, analogues with a large substituent had a lower affinity for the apoenzyme. Results with other analogues indicated that possession of the o-quinone/o-quinol moiety is not essential for binding but it is for activity.

Alcohol Oxidoreductases↗

PubMed1996

Characterization of the gene encoding quinohaemoprotein ethanol dehydrogenase of Comamonas testosteroni.

The gene encoding quinohaemoprotein ethanol dehydrogenase type I (QH-EDHI) from Comamonas testosteroni has been cloned and sequenced. Comparison of the amino acid sequence deduced from this with that determined for the N-terminal amino acid stretch of purified QH-EDHI, suggests that the gene also contains a leader sequence of 31 residues. Based on this information, the molecular mass of the apo-enzyme, i.e. the enzyme without the cofactors pyrroloquinoline quinone (PQQ) and haem c, and without the Ca2+, appears to be 73 200 Da. Alignment of the deduced amino acid sequence to that of other PQQ-containing dehydrogenases showed that good similarity (up to 43% identity) exists with most of them. This also showed that the amino acid residues presumed to be involved in PQQ and Ca2+ binding and in the typical features of structure and catalysis of methanol dehydrogenase, are conserved at the same positions in QH-EDHI. The C-terminal part of the protein, containing the haem c, exhibited some similarity to cytochromes C553 from cyanobacteria and algae. Correct processing of the qhedh gene appeared to occur in Escherichia coli strain JM 109 in which the gene was placed under control of the lac promoter, as judged from a positive reaction with antibodies raised against authentic QH-EDHI, the size of the protein, the presence of haem c in it, and the specific activity value obtained after reconstitution with PQQ. The qhedh gene seems to form part of an operon which is organized in a way different from that of the genes required for methanol oxidation in methylotrophic bacteria.

Alcohol Oxidoreductases↗

PubMed1996

Chemical structure of lipid A isolated from Comamonas testosteroni lipopolysaccharide.

The chemical structure of lipid A of lipopolysaccharide isolated from Comamonas testosteroni was determined by quantitative analysis, methylation analysis, mass spectrometry and NMR spectroscopy. The lipid A backbone was found to consist of 6-O-(2-deoxy-2-amino-beta-D-glucopyranosyl)-2-deoxy-2-amino-alpha-D-g luc ose which was phosphorylated in positions 1 and 4'. Hydroxyl groups at positions 4 and 6' were unsubstituted, and position 6' of the non-reducing terminal residue was identified as the attachment site of the polysaccharide part. Liquid secondary-ion/mass spectrometry revealed a pseudomolecular ion at m/z 1572 [M-H]- as a major diphosphoryl lipid component carrying six acyl groups. Fatty acid distribution analysis and electrospray ionization/mass spectrometry of the lipid A showed that positions 2,2',3, and 3' of the sugar backbone were N-acylated or O-acylated by (R)-3-hydroxydecanoic acid, and that the hydroxyl groups of the amide-linked residues attached to positions 2 and 2' were further O-acylated by tetradecanoic and dodecanoic acids, respectively.

Carbohydrate Sequence↗

PubMed1996

Crystallization and crystal packing of recombinant 3 (or 17) beta-hydroxysteroid dehydrogenase from Comamonas testosteroni ATTC 11996.

The enzyme 3 (or 17) beta-hydroxysteroid dehydrogenase from Comamonas testosteroni was crystallized. Crystals, of up to 0.6 mm in their longest dimension and suitable for a crystallographic analysis have been obtained by the vapour diffusion method. They belong to the orthorhombic lattice type and diffract to a maximum resolution of 0.23 nm. A final data set obtained by merging data from three crystals resulted in a completeness of 90% with an Rmerge of 6%. A molecular replacement search carried out by using 3 alpha (or 20 beta)-hydroxysteroid dehydrogenase from Streptomyces hydrogenans as a search model allowed us to assign I222 as the correct space group and to propose a model for the crystal packing, with one monomer per asymmetric unit. Thus, the whole unit cell contains two tetramers. The R-factor after rigid body refinement is 48.1%.

17-Hydroxysteroid Dehydrogenases↗

PubMed1999

Isolation of poly(beta-L-malic acid)-degrading bacteria and purification and characterization of the PMA hydrolase from Comamonas acidovorans strain 7789.

Several bacteria were isolated which were able to utilize poly(beta-L-malic acid) as sole carbon source for growth. The poly(beta-L-malic acid) hydrolyzing enzyme of Comamonas acidovorans strain 7789 was detected in the membrane fraction. The enzyme was purified by isolation of crude cell membranes by ultracentrifugation of disrupted cells, solubilization of the membrane fraction with octylglucoside, selective precipitation with 50% saturated ammonium sulfate and preparative isolectric focusing. SDS-PAGE analysis revealed a M(r) of 43,000. The pH optimum was 8.1 and the Km was 0.13 microM (in terms of monomeric units) and 0.0021 microM poly(beta-L-malic acid) at pH 8.1 (100 mM glycylglycine buffer). Addition of NaCl, KCl, CaCl2 or MgCl2 (from 25 to 100 mM) decreased the hydrolase activity, whereas EDTA or polymethane sulfonic acid fluoride had no influence on the enzyme. The depolymerization of poly(beta-L-malic acid) proceeded from the ends of the polyester resulting in the formation of L-malate. Esterase activity was not detectable with p-nitrophenyl acetate or p-nitrophenyl butyrate, which is used to determine for example poly(3-hydroxybutyric acid) depolymerase activity.

Biodegradation, Environmental↗

PubMed1992

Modelling of mixed chemostat cultures of an aerobic bacterium, Comamonas testosteroni, and an anaerobic bacterium, Veillonella alcalescens: comparison with experimental data.

A mathematical model of mixed chemostat cultures of the obligately aerobic bacterium Comamonas testosteroni and the anaerobic bacterium Veillonella alcalescens grown under dual limitation of L-lactate and oxygen was constructed. The model was based on Michaelis-Menten-type kinetics for the consumption of substrates, with noncompetitive inhibition of V. alcalescens by O2. The growth characteristics of the aerobic and anaerobic organisms were determined experimentally with pure cultures of the individual species in (oxygen-limited) chemostats. Using these pure-culture data in the model of the mixed culture resulted in a good description of the actual mixed cultures of the two bacteria. In the actual mixed-culture experiments, coexistence of the two species occurred only when the cultures were oxygen limited. With increasing oxygen supply (the actual oxygen concentration in the culture remaining at less than 0.2 microM), the biomass of C. testosteroni increased, whereas that of V. alcalescens decreased. Apparently, C. testosteroni protected V. alcalescens from inhibition by oxygen by maintaining sufficiently low oxygen concentrations. The model calculations indicated that competition between the aerobic and the anaerobic bacterium for common substrates (L-lactate and oxygen) occurred and that the anaerobe was the better competitor. Analysis of the culture fluid indicated that C. testosteroni grew primarily at the expense of the fermentation products of V. alcalescens, i.e., propionate and acetate. The model further indicated that with different values of several growth parameters (e.g., substrate affinity and/or inhibition constants), the affinity of the aerobic organism for oxygen and the sensitivity of the anaerobic organism for oxygen were the most important properties determining the coexistence of these two physiologically different types of bacteria.

Acetates↗

PubMed1994

Regulation of chloro- and methylphenol degradation in Comamonas testosteroni JH5.

Comamonas testosteroni JH5 was isolated from a mixed bacterial culture enriched on different chloro- and methylphenols. The strain completely mineralized a mixture consisting of 4-chlorophenol (4-CP) and 4-methylphenol (4-MP). During degradation of the mixture, 4-hydroxybenzyl alcohol, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, and 4-chlorocatechol were detected as short-lived intermediates. Mineralization of 4-CP and that of 4-MP occurred successively and were accompanied by diauxic growth, whereas 4-CP and 2-methylphenol were mineralized simultaneously. It was ascertained that neither a reversible enzyme inhibition nor potential toxic intermediates caused the observed diauxie. Some facts support the hypothesis that the successive degradation of 4-CP and 4-MP is regulated on the level of transcription. C. testosteroni JH5 contained a meta-cleaving enzyme when pregrown on 4-CP and the isomeric monomethylphenols. Inactivation of this enzyme in the presence of 3-chlorocatechol was observed.

Biodegradation, Environmental↗

PubMed1995

Oxidative Pathway for the Biodegradation of Nitrobenzene by Comamonas sp. Strain JS765.

Previous studies have shown that the biodegradation of nitrobenzene by Pseudomonas pseudoalcaligenes JS45 proceeds by the reduction of nitrobenzene through nitrosobenzene and hydroxylaminobenzene, followed by rearrangement to 2-aminophenol, which then undergoes meta ring cleavage. We report here the isolation of a Comamonas sp. that uses an oxidative pathway for the complete mineralization of nitrobenzene. The isolate, designated strain JS765, uses nitrobenzene as a sole source of carbon, nitrogen, and energy. Nitrobenzene-grown cells oxidized nitrobenzene, with the stoichiometric release of nitrite. Extracts of nitrobenzene-grown JS765 showed high levels of catechol 2,3-dioxygenase activity that were not abolished by heating the cell extracts to 60(deg)C for 10 min. The ring cleavage product had an absorbance maximum at 375 nm, consistent with that of 2-hydroxymuconic semialdehyde. Both NAD-dependent dehydrogenase and NAD-independent hydrolase activities towards 2-hydroxymuconic semialdehyde were induced in extracts of nitrobenzene-grown cells. Catechol accumulated in the reaction mixture when cells preincubated with 3-chlorocatechol were incubated with nitrobenzene. Conversion of nitrobenzene to catechol by induced cells in the presence of 3-chlorocatechol and (sup18)O(inf2) demonstrated the simultaneous incorporation of two atoms of oxygen, which indicated that the initial reaction was dioxygenation. The results indicate that the catabolic pathway involves an initial dioxygenase attack on nitrobenzene with the release of nitrite and formation of catechol, which is subsequently degraded by a meta cleavage pathway.

Journal Article↗

PubMed1996

Production of R-(-)-Ketoprofen from an Amide Compound by Comamonas acidovorans KPO-2771-4.

R-(-)-2-(3(prm1)-Benzoylphenyl)propionic acid [R-(-)-ketoprofen] was produced from racemic 2-(3(prm1)-benzoylphenyl)propionamide (keto-amide) by the isolated bacterial strain Comamonas acidovorans KPO-2771-4. Sodium fumarate as the carbon source and 2-azacyclononanone or isobutyronitrile as the enhancer in the culture medium were effective for bacterial growth and the enhancement of R-(-)-ketoprofen-producing activity. R-(-)-Ketoprofen produced from the keto-amide by resting cells was present in 99% enantiomeric exess. C. acidovorans KPO-2771-4 has an R-enantioselective amidase for keto-amide because the purified amidase from the bacterium hydrolyzed keto-amide, producing optically pure R-ketoprofen and ammonia.

Journal Article↗

PubMed1996

Molecular cloning of novel genes for polycyclic aromatic hydrocarbon degradation from Comamonas testosteroni GZ39.

Three strains of Comamonas testosteroni were isolated from river sediment for the ability to degrade phenanthrene; two of the strains also grew on naphthalene, and one strain also grew on anthracene. The homology of the genes for polycyclic aromatic hydrocarbon degradation in these strains to the classical genes (nah) for naphthalene degradation from Pseudomonas putida NCIB 9816-4 was determined. The three C. testosteroni strains showed no homology to the nah gene probe even under low-stringency conditions. The genes for naphthalene and phenanthrene degradation were cloned from one of the three C. testosteroni strains. Two cosmid clones expressing polycyclic aromatic hydrocarbon dioxygenase activity were identified from a library prepared with genomic DNA from C. testosteroni GZ39. The genes coding for the first two enzymes in the catabolic pathway, phenanthrene dioxygenase and cis-phenanthrene dihydrodiol dehydrogenase, were localized to a 5.4-kb NcoI-PstI fragment by subcloning and gene expression experiments. Further subcloning and analysis revealed a novel organization of the genes, with the gene for cis-phenanthrene dihydrodiol dehydrogenase located between the genes for the individual phenanthrene dioxygenase components. A Southern blot with the cloned genes from C. testosteroni GZ39 confirmed that these genes are distinct from those found in P. putida NCIB 9816-4. Southern blots also demonstrated that C. testosteroni GZ38A possesses genes for phenanthrene degradation that are similar to those cloned from C. testosteroni GZ39. However, C. testosteroni GZ42 possesses genes for phenanthrene degradation that are not homologous to those cloned from C. testosteroni GZ39. This suggests that there are at least two different sets of genes for the degradation of phenanthrene among the three C. testosteroni strains.

Biodegradation, Environmental↗

PubMed1996

Degradation of cocaine by a mixed culture of Pseudomonas fluorescens MBER and Comamonas acidovorans MBLF.

A mixed culture that could utilize cocaine as the sole source of carbon and energy for growth was isolated by selective enrichment. The individual microorganisms within this mixed culture were identified as Pseudomonas fluorescens (termed MBER) and Comamonas acidovorans (termed MBLF). Each microorganism was shown to be unable to grow to any appreciable extent on 10 mM cocaine in the absence of the other. C. acidovorans MBLF was found to possess an inducible cocaine esterase which catalyzed the hydrolysis of cocaine to ecgonine methyl ester and benzoate. C. acidovorans was capable of growth on benzoate at concentrations below 5 mM but was unable to metabolize ecgonine methyl ester. P. fluorescens MBER was capable of growth on either benzoate as the sole source of carbon or ecgonine methyl ester as the sole source of carbon and nitrogen. P. fluorescens MBER was found to initiate the degradation of ecgonine methyl ester via ecgonine, pseudoecgonine, and pseudoecgonyl-coenzyme A. Subcellular studies resulted in the identification of an ecgonine methyl esterase, an ecgonine epimerase, and a pseudoecgonyl-coenzyme A synthetase which were induced by growth on ecgonine methyl ester or ecgonine. Further metabolism of the ecgonine moiety is postulated to involve nitrogen debridging, with the production of carbonyl-containing intermediates.

Benzoates↗

PubMed1996

Comamonas testosteroni colony phenotype influences exopolysaccharide production and coaggregation with yeast cells.

A Comamonas testosteroni strain was isolated from activated sludge on the basis of its ability to coaggregate with yeast cells. On agar plates the following two types of colonies were formed: colonies with a mucoid appearance and colonies with a nonmucoid appearance. On plates this strain alternated between the two forms, making sectored colonies. In liquid medium with constant agitation no such change was observed. In the absence of agitation and in contact with a glass surface a culture with predominantly nonmucoid-colony-forming cells very rapidly shifted to a culture dominated by mucoid-colony-forming cells. In liquid medium the reverse was observed under stress conditions imposed by hydrogen peroxide, sodium dodecyl sulfate, or starvation. Nonmucoid cells formed very rapidly settling flocs with yeast cells, while coaggregation of mucoid cells with yeast cells did not occur. These findings may be relevant to the behavior of activated sludge microbial communities.

Gram-Negative Aerobic Bacteria↗

PubMed1996

Characterization of active recombinant 2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase from Comamonas testosteroni B-356 and sequence of the encoding gene (bphB).

2,3-Dihydro-2,3-dihydroxybiphenyl-2,3-dehydrogenase (B2,3D) catalyzes the second step in the biphenyl degradation pathway. The nucleotide sequence of Comamonas testosteroni B-356 bphB, which encodes B2,3D, was determined. Structural analysis showed that the dehydrogenases involved in the bacterial degradation of aromatic compounds are related to each other and that their phylogenetic relationships are very similar to the relationships observed for dioxygenases that catalyze the initial reaction in the degradation pathway. The bphB sequence was used to produce recombinant active His-tagged B2,3D, which allowed us to describe for the first time some of the main features of a B2,3D. This enzyme requires NAD+, its optimal pH is 9.5, and its native M(r) was found to be 123,000, which makes it a tetramer. These characteristics are very similar to those reported for the related enzyme cis-toluene dihydrodiol dehydrogenase. The Km value and maximum rate of metabolism for 2,3-dihydro-2,3-dihydroxybiphenyl were 73 +/- 16 microM and 46 +/- 4 nmol min-1 microgram-1, respectively. Compared with the cis-toluene dihydrodiol dehydrogenase, B2,3D appeared to be more substrate specific since it was unable to attack cis-1,2-dihydroxy-cyclohexa-3,5-diene.

Amino Acid Sequence↗

PubMed1997

Degradation of 4-Chlorophenol via the meta Cleavage Pathway by Comamonas testosteroni JH5.

Comamonas testosteroni JH5 used 4-chlorophenol (4-CP) as its sole source of energy and carbon up to a concentration of 1.8 mM, accompanied by the stoichiometric release of chloride. The degradation of 4-CP mixed with the isomeric 2-CP by resting cells led to the accumulation of 3-chlorocatechol (3-CC), which inactivated the catechol 2,3-dioxygenase. As a result, further 4-CP breakdown was inhibited and 4-CC accumulated as a metabolite. In the crude extract of 4-CP-grown cells, catechol 1,2-dioxygenase and muconate cycloisomerase activities were not detected, whereas the activities of catechol 2,3-dioxygenase, 2-hydroxymuconic semialdehyde dehydrogenase, 2-hydroxymuconic semialdehyde hydrolase, and 2-oxopent-4-enoate hydratase were detected. These enzymes of the meta cleavage pathway showed activity with 4-CC and with 5-chloro-2-hydroxymuconic semialdehyde. The activities of the dioxygenase and semialdehyde dehydrogenase were constitutive. Two key metabolites of the meta cleavage pathway, the meta cleavage product (5-chloro-2-hydroxymuconic semialdehyde) and 5-chloro-2-hydroxymuconic acid, were detected. Thus, our previous postulation that C. testosteroni JH5 uses the meta cleavage pathway for the complete mineralization of 4-CP was confirmed.

Journal Article↗

PubMed1997

Biochemical and molecular characterization of the polyhydroxybutyrate depolymerase of Comamonas acidovorans YM1609, isolated from freshwater.

Comamonas acidovorans YM1609 secreted a polyhydroxybutyrate (PHB) depolymerase into the culture supernatant when it was cultivated on poly(3-hydroxybutyrate) [P(3HB)] or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] as the sole carbon source. The PHB depolymerase was purified from culture supernatant of C. acidovorans by two chromatographic methods, and its molecular mass was determined as 45,000 Da by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme was stable at temperatures below 37 degrees C and at pH values of 6 to 10, and its activity was inhibited by diisopropyl fluorophosphonate. The liquid chromatography analysis of water-soluble products revealed that the primary product of enzymatic hydrolysis of P(3HB) was a dimer of 3-hydroxybutyric acid. Kinetics of enzymatic hydrolysis of P(3HB) film were studied. In addition, a gene encoding the PHB depolymerase was cloned from the C. acidovorans genomic library. The nucleotide sequence of this gene was found to encode a protein of 494 amino acids (M(r), 51,018 Da). Furthermore, by analysis of the N-terminal amino acid sequence of the purified enzyme, the molecular mass of the mature enzyme was calculated to be 48,628 Da. Analysis of the deduced amino acid sequence suggested a domain structure of the protein containing a catalytic domain, fibronectin type III module as linker, and a putative substrate-binding domain. Electron microscopic visualization of the mixture of P(3HB) single crystals and a fusion protein of putative substrate-binding domain with glutathione S-transferase demonstrated that the fusion protein adsorbed strongly and homogeneously to the surfaces of P(3HB) single crystals.

3-Hydroxybutyric Acid↗

PubMed1997

Conjugative plasmids and the degradation of arylsulfonates in Comamonas testosteroni.

Comamonas testosteroni T-2 degrades p-toluenesulfonate (TSA) via p-sulfobenzoate (PSB) and protocatechuate and degrades toluenecarboxylate via terephthalate (TER) and protocatechuate. The appropriate genes are expressed in at least five regulatory units, some of which are also found in C. testosteroni PSB-4 (F. Junker, R. Kiewitz, and A. M. Cook, J. Bacteriol. 179:919-927, 1997). C. testosteroni T-2 was found to contain two plasmids, pTSA (85 kbp) and pT2T (50 kbp); a TSA- mutant (strain TER-1) contained only plasmid pT2T. C. testosteroni PSB-4, which does not degrade TSA, contained one plasmid, pPSB (85 kbp). The type strain contained no plasmids. Conjugation experiments showed that plasmid pTSA (possibly in conjunction with pT2T) was conjugative, and the single copy of the TSA operon (tsaMBCD) with its putative regulator gene (tsaR) in strain T-2 was found on plasmid pTSA, which also carried the PSB genes (psbAC) and presumably transport for both substrates. Plasmid pTSA was assigned to the IncP1 beta group and was found to carry two copies of insertion element IS1071. Plasmid pPSB (of strain PSB-4), which could be maintained in strains with plasmid pTSA or pT2T, was also conjugative and was found to carry the PSB genes as well as to contain two copies of IS1071. In attempted conjugations with the type strain, no plasmid was recovered, but the PSB+ transconjugant carried two copies of IS1071 in the chromosome. We presume the PSB genes to be located in a composite transposon. The genes encoding the putative TER operon and degradation of protocatechuate, with the meta cleavage pathway, were attributed a chromosomal location in strains T-2 and PSB-4.

Amino Acid Sequence↗

PubMed1998

Purification and Properties of a Polyester Polyurethane-Degrading Enzyme from Comamonas acidovorans TB-35.

A polyester polyurethane (PUR)-degrading enzyme, PUR esterase, derived from Comamonas acidovorans TB-35, a bacterium that utilizes polyester PUR as the sole carbon source, was purified until it showed a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This enzyme was bound to the cell surface and was extracted by addition of 0.2% N,N-bis(3-d-gluconamidopropyl)deoxycholamide (deoxy-BIGCHAP). The results of gel filtration and SDS-PAGE showed that the PUR esterase was a monomer with a molecular mass of about 62,000 Da. This enzyme, which is a kind of esterase, degraded solid polyester PUR, with diethylene glycol and adipic acid released as the degradation products. The optimum pH for this enzyme was 6.5, and the optimum temperature was 45 degrees C. PUR degradation by the PUR esterase was strongly inhibited by the addition of 0.04% deoxy-BIGCHAP. On the other hand, deoxy-BIGCHAP did not inhibit the activity when p-nitrophenyl acetate, a water-soluble compound, was used as a substrate. These observations indicated that this enzyme degrades PUR in a two-step reaction: hydrophobic adsorption to the PUR surface and hydrolysis of the ester bond of PUR.

Journal Article↗