Abstract
The gene encoding the novel enzyme N-acylamino acid racemase (AAR) was cloned in recombinant phage λ-4 from the DNA library of Amycolatopsis sp. TS-1-60, a rare actinomycete, using antiserum against the enzyme. The cloned gene was subcloned and transformed in Escherichia coli JM105 using pUC118 as a vector. The AAR gene consists of an open-reading frame of 1104 nucleotides, which specifies a 368-amino-acid protein with a molecular mass of 39411Da. The molecular mass deduced from the AAR gene is in good agreement with the subunit molecular mass (40kDa) of AAR from Amycolatopsis sp. TS-1-60. The guanosine plus cytosine content of the AAR gene was about 70%. Although the AAR gene uses the unusual initiation codon GTG, the gene was expressed in Escherichia coli using the lac promoter of pUC118. The amount of the enzyme produced by the transformant was 16 times that produced by Amycolatopsis sp. TS-1-60. When the unusual initiation codon GTG was changed to ATG, the enzyme productivity of the transformant increased to more than 37 times that of Amycolatopsis sp. TS-1-60. In the comparison of the DNA sequence and the deduced amino acid sequence of AAR with those of known racemases and epimerases in data bases, no significant sequence homology was found. However, AAR resembles mandelate racemase in that requires metal ions for enzyme activity. Comparison of the deduced amino acid sequences of mandelate racemase and AAR revealed amino acid sequences in AAR similar to those of both the catalytic and metal-ion-binding sites of mandelate racemase.
Similar content being viewed by others
References
Galakatos NG, Daub E, Botstein, D, Walsh CT (1986) Biosynthetic alr alanine racemase from Salmonella typhimurium: DNA and protein sequence determination. Biochemistry 25:3255–3260
Gallo KA, Knowles JR (1993) Purification, cloning, and cofactor independence of glutamate racemase from Lactobacillus Biochemistry 32:3981–3990
Hopwood DA, Bibb MJ, Chater KF, Bruton CJ, Kieser HM, Lydiate DJ, Smith CP, Ward JM, Schrempf H (1985) Genetic manipulation of Streptomyces: a laboratory manual. John Innes Foundation, Norwich
Maniatis T, Fritsch EF, Smabrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Masaki T, Nakamura K, Isono M, Soejima M (1978) A new proteolytic enzyme from Achromobacter lyticus M497–1 Agric Biol Chem 42:1443–1445
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Munson LM, Stormo GD, Niece RN, Reznikoff WS (1984) lacZ Translation initiation mutations. J Mol Biol 177:663–683
Ouchterlony O (1985) Diffusion in gel methods for immunological analysis. Prog Allergy 5:1–9
Ranson SC, Gerlt JA, Powers VM, Kenyon GL (1988) Cloning, DNA sequence analysis, and expression in Escherichia coli of the gene for mandelate racemase from Pseudomonas putida. Biochemistry 27:540–545
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Shine J, Dalgano L (1974) The 3'-terminal sequence of E. coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci USA 71:1342–1346
Southern EN (1975) Detection of specific sequences among DNA fragments by gel electrophoresis. J Mol Biol 98:503–517
Tanizawa K, Ohshima A, Scheidegger A, Inagaki K, Tanaka H, Soda K (1988) Thermostable alanine racemase from Bacillus stearothermophilus: DNA and protein sequence determination and secondary structure prediction. Biochemistry 27:1311–1316
Tokuyama S, Hatano K (1995) Purification and properties of thermostable N-acylamino acid racemase from Amycolatopsis sp. TS-1-60. Appl Microbiol Biotechnol
Tokuyama S, Hatano K, Takahashi T (1994a) Discovery of a novel enzyme, N-acylamino acid racemase in an actinomycete: screening, isolation and identification. Biosci Biotechnol Biochem 58:24–27
Tokuyama S, Miya H, Hatano K, Takahashi T (1994b) Purification and properties of a novel enzyme, N-acylamino acid racemase, from Streptomyces atratus Y-53. Appl Microbiol Biotechnol 40:835–840
Tosa T, Mori T, Fuse N, Chibata I (1967) Studies on continuous enzyme reaction. IV. Preparation of a DEAE-Sephadex-aminoacylase column and continuous optical resolution of acyl-Dl-amino acids. Biotechnol Bioeng 9:603–615
Vieira J, Messing J (1987) Production of single-strand plasmid DNA. Methods Enzymol 153:3–11
Wasserman SA, Daub E, Grisafi P, Botstein D, Walsh CT (1984) Catabolic alanine racemase from Salmonella typhimurium: DNA sequence, enzyme purification, and characterization. Biochemistry 23:5182–5187
Watabe K, Ishikawa T, Mukohara Y, Nakamura H (1992) Identification and sequencing of a gene encoding a hydantoin racemase from the native plasmid of Pseudomonas sp.strain NS671. J Bacteriol 174:3461–3466
Weil-Malherbe H (1966) Some properties of mandelate racemase from Pseudomonas fluorescens. Biochem J 101:169–175
Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13 mp18 and pUC19 vectors. Gene 33:103–119
Young RA, Davis RW (1983) Yeast RNA polymerase II gene: isolation with antibody probes Science 222:778–782
Yohda M, Okada H, Kumagai H (1991) Molecular cloning and nucleotide sequencing of aspartate racemase gene from lactic acid
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Tokuyama, S., Hatano, K. Cloning, DNA sequencing and heterologous expression of the gene for thermostable N-acylamino acid racemase from Amycolatopsis sp. TS-1-60 in Escherichia coli . Appl Microbiol Biotechnol 42, 884–889 (1995). https://doi.org/10.1007/BF00191186
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00191186