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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.

6‐Aminohexanoic acid cyclic dimer hydrolase produced by Achromobacter guttatus cells grown on 6‐aminohexanoic acid cyclic dimer (1,8‐diazacyclotetradecane‐2,9‐dione) was purified until it was homogeneous according to polyacrylamide disc gel electrophoresis, sodium dodecylsulfate polyacrylamide gel electrophoresis, Sephadex G‐200 column chromatography, and analytical centrifugation. The following characteristics of the purified enzyme were observed. (1) The molecular weight of the enzyme, estimated to be 110 000 by column chromatography on Sephadex G‐200 and 100 000 by sedimentative equilibrium, together with the minimum molecular weight of 55 000 obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis, indicated it is a dimeric enzyme. The sedimentation constant (s20) was estimated to be 5.7 S from the sedimentation velocity. (2) The enzyme had an optimum pH of 7.3 when 6‐aminohexanoic acid cyclic dimer was used as substrate. It was stable in a pH range between 5.5 to 8.5, and 50% and 100% of activity was lost after 10 min of heating at 45°C and 50°C respectively. The optimum temperature for activity was 33°C. The Michaelis‐Menten constant (Km) was 6 mM toward the dimer, and the turnover number was 8 s−1 assuming a molecular weight of 100 000. (3) This enzyme was strongly inhibited by 1 μM diisoprpylphosphofluoridate and 10 μM p‐chloromercuribenzoate but scarcely inhibited by 100 mM ethylenediaminetetraacetic acid. The enzyme inhibited by p‐chloromercuribenzoate could be reactivated by 2‐mercaptoethanol. (4) This enzyme was only active on 6‐aminohexanoic acid cyclic dimer to form 6‐aminohexanoyl‐6‐aminohexanoic acid. It was inactive on 6‐aminohexanoic acid oligomers (degree of polymerization 2 to 6), various cyclic amides, cyclic diamides, amides, oligopeptides, and casein. (5) The absorption coefficient (A1%280 nm) of the enzyme was 19.4. The enzyme was composed of 500 amino acid residues including 3 cysteines; from this the molecular weight was calculated to be 103 200 as a dimer. (6) 6‐Aminohexanoic acid cyclic dimer hydrolase was classified as a new cyclic amide hydrolase.

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The nucleotide sequence of the bgaB gene, which encodes the thermostable ,-galactosidase I of Bacillus stearothermophilus, and its flanking region was determined. A 2,016-base-pair open reading frame observed was concluded to be for 13-galactosidase I (Mr 78,051) from observations that the amino acid composition of the elizyme,ahd the sequence of 14 amino acids from the amino-terminus of the enzyme coincided with those dedpcWd fromn this open frame. A 107-base-pair HaeIlH-AluI fragment just upstream of the estimated Shinke-Dilgarnio sequence of the bgaB gene had promoter activity toward cat-86 (chloramphenicol acetyltransferase gene) and produced the enzyme at a level equivalent to 7% of the total cellular protein of B. subtilis. Froth the base sequence of this DNA region and the transcriptional start site determined by Si nuclease mapping, the -35 and -10 sequences are estimated to be TTGACA and TAATTT, respectively, which are similar to the consensus sequence of B. subtilis cr RNA polymerase.Although extensive enzymatic and genetic studies have been made on the 3-galactosidases of Escherichia coli, little information is available on thermophilic f-galactosidases.We have cloned two ,B-galactosidase genes (bgaA and bgaB) of a thermophilic bacterium, Bacillus stearothermophilus IAM1i001, and revealed that two of the three 1-galactosidases (P-galactosidases II and III) produced by this bacterium are coded on the same gene (bgaA) but differ in quaternary structure (11). The other 1-galactosidase (,Bgalactosidase I [13-gall]) coded on bgaB is the most thermostable of the three, being stable up to 70°C (11), so its amino acid sequence will provide an example of the primary structure of a thermostable protein.Bacillus subtilis harboring a hybrid plasmid containing the bgaB gene produced about 50 times more 1-galI than B. stearothermophilus, and 1-galI accounted for 6% of the total protein of the host bacterium (12). This high production suggests that the promoter of the bgaB gene is effective in B. subtilis.In this paper we determined the nucleotide sequence of the bgaB gene, including its promoter region, and analyzed the amino acid sequence of 1-gall. The high expression of this promoter was confirmed by using the cat-86 gene. MATERIALS AND METHODSBacterial strains, plasmids, media, and culture conditions. B. stearothermophilus IAM11001 (ATCC 8005), B. subtilis MlIll (arg-15 leuA8 rM mM), and the plasmid pHG5 (a 2.9-kilobase [kb] EcoRI fragment containing the bgaB gene plus the 4.5-kb EcoRI fragment of pUB110) have been described previously (11). B. subtilis BR151, harboring the promoter cloning plasmid pPL603, was obtained from the Bacillus Genetic Stock Center (Ohio State University) (31). E. coli JM103 was used as host strain for the M13 phage derivatives mplO and mpll (20). Bacteria were grown on LL medium (11) or Penassay broth (Difco Laboratories). When necessary, kanamycin (5 ,ug/ml) or chloramphenicol (5 ,ug/ml) was added to the medium.Electrophoresis.