Article

Characterization of the 6-aminohexanoate-dimer hydrolase from Pseudomonas sp. NK87

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Abstract

The DNA base sequence of the Pseudomonas sp. NK87 gene (P-nylB) for 6-aminohexanoate-dimer hydrolase (P-EII), a xenobiotic-compound-degrading enzyme, was determined. It has an open reading frame of 1188 bp, initiated by ATG and terminated by TAG, and coding for 396 amino acids. The base sequence of the open reading frame has 53% sequence similarity to that of the gene for the same enzyme of Flavobacterium sp. KI72 (F-nylB) and 35% sequence similarity with respect to the deduced amino acid sequence. The P-EII enzyme was purified from an Escherichia coli clone in which the P-EII gene was highly expressed. The P-EII enzyme was inhibited by a serine protease inhibitor, diisopropyl fluorophosphate, as was the F-EII enzyme. Double reciprocal plots obtained from various concentrations of 6-aminohexanoate-dimer indicated that the kcat value of the P-EII enzyme (9.2 s-1) was approximately half that of the F-EII enzyme (19 s-1), and the P-EII enzyme had higher affinity toward this substrate (Km for P-EII, 0.6 mM; Km for F-EII, 15 mM). The P-EII enzyme had a temperature optimum of 48 degrees C, and a pH optimum of 7.5. It is speculated that since the P-nylB and F-nylB genes are more diverged from each other than the corresponding nylA genes, the latter may have evolved more recently.

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... However, to find suitable microorganisms does not ensure that such a polymer will become susceptible to organic recycling or will degrade in the environment. Nonetheless, biobased polyamides have a future and should become widespread displacing those from petrochemical sources [158,159]. ...
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Three kinds of improvements have been introduced into the M13-based cloning systems. (1) New Escherichia coli host strains have been constructed for the E. coli bacteriophage M13 and the high-copy-number pUC-plasmid cloning vectors. Mutations introduced into these strains improve cloning of unmodified DNA and of repetitive sequences. A new suppressorless strain facilitates the cloning of selected recombinants. (2) The complete nucleotide sequences of the M 13mp and pUC vectors have been compiled from a number of sources, including the sequencing of selected segments. The M13mp18 sequence is revised to include the G-to-T substitution in its gene II at position 6 125 bp (in M13) or 6967 bp in M13mp18. (3) M13 clones suitable for sequencing have been obtained by a new method of generating unidirectional progressive deletions from the polycloning site using exonucleases HI and VII.
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6-Aminohexanoate-dimer hydrolase (EII) and its analogous protein (EII′), of Flavobacterium sp. K172 are composed of 392 amino acids, in which 47 are different. The enzyme activity of EII′ toward 6-aminohexanoate dimer is approximately 0.5% of that of EII. We have constructed various hybrids of the two genes by exchanging fragments flanked by conserved restriction sites such as PvuII, BglII, SalI, and BamHI (respectively 74, 483, 771, and 1,141 bp downstream of the initiation codon), and purified their gene products to homogeneity. Hyb-12 protein, which was obtained by the replacement of the BglII-SalI region of the EII′ with the corresponding region of EII, had 12 times higher specific activity towards the 6-aminohexanoate dimer and its related substrates than EII′ protein. Hyb-10, which was composed of the N-terminal -BglII regions of EII′ and the BglII-C terminal region of EII, had activity toward these substrates nearly equal to the activity of the EII enzyme. Comparisons of the activity toward 6-aminohexanoate dimer and its analogues has demonstrated that EII, EII′, and their hybrid enzymes are highly active only toward the substrates that contain 6-aminohexanoate as the N-terminal residue, while the recognition of the C-terminal residue in the substrate was not stringent. The substrate specificity, pH-activity profile, and heat stability of these enzymes varied slightly.
Article
A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.
Article
A procedure for extracting plasmid DNA from bacterial cells 1s described. The method 1s simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day yet yields plasmid DNA which is pure enough to be digestible by restriction enzymes. The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is accomplished without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method.
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This chapter discusses the microbial transformations of herbicides and pesticides. Herbicide and pesticide usage has benefited modern society by improving the quality and quantity of the world's food supply while keeping the cost of that food supply reasonable. However, increased usage of chemicals has resulted in environmental concerns. The development and integration of microbes or their activities with the use of herbicides and pesticides can enhance the beneficial effects of chemical usage while eliminating some of the environmental concerns. Microbes can also provide a means to eliminate unwanted residues from the environment, protect previously susceptible crops from herbicide or pesticide damage, and provide a source of genetic material for the development of herbicide-resistant crops or pesticide-producing plants. A fundamental understanding of a microbe's degradative kinetics under various conditions, its biochemical systems, and its molecular biology are vital in maximizing the potential benefits of its use. The chapter describes the widespread use of xenobiotics with emphasis on the usage and detoxication of haloaromatic herbicides and pesticides.
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Publisher Summary This chapter reviews and evaluates the relevance and quality of existing xenobiotic data bases and test methods for evaluating (1) direct and indirect effects (both adverse and beneficial) of xenobiotics on the soil microbial community, (2) direct and indirect effects of the soil microbial community on xenobiotics, and (3) “adequacy of test methods used to evaluate these effects and interactions. The distribution of a xenobiotic between environmental compartments depends on the chemodynamic properties of the compound and on the physicochemical properties of the soil, and it occurs across soil–water and soil–air interfaces and across biological membranes. In assessing the effects of xenobiotic compounds on soil microorganisms, it is necessary to decide which microbial processes or properties should be evaluated. When exposed to xenobiotic compounds, various segments of the soil microbial community are affected to different extents. The degree to which a xenobiotic affects microbial activities is largely dependent on the chemical, its dosage, method of application, and the particular physicochemical characteristics of the soil, such as soil type, temperature, water content, and pH. A systemic examination of those classes of xenobiotics that have not been evaluated for their effects on microorganisms needs to be performed to allow development of a predictive model for environmental risk assessment.
Article
Diisopropylfluorophosphate, an inhibitor of serine proteinase, was used to label 6-aminohexanoate-dimer hydrolase, a nylon oligomer degradative enzyme of Flavobacterium sp. K172. More than 95% of the enzyme activity was lost upon incorporation of 1-1.5 molecules inhibitor/subunit of the enzyme. The tryptic peptide of the labeled enzyme was purified by HPLC (reverse-phase partition) and its amino acid sequence was identified. Radioactivity was found to be incorporated into an 8-amino-acid peptide (108His-Leu-Leu-Met-Ser-Val-Ser-Lys115). Amino acid alteration from Ser to Ala at the position 112 by site-directed mutagenesis caused loss of enzyme activity to below the detection threshold (1% of the activity of the parental enzyme). These results indicate that Ser112 is essential for the activity.
Article
A bacterial strain, Pseudomonas sp. strain NK87, that can use 6-aminohexanoate-cyclic dimer as the sole source of carbon and nitrogen was newly isolated from wastewater of a factory which produces nylon-6. Two responsible enzymes, 6-aminohexanoate-cyclic-dimer hydrolase (P-EI) and 6-aminohexanoate-dimer hydrolase (P-EII), were found in the NK87 strain, as is the case with Flavobacterium sp. strain KI72, another 6-aminohexanoate-cyclic-dimer-metabolizing bacterium (H. Okada, S. Negoro, H. Kimura, and S. Nakamura, Nature [London] 306:203-206, 1983). The P-EI enzyme is immunologically identical to the 6-aminohexanoate-cyclic-dimer hydrolase of KI72 (F-EI). However, antiserum against the 6-aminohexanoate-dimer hydrolase purified from KI72 (F-EII) did not react with cell extracts of NK87, indicating that the F-EII and P-EII enzymes are immunologically different. Restriction endonuclease analyses show that the NK87 strain harbors at least six plasmids ranging in size from 20 to 80 kilobase pairs (kbp). The P-EI and P-EII genes were cloned in Escherichia coli. Both the P-EI and F-EI probes strongly hybridized with a 23-kbp plasmid in Southern hybridization analyses. The P-EII probe hybridized specifically with an 80-kbp plasmid, but the F-EII probe hybridized with none of the plasmids harbored in NK87. These results indicate that the P-EI gene and P-EII gene are encoded on the 23-kbp and 80-kbp plasmids, respectively.
Article
A novel expression vector pKP1500 for synthesizing unfused protein in Escherichia coli was constructed. pKP1500 perserves the tac promoter, the lacZ SD sequence, unique restriction sites (EcoRI, SmaI, BamHI, SalI, PstI and HindIII) and the rrnB terminators of pKK223-3, but the replication origin is replaced with that of pUC9. Construction of this plasmid is based upon the observation that the copy number control of pUC9 is temperature dependent. At 28 degrees C, the copy number of pKP1500 is less than 25 per chromosome, approximately the same copy number as that of pKK223-3, which contains the replication origin of pBR322, whereas at 42 degrees C, the copy number increases about 10 times and reaches up to 230 copies per chromosome. The main advantage of this system is that the temperature-dependent copy control and regulatable expression of the tac promoter make cells carrying pKP1500 derivatives stable against selective pressure by detrimental overproduction of foreign proteins at a low temperature and permits high expression of cloned DNAs at a high temperature. When chicken lysozyme cDNA carrying the initiation codon (ATG) immediately upstream from the Lys1 codon was inserted downstream from the tac promoter and the SD sequence, the pKP1500 derivative produced lysozyme at about 25% of the total cellular proteins. This value is more than 10 times higher than that obtained with the pKK223-3 derivative carrying the same lysozyme cDNA. By comparison, the expression of eukaryotic genes from the tac promoter reported by others has usually been less than a few % of the total cellular protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Article
The dideoxy chain termination method using deoxy-7-deaza-guanosine triphosphate (dc7GTP)in place of dGTP was found to be very useful. Sequencing of a part of the human N-myc gene having 85% GC content is impossible by the original method using dGTP, because of compression of bands. However, the nucleotide sequence of this part was unambiguously determined by analysis of both strands by the modified method. Use of dc7GTP is concluded to improve the dideoxy chain termination method for DNA sequencing.
Article
Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
Article
The nylon oligomer (6-aminohexanoic acid cyclic dimer) degradation genes on plasmid pOAD2 of Flavobacterium sp. KI72 were cloned into Escherichia coli vector pBR322. The locus of one of the genes, the structural gene of 6-aminohexanoic acid linear oligomer hydrolase, was determined by constructing various deletion plasmids and inserting the lacUV5 promoter fragment of E. coli into the deletion plasmid. Two kinds of repeated sequences (RS-I and RS-II) were detected on pOAD2 by DNA-DNA hybridization experiments. These repeated sequences appeared five times (RS-I) or twice (RS-II) on pOAD2. One of the RS-II regions and the structural gene of the hydrolase overlapped.
Article
With the development of large data banks of protein and nucleic acid sequences, the need for efficient methods of searching such banks for sequences similar to a given sequence has become evident. We present an algorithm for the global comparison of sequences based on matching k-tuples of sequence elements for a fixed k. The method results in substantial reduction in the time required to search a data bank when compared with prior techniques of similarity analysis, with minimal loss in sensitivity. The algorithm has also been adapted, in a separate implementation, to produce rigorous sequence alignments. Currently, using the DEC KL-10 system, we can compare all sequences in the entire Protein Data Bank of the National Biomedical Research Foundation with a 350-residue query sequence in less than 3 min and carry out a similar analysis with a 500-base query sequence against all eukaryotic sequences in the Los Alamos Nucleic Acid Data Base in less than 2 min.
Article
Flavobacterium sp. KI72 metabolizes 6-aminohexanoic acid cyclic dimer, a by-product of nylon manufacture, through two newly evolved enzymes, 6-aminohexanoic acid cyclic dimer hydrolase (EI) and 6-aminohexanoic acid linear oligomer hydrolase (EII). These enzymes are active towards man-made compounds, the cyclic dimer and linear oligomers of 6-aminohexanoic acid respectively, but not towards any of the natural amide bonds tested. The structural genes of EI (nylA) and EII (nylB) are encoded on pOAD2, one of three plasmids harboured in Flavobacterium sp. KI72. This plasmid contains two kinds of repeated sequence (RS-I and RS-II); one of the two RS-II sequences, RS-IIA, contains the nylB gene, while the other, RS-IIB, contains a homologous nylB' gene. From comparisons of the nucleotide sequences and gene products of the nylB and nylB' genes, we now conclude that EII enzyme is newly evolved by gene duplication followed by base substitutions on the same plasmid.
Article
6-Aminohexanoic-oligomer hydrolase of Flavobacterium sp. KI72 was purified to homogeneity by column chromatography three times, and by preparation polyacrylamide gel electrophoresis twice. The purified enzyme had the following characteristics. 1. The molecular weight was estimated to be 84000 by Sephadex G-200 molecular-sieve chromatography. The enzyme consisted of two homologous subunits of 42000, judged from sodium dodecylsulfate/polyacrylamide gel electrophoresis. 2. The optimum pH for activity was between 8 and 9, the optimum temperature was 40 degrees C for a 1-h reaction. The Michaelis-Menten constants and turnover numbers for the 6-aminohexanoic acid dimer and trimer were 5.9 mM and 2.4 s-1, and 6.2 mM and 2.0 s-1 respectively. 3. The enzyme was inhibited by 0.37 mM diisopropylfluorophosphate and by 0.013 mM p-chloromercuribenzoate. 4. The enzyme was active on 6-aminohexanoic acid oligomers from dimer to hexamer and icosamer but not on hectamer, and the activity decreased with the increase of the polymerization number of the oligomer. The oligomers were hydrolyzed so as to remove the 6-aminohexanoic acid residue successively from the amino terminus. The enzyme could not hydrolyze other linear amides, cyclic amides, dipeptides, tripeptides or casein. 5. 6-aminohexanoic-acid-oligomer hydrolase was classified as a new member of the linear amidases (EC 3.5.1.-).
Plasmid-determined enzymatic degradation of nylon oligo-mers Journal o f Bacteriology 155, 22-3 1 Determination of the active-site serine of 6-aminohexanoate-dimer hydrolase Evolutionary adaptation of plasmid-encoded enzymes for degrading nylon oligomers
  • S Negoro
  • T Taniguchi
  • S Negoro
  • T Mitamura
  • K Oka
  • Kanagawa
  • H Okada
  • S Negoro
  • H Kimura
  • N Nakamura
  • H Okada
Journal of Bacteriology 143, 238-245. NEGORO, S., TANIGUCHI, T. KANAOKA, (1 983). Plasmid-determined enzymatic degradation of nylon oligo-mers. Journal o f Bacteriology 155, 22-3 1. NEGORO, S., MITAMURA, T., OKA, K., KANAGAWA, (1 989). Determination of the active-site serine of 6-aminohexanoate-dimer hydrolase. European Journal o f Biochemistry 185, 52 1-524. OKADA, H., NEGORO, S., KIMURA, H. & NAKAMURA, Evolutionary adaptation of plasmid-encoded enzymes for degrading nylon oligomers. Nature, London 306, 203-206. N. & OKADA, H. (1989). Plasmid M., BISARIA, V. S., SAWADA, A., KINOSHITA, S., HATOZAKI, M., KIMURA, H. & OKADA, H
New M13 vectors for cloning
MESSING, J. (1983). New M13 vectors for cloning. Methods in Enzymology 101, 20-78.
High homology between
  • K Fukwama
  • S Kanzaki
  • N Kanagawa
  • K Negoro
  • S Okada
TSUCHIYA, K., FUKWAMA, S., KANZAKI, N., KANAGAWA, K., NEGORO, S. & OKADA, H. (1989). High homology between