Cloning, sequencing, and high expression of the proline iminopeptidase gene from Bacillus coagulans

Abstract

The gene coding for proline iminopeptidase in BaciUus coagulans was cloned and expressed in Escherichia coli. Nucleotide sequencing revealed an 861-bp open reading frame with an unusual TTG initiation codon, encoding a 287-amino-acid protein. The calculated molecular weight of the product was 32,415. The amino acid sequences of the amino-terminal region and those of some peptide fragments obtained by endoproteinase Asp-N digestion of the purified enzyme completely coincided with those deduced from the nucleotide sequence. The rare TTG initiation codon that normally codes for leucine was translated as a formal initiation codon; a methionine residue was found at the amino terminus of the enzyme. By using a vector bearing the strong tac promoter, an expression level as high as 200-fold that of the first clone was achieved. The replacement of the TTG initiation codon with ATG and a simultaneous reduction of the distance to the tac promoter resulted in a further increase of 2.5-fold. The expressed enzyme was easily purified to homogeneity by hydrophobic chromatography on a Toyopearl HW-65C column and crystallization, with a recovery of activity of 36%. The molecular weight was found to be 33,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration on a Hi-Load 16/60 Superdex 200 fast protein liquid chromatography column. The expressed enzyme showed the same catalytic and physicochemical properties as those of the wild type, specifically cleaving the N-terminal proline from small substrates.Proline iminopeptidase (EC 3.4.11.5) activity was reported for the first time by Sarid et al. (24,25), who found it in a prolineless mutant of Escherichia coli that could utilize poly-L-proline in place of the required amino acid. After these two reports, however, there have not been any others about proline iminopeptidase activity in E. coli. Recently, Fanghanel et al. (3) reported the use of L-proline iminopeptidase activity for the identification of Serratia and Hafnia species. These strains hydrolyzed L-proline-4-nitroanilide, in contrast to 18 other Enterobacteriaceae species including E. coli. The enzyme activity has also been detected in a variety of organisms, i.e., some human oral cavity microorganisms (14), Neisseria gonorrhoeae (2), Bacillus megatenium (36), Bacillus coagulans (38), Lyophyllum cinerascens (26), and apricot seeds (18). Furthermore, a proline iminopeptidase was partially purified from Bacillus brevis and used for the enzymatic synthesis of proline-containing peptides (20). However, only the enzymes from B. coagulans and apricot seeds have been purified homogeneously.In mammals, an activity that cleaved the N-terminal proline of melanocyte-stimulating-hormone-release-inhibiting factor (Pro-Leu-GlyNH2) from pig (19) and bovine kidney (10), and recently, the detection of a proline iminopeptidase activity in rat liver and kidney have also been reported (9). However, the presence of a real proline iminopeptidase in mammalian tissues has remained uncertain, since other aminopept...

Prolylcarboxypeptidase (Angiotensinase C): Purification and Characterization of the Enzyme fromXanthomonas maltophilia

“…The molecular weight of the native enzyme was estimated by gel filtration on a column 0.6 x 60 cm) of Hiload Superdex 200 pg (FPLC system, Pharmacia) using 20 mM Tris-HCI buffer (pH 8.0) containing 0.15 M NaCl. Aminopeptidase P from E. co/i14.15) (M r 200,000), yeast alcohol dehydrogenase (M r 140,000), bovine serum albumin (M r 67,000), and proline iminopeptidase from B. coagulants 16 ) (M f 32,000) were used as reference proteins.…”

Prolidase fromXanthomonas maltophilia: Purification and Characterization of the Enzyme

“…buffer (pH 8.0) containing 0.15M NaC!. Aminopeptidase P from E. COli 16 • 17 ) (M r 200,000), yeast alcohol dehydrogenase (M r 140,000), bovine serum albumin (M r 67,000), and proline iminopeptidase from B. coagulants 18 ) (M r 32,000) were used as reference proteins. The purity and molecular weight of the isolated enzyme with or without reduction with 2-mercaptoethanol were measured by SDS-PAGE with a Phast Gel gradient 10 to 15% (Phast system, Pharmacia).…”

Purification and Characterization of a Novel Prolyl Aminopeptidase fromMaitake(Grifola frondosa)

“…The presence of this enzyme has been reported mainly in micooroganisms. [1][2][3] Hence, we screened more than 30 kinds of fruiting bodies of edible mushrooms and found that Maitake (Grifola frondosa) had high PAP activity. G. frondosa is one of the most popular edible mushrooms in Japan.…”

“…It is commonly assumed that these PAPs are sulfhydryl enzymes. 2,3) But the enzymes were identified as a serine peptidase by sitedirected mutagenesis. 10) For the above reason, further studies are necessary to identify the active site residue(s) of GfPAP.…”