Purification and Characterization of l-Methionine γ-Lyase from Brevibacterium linens BL2

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 from Pseudomonas putida. Its K_m 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 is associated with desirable Cheddar-type sulfur notes in good-quality Cheddar cheese (2, 27). The mechanism for the production of methanethiol in cheese is unknown, but it is linked to the catabolism of methionine (1, 15). l-Methionine γ-lyase (EC 4.4.1.11; MGL), also known as methionase, l-methionine γ-demethiolase, and l-methionine methanethiollyase (deaminating), is a pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the direct conversion of l-methionine to α-ketobutyrate, methanethiol, and ammonia by an α,γ-elimination reaction (26). It does not catalyze the conversion of d enantiomers (24–26). MGL in Pseudomonas putida is a multifunctional enzyme system since it catalyzes the α,γ- and α,β-elimination reactions of methionine and its derivatives (24). In addition, the enzyme also catalyzes the β-replacement reactions of sulfur amino acids (24). Since its discovery in Escherichia coli and Proteus vulgaris by Onitake (19), this enzyme has been found in various bacteria and is regarded as a key enzyme in the bacterial metabolism of methionine. However, this enzyme has not been purified to homogeneity from any food-grade microorganisms.MGL is widely distributed in bacteria, especially in pseudomonads, and is induced by the addition of l-methionine to the culture medium (9, 28). The enzyme has been purified from Pseudomonas putida (25), Aeromonas sp. (26), Clostridium sporogenes (11), and Trichomonas vaginalis (16) and partially purified from and characterized for Brevibacterium linens NCDO 739 (4).B. linens is a nonmotile, non-spore-forming, non-acid-fast, gram-positive coryneform bacterium normally found on the surfaces of Limburger and other Trappist-type cheeses. This organism tolerates salt concentrations ranging between 8 and 20% and is capable of growing in a broad pH range from 5.5 to 9.5, with an optimum pH of 7.0 (20). In Trappist-type cheeses, brevibacteria depend on Saccharomyces cerevisiae to metabolize lactate, which increases the pH of the curd, as well as to produce growth factors that are important for their growth (20). Interest in B. linens has focused around its ability to produce an extracellular protease, which has recently been isolated (21), and its ability to produce high levels of methanethiol (3, 9, 10, 22).B. linens produces various sulfur compounds, including methanethiol, that are thought to be important in Cheddar-like flavor and aroma (3, 9, 10, 22). Ferchichi et al. (9) suggested that MGL is responsible for the methanethiol-producing capability of B. linens but did not provide definitive evidence. Weimer et al. (28) proposed that B. linens BL2 is responsible for Cheddar-type flavor development in low-fat cheese, but again conclusive evidence was lacking. In this study, MGL was purified to homogeneity from B. linens BL2 and its physical and chemical properties were examined.