A new nylon oligomer degradation gene (nylC) on plasmid pOAD2 from a Flavobacterium sp.

Flavobacterium sp. strain KI725 harbors plasmid pOAD21, a derivative of nylon oligomer-degradative plasmid pOAD2, in which all of nylA (the gene for 6-aminohexanoate cyclic dimer hydrolase [EI]) was deleted but nylB (the gene for 6-aminohexanoate dimer hydrolase [EII]) was retained. KI725 showed no growth on unfractionated nylon oligomers (Nom1) obtained from a nylon factory as a sole carbon and nitrogen source (Nom1 minimum plate). Extracts of KI725 cells possessed hydrolytic activity for Nom1 (approximately 5% of the activity of KI72), but pOAD2-cured strains (KI722 and KI723) showed no activity. KI725R strains which grew on the Nom1 minimum plate were spontaneously isolated from KI725 at a frequency of 10(-7) per cell. Activity toward Nom1 was enhanced in KI725R strains (10 to 30% of the activity of KI72). This new Nom1 degrading enzyme (EIII, the nylC gene product) hydrolyzed not only Nom1 but also the N-carbobenzoxy-6-aminohexanoate trimer, a substrate which was not hydrolyzed by either EI or EII. Cloning and sequence analysis showed that the nylC gene is located close to nylB on pOAD21 and is a 1,065-bp open reading frame corresponding to 355 amino acid residues. The nucleotide sequence of the nylC gene and the deduced amino acid sequence of EIII had no detectable homology with the sequences of nylA (EI) and nylB (EII).     

Enzymatic hydrolysis of nylons: quantification of the reaction rate of nylon hydrolase for thin-layered nylons

Nylon hydrolase degrades various aliphatic nylons, including nylon-6 and nylon-66. We synthesized a nylon-66 copolymer (M _w?=?22,900, M _n?=?7,400), in which a part of an adipoyl unit (32 % molar ratio) of nylon-66 was replaced with a succinyl unit by interfacial polymerization. To quantify the reaction rate of the enzymatic hydrolysis of nylons at the surface of solid polymers, we prepared a thin layer of nylons on the bottom surface of each well in a polystyrene-based micro-assay plate. The thickness of the nylon layer was monitored by imaging analysis of the photographic data. More than 99 % of the copolymer with thicknesses of 260 nm (approximately 600 layers of polymer strands) were converted to water-soluble oligomers by nylon hydrolase (3 mg enzyme ml^?1) at 30 °C within 60 h. These results were further confirmed by TLC analysis of the reaction products and by assay of liberated amino groups in the soluble fractions. The degradation rate of the thin-layered nylon-6 was similarly analyzed. We demonstrate that this assay enables a quantitative evaluation of the reaction rate of hydrolysis at the interface between the solid and aqueous phases and a quantitative comparison of the degradability for various polyamides.     

Three-dimensional structure of nylon hydrolase and mechanism of nylon-6 hydrolysis

We performed x-ray crystallographic analyses of the 6-aminohexanoate oligomer hydrolase (NylC) from Agromyces sp. at 2.0 Å-resolution. This enzyme is a member of the N-terminal nucleophile hydrolase superfamily that is responsible for the degradation of the nylon-6 industry byproduct. We observed four identical heterodimers (27 kDa + 9 kDa), which resulted from the autoprocessing of the precursor protein (36 kDa) and which constitute the doughnut-shaped quaternary structure. The catalytic residue of NylC was identified as the N-terminal Thr-267 of the 9-kDa subunit. Furthermore, each heterodimer is folded into a single domain, generating a stacked αββα core structure. Amino acid mutations at subunit interfaces of the tetramer were observed to drastically alter the thermostability of the protein. In particular, four mutations (D122G/H130Y/D36A/E263Q) of wild-type NylC from Arthrobacter sp. (plasmid pOAD2-encoding enzyme), with a heat denaturation temperature of T_m = 52 °C, enhanced the protein thermostability by 36 °C (T_m = 88 °C), whereas a single mutation (G111S or L137A) decreased the stability by ∼10 °C. We examined the enzymatic hydrolysis of nylon-6 by the thermostable NylC mutant. Argon cluster secondary ion mass spectrometry analyses of the reaction products revealed that the major peak of nylon-6 (m/z 10,000–25,000) shifted to a smaller range, producing a new peak corresponding to m/z 1500–3000 after the enzyme treatment at 60 °C. In addition, smaller fragments in the soluble fraction were successively hydrolyzed to dimers and monomers. Based on these data, we propose that NylC should be designated as nylon hydrolase (or nylonase). Three potential uses of NylC for industrial and environmental applications are also discussed.     

Biodegradation of nylon oligomers

This mini-review is a compendium of the degradation of a man-made compound, 6-aminohexanoate-oligomer, in Flavobacterium strains. The results are summarized as follows: 1. Three enzymes, 6-aminohexanoate-cyclic-dimer hydrolase (EI), 6-aminohexanoate-dimer hydrolase (EII), and endotype 6-aminohexanoate-oligomer hydrolase (EIII) were responsible for degradation of the oligomers. 2. The genes coding these enzymes were located on pOAD2, one of three plasmids harbored in Flavobacterium sp. KI72, which comprised 45,519 bp. 3. The gene coding the EII′ protein (a protein having 88% homology with EII) and five IS6100 elements were identified on pOAD2. 4. The specific activity of EII was 200-fold higher than that of EII′. However, altering two amino acid residues in the EII′ enzyme enhanced the activity of EII′ to the same level as that of the EII enzyme. 5. The deduced amino acid sequences from eight regions of pOAD2 had significant similarity with the sequences of gene products such as oppA-F (encoding oligopeptide permease), ftsX (filamentation temperature sensitivity), penDE (isopenicillin N-acyltransferase) and rep (plasmid replication). 6. The EI and EII genes of Pseudomonas sp. NK87 (another nylon oligomer-degrading bacterium) were also located on plasmids. 7. Through selective cultivation using nylon oligomers as a sole source of carbon and nitrogen, two strains which initially had no metabolic activity for nylon oligomers, Flavobacterium sp. KI725 and Pseudomonas aeruginosa PAO1, were given the ability to degrade xenobiotic compounds. A molecular basis for the adaptation of microorganisms toward xenobiotic compounds was described. Received: 25 February 2000 / Received revision: 22 May 2000 / Accepted: 26 May 2000     

Emergence of nylon oligomer degradation enzymes in Pseudomonas aeruginosa PAO through experimental evolution.

Through selective cultivation with 6-aminohexanoate linear dimer, a by-product of nylon-6 manufacture, as the sole source of carbon and nitrogen, Pseudomonas aeruginosa PAO, which initially has no enzyme activity to degrade this xenobiotic compound, was successfully expanded in its metabolic ability. Two new enzyme activities, 6-aminohexanoate cyclic dimer hydrolase and 6-aminohexanoate dimer hydrolase, were detected in the adapted strains.     

6-Aminohexanoate Oligomer Hydrolases from the Alkalophilic Bacteria Agromyces sp. Strain KY5R and Kocuria sp. Strain KY2

Alkalophilic, nylon oligomer-degrading strains, Agromyces sp. and Kocuria sp., were isolated from the wastewater of a nylon-6 factory and from activated sludge from a sewage disposal plant. The 6-aminohexanoate oligomer hydrolases (NylC) from the alkalophilic strains had 95.8 to 98.6% similarity to the enzyme in neutrophilic Arthrobacter sp. but had superior thermostability, activity under alkaline conditions, and affinity for nylon-related substrates, which would be advantageous for biotechnological applications.     

Alteration of catalytic function of 6-aminohexanoate-dimer hydrolase by site-directed mutagenesis

Alteration of Asp181 in a nylon oligomer-degrading enzyme, 6-aminohexanoate-dimer hydrolase (EII) of Flavobacterium sp. KI72, to Asn and to Glu by site-directed mutagenesis increased K_m values toward 6-aminohexanoate-dimer 4 times and 11 times, respectively. Replacement to His or to Lys caused complete loss of the activity (less than 0.02% of the activity of the EII enzyme). Thus, a single amino acid alteration at position 181 of the enzyme drastically affects the catalytic function.     

X-ray crystallographic analysis of 6-aminohexanoate-dimer hydrolase: molecular basis for the birth of a nylon oligomer-degrading enzyme

6-Aminohexanoate-dimer hydrolase (EII), responsible for the degradation of nylon-6 industry by-products, and its analogous enzyme (EII') that has only approximately 0.5% of the specific activity toward the 6-aminohexanoate-linear dimer, are encoded on plasmid pOAD2 of Arthrobacter sp. (formerly Flavobacterium sp.) KI72. Here, we report the three-dimensional structure of Hyb-24 (a hybrid between the EII and EII' proteins; EII'-level activity) by x-ray crystallography at 1.8 A resolution and refined to an R-factor and R-free of 18.5 and 20.3%, respectively. The fold adopted by the 392-amino acid polypeptide generated a two-domain structure that is similar to the folds of the penicillin-recognizing family of serine-reactive hydrolases, especially to those of d-alanyl-d-alanine-carboxypeptidase from Streptomyces and carboxylesterase from Burkholderia. Enzyme assay using purified enzymes revealed that EII and Hyb-24 possess hydrolytic activity for carboxyl esters with short acyl chains but no detectable activity for d-alanyl-d-alanine. In addition, on the basis of the spatial location and role of amino acid residues constituting the active sites of the nylon oligomer hydrolase, carboxylesterase, d-alanyl-d-alanine-peptidase, and beta-lactamases, we conclude that the nylon oligomer hydrolase utilizes nucleophilic Ser(112) as a common active site both for nylon oligomer-hydrolytic and esterolytic activities. However, it requires at least two additional amino acid residues (Asp(181) and Asn(266)) specific for nylon oligomer-hydrolytic activity. Here, we propose that amino acid replacements in the catalytic cleft of a preexisting esterase with the beta-lactamase fold resulted in the evolution of the nylon oligomer hydrolase.     

Purification and Characterization of a Nylon-Degrading Enzyme

A nylon-degrading enzyme found in the extracellular medium of a ligninolytic culture of the white rot fungus strain IZU-154 was purified by ion-exchange chromatography, gel filtration chromatography, and hydrophobic chromatography. The characteristics of the purified protein (i.e., molecular weight, absorption spectrum, and requirements for 2,6-dimethoxyphenol oxidation) were identical to those of manganese peroxidase, which was previously characterized as a key enzyme in the ligninolytic systems of many white rot fungi, and this result led us to conclude that nylon degradation is catalyzed by manganese peroxidase. However, the reaction mechanism for nylon degradation differed significantly from the reaction mechanism reported for manganese peroxidase. The nylon-degrading activity did not depend on exogenous H₂O₂ but nevertheless was inhibited by catalase, and superoxide dismutase inhibited the nylon-degrading activity strongly. These features are identical to those of the peroxidase-oxidase reaction catalyzed by horseradish peroxidase. In addition, α-hydroxy acids which are known to accelerate the manganese peroxidase reaction inhibited the nylon-degrading activity strongly. Degradation of nylon-6 fiber was also investigated. Drastic and regular erosion in the nylon surface was observed, suggesting that nylon is degraded to soluble oligomers and that nylon is degraded selectively.     

6-Aminohexanoate oligomer hydrolases from the alkalophilic bacteria Agromyces sp. strain KY5R and Kocuria sp. strain KY2

Alkalophilic, nylon oligomer-degrading strains, Agromyces sp. and Kocuria sp., were isolated from the wastewater of a nylon-6 factory and from activated sludge from a sewage disposal plant. The 6-aminohexanoate oligomer hydrolases (NylC) from the alkalophilic strains had 95.8 to 98.6% similarity to the enzyme in neutrophilic Arthrobacter sp. but had superior thermostability, activity under alkaline conditions, and affinity for nylon-related substrates, which would be advantageous for biotechnological applications.     

6-aminohexanoate and chloride ion in the activation by urokinase of porcine plasminogens

The rate of activation by urokinase of porcine plasminogen is accelerated by 6-aminohexanoate, although the maximally enhanced rate is 10-fold less than that of human plasminogen without the amino acid. 6-Aminohexanoate facilitates only activation of native porcine plasminogen (asp-plasminogen), but has no effect on activation of des-kringle1-4-plasminogen. Sodium chloride, on the other hand, inhibits activation by urokinase of both porcine asp-plasminogen and des-kringle1-4-plasminogen. It is concluded that 6-aminohexanoate exerts its effect via kringle1-4 domains of plasminogen, whereas Cl- acts, at least in part, through effects on the kringle5 or proteinase domains.     

Improved production of adipate with Escherichia coli by reversal of β-oxidation

The linear C₆ dicarboxylic acid adipic acid is an important bulk chemical in the petrochemical industry as precursor of the polymer nylon-6,6-polyamide. In recent years, efforts were made towards the biotechnological production of adipate from renewable carbon sources using microbial cells. One strategy is to produce adipate via a reversed β-oxidation pathway. Hitherto, the adipate titers were very low due to limiting enzyme activities for this pathway. In most cases, the CoA intermediates are non-natural substrates for the tested enzymes and were therefore barely converted. We here tested heterologous enzymes in Escherichia coli to overcome these limitations and to improve the production of adipate via a reverse β-oxidation pathway. We tested in vitro selected enzymes for the efficient reduction of the enoyl-CoA and in the final reaction for the thioester cleavage. The genes encoding the enzymes which showed in vitro the highest activity were then used to construct an expression plasmid for a synthetic adipate pathway. Expression of paaJ, paaH, paaF, dcaA, and tesB in E. coli BL21(DE3) resulted in the production of up to 36 mg/L of adipate after 30 h of cultivation. Beside the activities of the pathway enzymes, the availability of metabolic precursors may limit the synthesis of adipate, providing another key target for further strain engineering towards high-yield production of adipate with E. coli.

     

Amino acid alterations essential for increasing the catalytic activity of the nylon-oligomer-degradation enzyme of Flavobacterium sp

The structural genes of two homologous enzymes, 6-aminohexanoate-dimer hydrolase (EII; nylB) and its evolutionally related protein EII' (nylB') of Flavobacterium sp. KI72 have an open reading frame encoding a peptide of 392 amino acids, of which 47 are different, and conserved restriction sites. The specific activity of EII towards 6-aminohexanoate dimer is about 1000-fold that of EII'. Construction of various hybrid genes obtained by exchanging fragments flanked by conserved restriction sites of the two genes demonstrated that two amino acid replacements in the EII' enzyme, i.e. Gly181----Asp (EII type) and His266----Asn (EII type), enhanced the activity toward 6-aminohexanoate dimer 1000-fold.     

Nylon oligomer degradation gene, nylC, on plasmid pOAD2 from a Flavobacterium strain encodes endo-type 6-aminohexanoate oligomer hydrolase: purification and characterization of the nylC gene product.

A new type of nylon oligomer degradation enzyme (EIII) was purified from an Escherichia coli clone harboring the EIII gene (nylC). This enzyme hydrolyzed the linear trimer, tetramer, and pentamer of 6-aminohexanoate by an endo-type reaction, and this specificity is different from that of the EI (nylA gene product) and EII (nylB gene product). Amino acid sequencing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified EIII demonstrated that the enzyme is made of two polypeptide chains arising from an internal cleavage between amino acid residues 266 and 267.     

Purification and some properties of human DNA-O6-methylguanine methyltransferase

DNA-O⁶-methylguanine methyltransferase was purified from the nuclear fraction of fresh human placenta using ammonium sulphate precipitation, gel filtration, affinity chromatography on DNA-cellulose and hydroxyapatite. The methyltransferase preparation was approximately 1–2% pure based on specific activity, and was free of nucleic acids. The protein reacts stoichiometrically with O⁶-methylguanine in DNA with apparent second-order kinetics. The human methyltransferase has a pH optimum of about 8.5, similar to that of the corresponding rat and mouse proteins. NaCl inhibits the reaction in a concentration-dependent fashion. The human protein, like the rodent andE. coli methyltransferases, needs no cofactor. While lmM MnCl₂, lmM spermidine, 5mM MgCl₂ and 10 mM EDTA individually do not significantly inhibit the initial rate of reaction, the protein is nearly completely inactive in 5 mM A1Cl₃ or FeCl₂ or 10 mM spermidine. The initial rate of reaction increases as a function of temperature at least up to 42°. The reaction is inhibited by DNA in a concentration-dependent manner, with single-stranded DNA being more inhibitory than duplex DNA.     

The nylon oligomer biodegradation system ofFlavobacterium andPseudomonas

This review article is a compendium of the available information on the degradation of a man-made compound, 6-aminohexanoate-oligomer, inFlavobacterium andPseudomonas strains, and discusses the molecular basis for adaptation of microorganisms toward these xenobiotic compounds. Three plasmid-encoded enzymes, 6-aminohexanoate-cyclic-dimer hydrolase (EI), 6-aminohexanoate-dimer hydrolase (EII), and endo-type 6-aminohexanoate-oligomer hydrolase (EIII) are responsible for the degradation of the oligomers. Two repeated sequences, designated RS-I and RS-II, are found on plasmid pOAD2, which is involved in 6-aminohexanoate degradation inFlavobacterium. RS-I appears 5 times on the pOAD2, and all copies have the same sequences as insertion sequence IS6100. RS-II appears twice on the plasmid. RS-IIA contains the gene encoding EII, while RS-IIB contains the gene for the analogous EII' protein. Both EII and EII' are polypeptides of 392 amino acids, which differ by 46 amino acid residues. The specific activity of the EII enzyme is 200-fold higher than that of EII'. Construction of various hybrid genes demonstrated that only the combination of two amino acid residues in the EII' enzyme can enhance the activity of the EII' to the same level as that of EII enzyme.Abbreviations EI 6-aminohexanoate-cyclic-dimer hydrolase - EII 6-aminohexanoate-dimer hydrolase - EIII endo-type 6-aminohexanoate-oligomer hydrolase - F-EI EI fromFlavobacterium - F-EII EII fromFlavobacterium - P-EI EI fromPseudomonas - P-EII EII fromPseudomonas - EII' a protein having 88% homology to the EII encoded on the RS-IIB region of pOAD2 - nylA gene for the EI enzyme - nylB gene for the EII enzyme - nylC gene for the EIII enzyme - nylB' gene for the EII' protein - kb kilo-base-pairs     

Biological treatment of the components of solid oligomeric waste from a nylon-6 production plant

On partial analysis of the solid oligomeric waste of a nylon-6 production plant, it was found to contain ε-caprolactam, 6-aminocaproic acid (6-ACA) and its linear and cyclic oligomers. Out of four bacterial isolates capable of utilizing caprolactam as the sole growth substrate, Alcaligenes faecalis was found to be the most potent and utilized 90% of caprolactam in 24 h. In shake flask experiments, when the solid waste after solubilization was treated with a consortium of bacteria of four different genera, except the cyclic oligomers, all the other constituents were found to be degraded. A reduction of the chemical oxygen demand (COD) of the solid waste to the level of 63–66% was obtained when it was treated with either a consortium of the bacterial isolates or only a single isolate, A. faecalis. Alcaligenes faecalis could bring about a decrease of 95% in the caprolactam content of the solid waste, while 6-ACA and its linear oligomers were almost completely degraded. Alcaligenes faecalis cells adapted on solid waste could degrade the linear oligomers at a faster rate as compared to cells adapted on caprolactam. However, cyclic oligomers could not be degraded in either case. When solid waste, partially hydrolysed with acid to yield 6-ACA as the major constituent, was treated with the consortium of bacterial isolates, a 95% reduction in the COD was achieved. This revised version was published online in November 2006 with corrections to the Cover Date.     

DNA-DNA hybridization analysis of nylon oligomer-degradative plasmid pOAD2: identification of the DNA region analogous to the nylon oligomer degradation gene.

Fine structure of the gene of 6-aminohexanoic acid cyclic dimer hydrolase, one of the enzymes responsible for the degradation of the nylon oligomer (6-aminohexanoic acid cyclic dimer), on the plasmid pOAD2 harbored in Flavobacterium sp. KI72 was determined by constructing miniplasmids from plasmid pNDH5 (a hybrid plasmid consisting of pBR322 and a 9.1-kilobase-pair HindIII fragment of pOAD2 ). The 6-aminohexanoic acid cyclic dimer hydrolase produced by cells of Escherichia coli C600 harboring pNDH5 or its miniplasmid was examined immunologically and electrophoretically and was found to be identical to that of Flavobacterium sp. KI72 . A fragment of pOAD2 (17.2- to 19.1-kilobase-pair region on pOAD2 ) was detected as hybridized fragment by Southern blotting experiments, indicating the presence of the DNA region analogous to the 6-aminohexanoic acid cyclic dimer hydrolase gene on the plasmid.     

Assessing the sequence specificity in the binding of Co(III) to DNA via a thermodynamic approach

The interaction specificities of Co(III) with DNA were investigated via consideration of thermodynamic characteristics of the duplex to single strand transition for DNA oligomers incubated in the presence of [Co(NH₃)₅(OH₂)] (ClO₄)₃. It has previously been demonstrated that incubation of the DNA oligomer [(5medC-dG)₄]₂ with this cobalt complex leads to coordination of the cobalt center to the DNA, presumably at N7 of guanine bases [D. C. Calderone, E. J. Mantilla, M. Hicks, D. H. Huchital, W. R. Murphy, Jr. and R. D. Sheardy, (1995) Biochemistry 34, 13841]. In this report, DNA oligomers of different sequence were incubated with [Co(NH₃)₅(OH₂)] (ClO₄)₃ via protocols developed previously and the treated oligomers were subjected to thermal denaturation for comparison to the untreated oligomers. The DNA oligomers were designed in order to investigate the sequence specificity, if any, in the reaction of the cobalt complex with DNA. The values of T_m, ΔH_uH, and Δn (the differential ion binding term) obtained from the thermal denaturations were used to assess the sequence specificity of the interaction. For all oligomers, treated or untreated, T_m and Δ_uH vary linearly with log [Na⁺] and hence the value of Δn is a function of the Na⁺ concentration. The results indicate no significant reaction between the cobalt complex and oligomers possessing isolated -GA- or -CG- sites; however, the thermodynamic characteristics of DNA oligomers possessing either an isolated -GG- site or an isolated -GC- site were altered by the treatment. Atomic absorption studies of the treated oligomers demonstrate that only the DNA oligomers possessing isolated -GG- or -GC- sites bind cobalt. Hence, the changes in the thermodynamic properties of these oligomers are a result of cobalt binding with a remarkable sequence specificity. © 1997 John Wiley & Sons, Inc. Biopoly 42: 549–599, 1997