GENE CLONING, SEQUENCE-ANALYSIS, PURIFICATION, AND CHARACTERIZATION OF A THERMOSTABLE AMINOACYLASE FROM BACILLUS-STEAROTHERMOPHILUS

A genomic DNA fragment encoding aminoacylase activity of the eubacteri um Bacillus stearothermophilus was cloned into Escherichia coli. Trans formants expressing aminoacylase activity were selected by their abili ty to complement E. coli mutants defective in acetylornithine deacetyl ase activity, the enzyme that converts N-acetylornithine to ornithine in the arginine biosynthetic pathway. The 2.3-kb cloned fragment has b een entirely sequenced. Analysis of the sequence revealed two open rea ding frames, one of which encoded the aminoacylase. B. stearothermophi lus aminoacylase, produced in E. coli, was purified to near homogeneit y in three steps, one of which took advantage of the intrinsic thermos tability of the enzyme. The enzyme exists as homotetramer of 43-kDa su bunits as shown by cross-linking experiments. The deacetylating capaci ty of purified aminoacylase varies considerably depending on the natur e of the amino acid residue in the substrate. The enzyme hydrolyzes N- acyl derivatives of aromatic amino acids most efficiently. Comparison of the predicted amino acid sequence of B. stearothermophilus aminoacy lase with those of eubacterial acetylornithine deacylase, succinyldiam inopimelate desuccinylase, carboxypeptidase G2, and eukaryotic aminoac ylase I suggests a common origin for these enzymes.