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4-Nitrophenol (4-NP) is a toxic product of the hydrolysis of organophosphorus pesticides such as parathion in soil. Rhodococcus sp. strain PN1 degrades 4-NP via 4-nitrocatechol (4-NC) for use as the sole carbon, nitrogen, and energy source. A 5-kb EcoRI DNA fragment previously cloned from PNI contained a gene cluster (nphRA1A2) involved in 4-NP oxidation. From sequence analysis, this gene cluster is expected to encode an AraC/XylS family regulatory protein (NphR) and a two-component 4-NP hydroxylase (NphA1 and NphA2). A transcriptional assay in a Rhodococcus strain revealed that the transcription of nphA1 is induced by only 4-NP (of several phenolic compounds tested) in the presence of nphR, which is constitutively expressed. Disruption of nphR abolished transcriptional activity, suggesting that nphR encodes a positive regulatory protein. The two proteins of the 4-NP hydroxylase, NphA1 and NphA2, were independently expressed in Escherichia coli and purified by ion-exchange chromatography or affinity chromatography. The purified NphA2 reduced flavin adenine dinucleotide (FAD) with the concomitant oxidation of NADH, while the purified NphA1 oxidized 4-NP into 4-NC almost quantitatively in the presence of FAD, NADH, and NphA2. This functional analysis, in addition to the sequence analysis, revealed that this enzyme system belongs to the two-component flavin-diffusible monooxygenase family. The 4-NP hydroxylase showed comparable oxidation activities for phenol and 4-chlorophenol to that for 4-NP and weaker activities for 3-NP and 4-NC.

The amino acid sequence of the Flavobacterium sp. K1723T1 plasmid pOAD2, which forms nylon degrading enzymes, is given. The plasmid consists of 45519 bp and contains 876 termination codons in the clockwise direction and 778 termination codons in the anticlockwise direction. The G+C content of the plasmid is high. The structure of the plasmid is similar to substances such as oligopeptide permeases and isopenicillin N-acyltransferase. The position of the genes forming the degradation enzymes on the genome map is given.

The nucleotide sequence I reveals that the five constituent regions possess identical 880 bp sequences that resemble the IS6100 insertion sequence of the IS6 family. The nylon oligomer-degrading plasmid pNAD2, seen in Pseudomonas sp. strain NK87, also exhibits sequences homologous to IS6100 of the Mycobacterium fortuitum.

The 6-aminohexanoate-dimer hydrolase (P-EII) from Pseudomonas sp. was characterizedand its base sequence described. The DNA sequence contained 396 amino acids, was initiated by ATG and terminated by TAG. The F-nylB and P-nylB genes had 35%similarity to amino acid sequences in the whole coding region. The F-EII and P-EII enzymes showed similar DFP inhibition profiles. No significant sequence similarities were observed for F-E1 and F-EII enzymes. Therefore, the nylA and nylB families could have evolved independently.

Bacillus megaterium cells have more than one type of glucose dehydrogenase (GlcDH) genes which are regulated differently. To understand the expression mechanisms and physiological role of each isozyme, the genes coding for GlcDH were isolated. Two new genes were identified and were designated as gdhIII and gdhIV, bring to four the total number of gdh genes in B. megaterium IAM1030. Characterization of the four different isozymes showed that they differ in electrophoretic mobility, immunoreactivity, salt stability and coenzyme preference.

When Pseudomonas aeruginosa PAO is grown in a culture containing 6-aminohexanoate (Ahx) which is a by-product of nylon-6, it acquires the ability to obtain carbon and nitrogen from the Ahx cyclic dimer (Acd). This ability of the bacteria to use Ahx is due to a genetic change and two new enzymes are produced which can hydrolyse the cyclic dimer of Ahx. This metabolic adaptation of Pseudomonas aeruginosa PAO is useful in the biodegradation of nylon-6.

The isolation of alkalophilic, nylon oligomer-degrading bacteria, their genetic cloning and the characterization of their nylon oligomer-degrading enzymes are described. The 6-aminohexanoate oligomer hydrolases (NylC) from the alkalophilic strains are similar to the enzyme in neutrophilic Arthrobacter sp. but has superior thermostability and affinity for nylon-related substrates.

Arthrobacter sp. strain KI72 grows on a 6-aminohexanoate oligomer, which is a by-product of nylon-6 manufacturing, as a sole source of carbon and nitrogen. We cloned the two genes, nylD and nylE, responsible for 6-aminohexanoate metabolism on the basis of the draft genomic DNA sequence of strain KI72. We amplified the DNA fragments that encode these genes by polymerase chain reaction using a synthetic primer DNA homologous to the 4-aminobutyrate metabolic enzymes. We inserted the amplified DNA fragments into the expression vector pColdI in Escherichia coli, purified the His-tagged enzymes to homogeneity, and performed biochemical studies. We confirmed that 6-aminohexanoate aminotransferase (NylD) catalyzes the reaction of 6-aminohexanoate to adipate semialdehyde using α-ketoglutarate, pyruvate, and glyoxylate as amino acceptors, generating glutamate, alanine, and glycine, respectively. The reaction requires pyridoxal phosphate (PLP) as a cofactor. For further metabolism, adipate semialdehyde dehydrogenase (NylE) catalyzes the oxidative reaction of adipate semialdehyde to adipate using NADP as a cofactor. Phylogenic analysis revealed that NylD should be placed in a branch of the PLP-dependent aminotransferase sub III, while NylE should be in a branch of the aldehyde dehydrogenase superfamily. In addition, we established a NylD/NylE coupled system to quantify the aminotransferase activity and to enable the conversion of 6-aminohexaoate to adipate via adipate semialdehyde with a yield of > 90%. In the present study, we demonstrate that 6-aminohexanoate produced from polymeric nylon-6 and nylon oligomers (i.e., a mixture of 6-aminohexaoate oligomers) by nylon hydrolase (NylC) and 6-aminohexanoate dimer hydrolase (NylB) reactions are sequentially converted to adipate by metabolic engineering technology. [ABSTRACT FROM AUTHOR]