A new, broad-substrate-specificity aminopeptidase from the dairy organism Lactobacillus helveticus SBT 2171

Abstract

An aminopeptidase with a very broad substrate specificity was purified to homogeneity from Lactobacillus helveticus SBT 2171 by FPLC. The enzyme was purified 144-fold from a cell-free extract with a yield of 16%. The purified enzyme appeared as a single band on an SDS-PAGE gel. It had a molecular mass of 95 kDa and an isoelectric point of 4.9. The enzyme hydrolysed a large range of naphthylamide-and nitroanilide-substituted amino acids, as well as several di-, tri-and oligopeptides. It also exhibited significant prolineiminopeptidase-like activity, since it hydrolysed several proline-containing peptides. Prolyl-p-nitroanilide was hydrolysed with a low affinity (Michaelis-Menten constant 0.6 mM) and a Vmex of 2.5 pmol min-l (mg protein)-' while lysyl-p-nitroanilide was hydrolysed with a high affinity [K, 0003 mM; VmaX 37.5 pmol min-l (mg protein)-l]. The aminopeptidase activity, which was optimal between pH 6.0 and 8 0 and at 50 "C, was very stable a t 30 "C for more than 7 d. The activity lost by treatment with the thiol-blocking reagents could be restored with P-mercaptoethanol, while Co2+ and Mn2+ restored the activity of the EDTA-treated enzyme. Immunological experiments with antibodies raised against the aminopeptidases from Lactococcus lactis and Lb. helveticus clearly showed that both aminopeptidases are a t least immunologically different from each other.

Purification and properties of phenylalanyl aminopeptidase synthesised by Pseudomonas sp.

“…Moreover, a strong inhibition of enzymatic activity in the presence of cysteine enzyme inhibitors was also observed in aminopeptidases synthesised by the strains of Streptococcus , Pseudomonas fluorescens (GONZALES and ROBERT-BAUDOUY 1994), Bacillus (SZEWCZUK and MULCZYK 1969), Xanthomonas citri (OSADA and ISONO 1986), as well as in aminopeptidase synthesised by Neisseria catarrhalis (BEHAL and COX 1968). Inhibited activity in the presence of iodoacetamide and p-CMB was also obtained in some metallo-aminopeptidases (MIYA- KAWA et al 1992, SASAKI et al 1996, MAGBOUL and SWEENEY 1999. In the present investigations, metalloenzyme inhibitors: (EDTA), bestatin and amastatin did not effect losses in phenylalanyl aminopeptidase activity, which suggested the enzyme in question was not a metallo-aminopeptidase.…”

Variation in aminopeptidase and aminotransferase activities of six cheese related Lactobacillus helveticus strains

“…Thiol-enzyme inhibitors, such as pCMB, may inhibit PepC completely while no inhibition has been found using metallo-inhibitors such as EDTA (Ferná ndez de Palencia et al, 2000;Vesanto et al, 1994). On the other hand, EDTA efficiently inhibits PepN, but inhibitors for thiol-enzymes have also been shown to partly inhibit PepN from several but not all L. helveticus strains (Blanc et al, 1993;Khalid & Marth, 1990b;Miyakawa et al, 1992;Sasaki et al, 1996;. The aminopeptidase activities in this study against all individual amino acids except Pro were reduced by both pCMB and EDTA indicating that both PepN and PepC contribute to these activities.…”

“…Type a Substrate b Reference (Blanc et al, 1993) 53/7 M Lys-> Leu-> Met-pNA SBT 2171 M Lys-> Arg-> Ala-> Leu-pNA (Sasaki et al, 1996) JCM 1004 M Lys-> Arg-> Leu-> Ala-pNA…”

“…Two well-characterised general aminopeptidases, the thiolenzyme PepC and the metallo-enzyme PepN, have been isolated and physiologically as well as genetically characterised in several L. helveticus strains and both enzymes appear to be well conserved among lactobacilli (Blanc, Laloi, Atlan, Gilbert, & Portalier, 1993;Ferná ndez, Bhowmik, & Steele, 1994; Ferná ndez de Palencia, Ló pez de Felipe, Requena, & Pelá ez, 2000;Khalid & Marth, 1990b;Miyakawa, Kobayashi, Shimamura, & Tomita, 1992;Pan & Tanokura, 2004;Sasaki, Bosman, & Tan, 1996;. Both aminopeptidases have been shown to have high activity against the same chromogenic substrates but with somewhat different affinity (Table 1), and they have generally no or very low activity against Pro containing aminopeptidase substrates.…”

Preventive Effect of Lactobacillus helveticus SBT2171 on Collagen-Induced Arthritis in Mice

“…To contribute toward resolving these issues, in this study, we focused on Lactobacillus helveticus SBT2171, a strain of lactobacilli that is derived from dairy products, possesses high protease activity, and is commonly used as a starter bacterial strain in the production of a Gouda-type cheese (Sasaki et al, 1996). Our previous study clarified that L. helveticus SBT2171 significantly suppressed the proliferation of primary murine immune cells among 41 LAB strains of various species, and decreased the production of lipopolysaccharide (LPS)-stimulated inflammatory cytokines [interleukin (IL)-6 and IL-1β] from the immune cells in vitro (Yamashita et al, 2014).…”