Cloning, DNA sequencing and heterologous expression of the gene for thermostable N-acylamino acid racemase from Amycolatopsis sp. TS-1-60 in Escherichia coli.
The gene encoding the novel enzyme N-acylamino acid racemase (AAR) was cloned in recombinant phage lambda-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.(ABSTRACT TRUNCATED AT 250 WORDS)
Tokuyama
S
S
Technology Development Laboratories, Takeda Chemical Industries Ltd., Osaka, Japan.
Hatano
K
K
D039903:Actinobacteria / Q000201:enzymology* / Q000235:genetics*; D000591:Amino Acid Isomerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics; D019998:Racemases and Epimerases / Q000235:genetics; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D000596:Amino Acids; D004271:DNA, Fungal; D010446:Peptide Fragments; D019998:Racemases and Epimerases; D000591:Amino Acid Isomerases; C021540:mandelate racemase