Purification and characterization of an extracellular proline iminopeptidase from Corynebacterium variabilis NCDO 2101

Abstract

Aims: To screen the extracellular proteolytic and lipolytic activities of Corynebacterium variabilis NCDO 2101 and to purify and characterize a proline iminopeptidase enzyme in order to investigate the role of the major component of the smear of bacterial surface-ripened cheeses. Methods and Results: Four chromatographic steps were used to purify the enzyme and a three-factor, ®ve-level Central Composite Design was used to study the interactive effects of cheese-related values of pH, NaCl and temperature. The proline iminopeptidase showed some biochemical properties different from the same enzyme puri®ed from lactic acid bacteria and other smear bacteria. It tolerated NaCl concentrations up to 7á5% and was sensitive to low values of pH especially when they were combined with low temperature. Conclusions: The proline iminopeptidase of C. variabilis NCDO 2101 may have a role in proteolysis during ripening of smear surface-ripened cheeses. Signi®cance and Impact of the Study: The ®ndings of this work contribute to the knowledge of the enzymology of smear bacteria in order to improve the ripening of bacterial surface-ripened cheeses.

Complete genome sequence of Corynebacterium variabile DSM 44702 isolated from the surface of smear-ripened cheeses and insights into cheese ripening and flavor generation

“…In addition, amino-terminal prolines are released from peptides by the enzymatic action of proline iminopeptidase (EC 3.4.11.5) encoded by the pepI gene. The enzymatic activity of an extracellular proline iminopeptidase from C. variabile NCDO 2101 was demonstrated previously [122]. Since caseins are very rich in proline residues and proline is the most abundant amino acid in cheese, it may constitute a prominent substrate for C. variabile .…”

“…Likewise, caseins are the major protein sources in milk and cheese [1] and are thus prominent substrates for proteolysis by C. variabile , presumably carried out by secreted proteases. Moreover, proline iminopeptidase can contribute to proteolysis [122], as prolines are the major amino acid constituents of caseins [1]. It is obvious that these metabolic specificities of C. variabile are closely linked to its niche adaption.…”

Mammals, Milk, Molecules, and Micelles

“…(Stepaniak et al 1982;Fox et al 1989;Garcia de Fernandez & Fox 1991;Requena et al 1993;Gobbetti et al 1995Gobbetti et al , 1997aGobbetti et al , 2001Baral et al 1995;Stepaniak & Fox 1995;Rattray et al 1996FernandezEspla et al 1997;Smacchi et al 1999).…”

Dairy enzymology

“…At a later stage of ripening, aerobic, halotolerant Brevibacterium linens become a dominating bacterium. Other corynobacteria species such as Arthrobacter nicotianae and micrococci also comprise a large part of the smear microflora (Rattray and Fox 1997, 1999; Gobbetti et al . 2001).…”

“…Purified serine extracellular PepI from Corynobacterium variabilis was also active at the temperature of cheese ripening and an NaCl concentration of 7.5%. The monomeric enzyme had a molecular mass of 45 kDa (Gobbetti et al . 2001).…”