Antilisterial activity by enterococci in a model predicting the temperature evolution of Taleggio, an Italian soft cheese.

Enterococcus ssp. produced broad-spectrum bacteriocins, which were active against Listeria innocua and Listeria monocytogenes. Inhibition of L. innocua in milk by different starter cultures was evaluated. Milk was inoculated with a conventional starter (Streptococcus salivarius ssp. thermophilus and Lactobacillus delbrueckii ssp. bulgaricus mixed cultures) as a sole starter or in combination with inhibitory starter cultures (Enterococcus ssp., bacteriocin-producing strains). Tests were performed under a temperature gradient that reproduced the first 55 h of Taleggio cheese manufacturing and in the presence of rennet. Results indicated that satisfactory inhibition of L. innocua was linked to the combined synergistic antimicrobial effect of pH decrease and bacteriocin production. Overall, data suggested that a potential existed for use of the antagonistic activity from enterococci for protection of Taleggio cheese during ripening, when L. monocytogenes may often reach dangerous concentrations.

Characterization of a fully active N-terminal 37-kDa polypeptide obtained by limited tryptic cleavage of pig kidney D-amino acid oxidase.

In order to obtain further information on the structure of D-amino acid oxidase (EC 1.4.3.3), limited proteolysis experiments have been carried out on its apo-, holo-, and holoenzyme-benzoate forms. The enzyme is unsensitive to 10% (w/w) chymotrypsin, while incubation with 10% (w/w) trypsin, under nondenaturating conditions, produces inactivation and proteolysis patterns which are different for the three forms of enzyme analyzed. These results confirm the previously reported conformational changes which occur upon binding of coenzyme to the apoprotein, and of benzoate to holoenzyme. The stable 37.0-kDa polypeptide, obtained from the apo- and holoenzyme-benzoate complex upon cleavage of a C-terminal 2.0-kDa fragment, retains full catalytic activity with unaltered kinetic parameters, and the coenzyme binding properties of the native enzyme. These results are in agreement with the tentative localization of the FAD-binding domain in the N-terminal region of the enzyme, and with the hypothesis that the function of the C-terminal region of D-amino acid oxidase could be related to the import of the enzyme into the peroxisomes, as suggested by Gould et al. (Gould, S. J., Keller, G. A., and Subramani, S. (1988) J. Cell. Biol. 107, 897-905).

cDNA cloning, sequence analysis, and chromosomal localization of the gene for human carnitine palmitoyltransferase.

We have cloned and sequenced a cDNA encoding human liver carnitine palmitoyltransferase (CPTase; palmitoyl-CoA:L-carnitine O-palmitoyltransferase, EC 2.3.1.21), an inner mitochondrial membrane enzyme that plays a major role in the fatty acid oxidation pathway. Mixed oligonucleotide primers whose sequences were deduced from one tryptic peptide obtained from purified CPTase were used in a polymerase chain reaction, allowing the amplification of a 0.12-kilobase fragment of human genomic DNA encoding such a peptide. A 60-base-pair (bp) oligonucleotide synthesized on the basis of the sequence from this fragment was used for the screening of a cDNA library from human liver and hybridized to a cDNA insert of 2255 bp. This cDNA contains an open reading frame of 1974 bp that encodes a protein of 658 amino acid residues including 25 residues of an NH2-terminal leader peptide. The assignment of this open reading frame to human liver CPTase is confirmed by matches to seven different amino acid sequences of tryptic peptides derived from pure human CPTase and by the 82.2% homology with the amino acid sequence of rat CPTase. The NH2-terminal region of CPTase contains a leucine-proline motif that is shared by carnitine acetyl- and octanoyltransferases and by choline acetyltransferase. The gene encoding CPTase was assigned to human chromosome 1, region 1q12-1pter, by hybridization of CPTase cDNA with a DNA panel of 19 human-hamster somatic cell hybrids.