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

Abstract

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.

Identification and characterization of a cystathionine β/γ-lyase from Lactococcus lactis ssp. cremoris MG1363

“…This is similar to other known PLP-dependent enzymes from various organisms (e.g. [30,31]). PLP-dependent enzymes have been classi¢ed into the K, L and Q families on the basis of sequence alignments and construction of protein pro¢les [15].…”

Flavour formation by lactic acid bacteria and biochemical flavour profiling of cheese products

“…All cystathionine lyases are able to convert several substrates, use PLP as cofactor and have a pH optimum close to 8. The relative activity towards methionine is below 10% compared to the rate for cystathionine [126]. Although cystathionine lyases are active under cheese-ripening conditions [42,129,130], their activity towards methionine could not be detected in lactococci using 13 C nuclear magnetic resonance [86].…”

“…Cystathionine b-lyase (CBL, EC 4.4.1.8) and cystathionine c-lyase (CGL, EC 4.4.1.1) posses this activity and are found in and isolated from several LAB [42,[125][126][127][128][129]. However, the main physiological function of these enzymes is the a-b and a-c elimination of cystathionine in homoserine and cysteine in the anabolism (Fig.…”

“…In Brevibacterium linens a similar enzyme, methionine c-lyase (MGL, EC 4.1.1.11) has been identified and characterised [126,132]. This highly specific enzyme plays a central role in the methionine and cystathionine metabolism of Brevibacterium linens.…”

“…This highly specific enzyme plays a central role in the methionine and cystathionine metabolism of Brevibacterium linens. The homotetrameric enzyme with a M w of 170 kDa is PLP dependent and has a pH optimum of 7.5 [126]. The enzyme is also slightly active under cheese ripening conditions, but is also susceptible for proteolysis [41,126].…”