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l-Methionine γ-lyase (EC 4.4.1.11 ) was purified to homogeneity from Brevibacterium linens BL2, a coryneform bacterium which has been used successfully as an adjunct bacterium to improve the flavor of Cheddar cheese. The enzyme catalyzes the α,γ elimination of methionine to produce methanethiol, α-ketobutyrate, and ammonia. It is a pyridoxal phosphate-dependent enzyme, with a native molecular mass of approximately 170 kDa, consisting of four identical subunits of 43 kDa each. The purified enzyme had optimum activity at pH 7.5 and was stable at pHs ranging from 6.0 to 8.0 for 24 h. The pure enzyme had its highest activity at 25°C but was active between 5 and 50°C. Activity was inhibited by carbonyl reagents, completely inactivated by dl-propargylglycine, and unaffected by metal-chelating agents. The pure enzyme had catalytic properties similar to those of l-methionine γ-lyase fromPseudomonas putida. Its Km for the catalysis of methionine was 6.12 mM, and its maximum rate of catalysis was 7.0 μmol min−1 mg−1. The enzyme was active under salt and pH conditions found in ripening Cheddar cheese but susceptible to degradation by intracellular proteases.

Methanethiol has been strongly associated with desirable Cheddar cheese flavor and can be formed from the degradation of methionine (Met) via a number of microbial enzymes. Methionine γ-lyase is thought to play a major role in the catabolism of Met and generation of methanethiol in several species of bacteria. Other enzymes that have been reported to be capable of producing methanethiol from Met in lactic acid bacteria include cystathionine β-lyase and cystathionine γ-lyase. The objective of this study was to determine the production, stability, and activities of the enzymes involved in methanethiol generation in bacteria associated with cheese making. Lactococci and lactobacilli were observed to contain high levels of enzymes that acted primarily on cystathionine. Enzyme activity was dependent on the concentration of sulfur amino acids in the growth medium. Met aminotransferase activity was detected in all of the lactic acid bacteria tested and α-ketoglutarate was used as the amino group acceptor. In Lactococcus lactis subsp. cremorisS2, Met aminotransferase was repressed with increasing concentrations of Met in the growth medium. While no Met aminotransferase activity was detected in Brevibacterium linens BL2, it possessed high levels of l-methionine γ-lyase that was induced by addition of Met to the growth medium. Met demethiolation activity at pH 5.2 with 4% NaCl was not detected in cell extracts but was detected in whole cells. These data suggest that Met degradation in Cheddar cheese will depend on the organism used in production, the amount of enzyme released during aging, and the amount of Met in the matrix.

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Summary-The influence of NaCl and reduced pH was determined for aminopeptidase, lipase/esterase and methanethiol-producing capability in selected lactic acid bacteria and brevibacteria in simulated cheese-like conditions. The observations on simulated cheese-like conditions were confirmed in 60% reduced-fat Cheddar cheese. The activity of each enzyme decreased with NaCI addition and when the pH was reduced to approximate Cheddar chee se conditions (5% NaCI, pH 5.2). Residual intracellular aminopeptidase activity was dominated by general aminopeptidase activity (aminopeptidase N and/or aminopeptidase C) in laboratory, simulated cheese-like conditions, and 60% reduced-fat Cheddar cheese curd. During chee se aging, totallipase/esterase activity peaked at 120 d then decreased, ev en though starter culture populations remained high. Methanethiol-producing capability occurred under cheese-like conditions in whole cells, but not in cell-free extracts. Met and Met-containing peptides induced methanethiol-producing capability for 2-3 generations and could be re-induced later in the growth cycle of Brevibacterium linens BL2. Aminopeptidase and lipase/esterase activity in reduced fat cheese were not correlated to an increase in Cheddar-type flavar, but a culture's methanethiol-producing capability was associated with higher chee se consumer preference scores. Results suggest that use of cheese-like conditions may aid in selecting cultures to cheddar cheese / ripenlng / flavor /Iactococcus /Iactobacillus / brevibacterlum / methanethiol production Résumé -Influence du NaCI et du pH sur les enzymes intracellulaires qui affectent l'affinage du cheddar. L'influence du NaCI et d'une réduction du pH a été déterminée sur les ami nopeptidases, les lipases/esterases, ainsi que l'aptitude à produire du méthanéthiol de bactéries lactiques et de brevibactéries sélectionnées dans des conditions de fabrications fromagères simulées. Les observations, dans ces conditions, étaient confirmées, dans le cas du cheddar à teneur en matières grasses réduites de 60 %. Les activités de chaque enzyme diminuaient avec l'addition de NaCI et lorsque le pH était abaissé à des niveaux proches des conditions de fabrication du cheddar (NaCI5 % et pH 5,2). L'activité aminopeptidasique intracellulaire résiduelle était dominée par l'activité aminopeptidasique générale (aminopeptidase Net/ou aminopeptidase C) au laboratoire, en conditions fromagères simulées, et dans un caillé de cheddar à teneur en MG réduite de 60 %. Pendant le vieillissement des fromages, l'activité totale lipase/esterase atteignait un pic à 120 jours puis diminuait, même si les populations de levains demeuraient élevées. L'aptitude à produire du métha-néthiol se manifestait en conditions fromagères simulées dans les cellules entières, mais non dans les extraits dépourvus de cellules. Les peptides Met et Met-contenant induisaient une aptitude à produire du méthanéthiol sur deux ou trois générations, et pouvaient être réinduits ultérieurement dans le cycle de croissance de Brevibactérium lin...