Purification and characterization of an extracellular proline iminopeptidase from Arthrobacter nicotianae 9458

Abstract

An extracellular proline iminopeptidase, with a molecular mass of about 53 kDa, was purified from Arthrobacter nicotianae 9458 and characterized. The enzyme had temperature and pH optima of 37³C and 8.0, respectively, was completely inactivated by heating for 1 min at 80³C and showed highest activity on Pro-pNA. The proline iminopeptidase was characterized by activity at low temperature, NaCl concentrations up to 7,5% and by high sensitivity to pH values 6.0, serine enzyme inhibitor PMSF and divalent cations, Fe 2 , Sn 2 , Cu 2 , Zn 2 , Hg 2 , Co 2 and Ni 2 . The extracellular proline iminopeptidase from A. nicotianae 9458 was able to hydrolyze proline-containing peptides at the pH, temperature and NaCl concentration typical of the surface of smear-ripened cheeses and may contribute to proteolysis of these cheeses during ripening. ß

Purification and Characterization of a Prolyl Aminopeptidase from Debaryomyces hansenii

“…Most of the purified PAPs have optimal pHs of between 7 and 8 (7,14,16,32,35). The optimum temperature of the majority of PAPs is between 37 and 55°C (17,32).…”

“…PAP of P. camemberti is inhibited by thiol reagents but also exhibits inhibition by di-isopropylfluorophosphate, indicating that serine residues are important for the catalytic activity (7). In contrast, PAPs from lactic acid bacteria such as Propionibacterium shermanii and Lactobacillus delbrueckii and from Arthrobacter nicotianae and Hafnia alvei are considered serine proteases on the basis of 3,4-DCI inhibition (8,16,25,32). Studies by sitedirected mutagenesis in Bacillus coagulans and Aeromonas so- bria also demonstrated that the enzyme from prokaryotes is indeed a serine protease (15).…”

“…The unique structure of proline in polypeptide chains restricts their susceptibility to the action of most proteases, and the activity of specific enzymes is required to avoid accumulation of proline-containing polypeptides. There are two major groups of specialized enzymes that are able to cleave peptide bounds involving proline: (i) aminopeptidase P and prolidase, which cleave X-Pro bonds from oligopeptides and dipeptides, respectively, and are both metalloenzymes, and (ii) prolyl oligopeptidase, prolinase, prolyl carboxypeptidase, and prolyl aminopeptidase, which cleave Pro-X bonds and are either serine (8,15,17,25,32) or cysteine (7,14,35,36) proteases. The PAP of D. hansenii releases proline from the N-terminal position of peptides of at least six amino acid residues (i.e., Pro-Pro-Gly-Phe-Ser-Pro) and shows higher preference for oligopeptides than for dipeptides.…”

Proline-Specific Extracellular Aminopeptidase Purified from Streptomyces lavendulae

“…3). Most of the purified PAPs have an optimal pH of between 7 and 8 [15,[18][19][20][21]. The optimum temperature of the majority of PAPs is between 37 and 55°C [19,20].…”

Arthrobacter

“…Both enzyme functions, poly(P)-and ATP-dependent gluco-and mannokinase activities require Mg 2+ or Mn 2+ . Zn 2+ and Co 2+ mediate high activity only for poly(P)-dependent glucomannokinase activity (Mukai et al, 2003 Two extracellular proteinases and an extracellular proline iminopeptidase have been characterized from Arthrobacter nicotianae 9458, isolated from the bacterial smear of surface ripened cheeses (Smacchi et al, 1999a(Smacchi et al, , 1999b. The proteinases have molecular masses of approximately 54 kDa (proteinase P1) and 71 kDa (proteinase P2).…”