Lactobacillus reuteri DSM 20016: purification and characterization of a cystathionine gamma-lyase and use as adjunct starter in cheesemaking.
A homo-tetrameric approximately 160-kDa cystathionine gamma-lyase was purified to homogeneity from Lactobacillus reuteri DSM 20016 by four chromatographic steps. The activity was pyridoxal-5'-phosphate dependent and the enzyme catalyzed the alpha,gamma-elimination reaction of L-cystathionine, producing L-cysteine, ammonia and alpha-ketobutyrate. The enzyme was active towards a range of amino acids and amino acid derivatives, including methionine. The pH and temperature optima were found to be 8.0 and 35 degrees C. respectively. Isoelectric pH (pI) was approximately 5.0 as determined by two-dimensional electrophoresis. Sensitivity to chemical inhibitors was typical of lactococcal cystathionine gamma- and beta-lyases, except it was inhibited by sulphydryl reagents. The N-terminal sequence was MKFNTQLIHGGNSED, which had 100% homology with cystathionine beta-lyase of Lb. reuteri 104R (Accession Number (CAC05298). Lb. reuteri DSM 20016. together with 10 other strains of non-starter lactic acid bacteria, was used as adjunct starter in the production of miniature ('anestrato Pugliese-like cheeses. After 40 d ripening, the water-soluble extract of the cheeses with added Lactobacillus fermentum DT41 and Lb. reuteri DSM 20016 contained the highest enzyme activities on cystathionine and methionine substrates. Determinations of methanethiol, dimethyl sulphide, dimethyl disulphide and dimethyl trisulphide in the miniature cheeses confirmed the findings of enzyme activities.
De Angelis
Maria
M
Dipartimento di Scienze degli Alimenti, Università degli Studi di Perugia, Italy.
Curtin
Aine C
AC
McSweeney
Paul L H
PL
Faccia
Michele
M
Gobbetti
Marco
M
D000596:Amino Acids / Q000378:metabolism*; D002611:Cheese / Q000382:microbiology*; D003540:Cystathionine / Q000378:metabolism; D003542:Cystathionine gamma-Lyase / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005516:Food Microbiology; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007778:Lactobacillus / Q000201:enzymology*; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D013379:Substrate Specificity; D013696:Temperature
D000596:Amino Acids; D003540:Cystathionine; D008715:Methionine; D003542:Cystathionine gamma-Lyase