L-methionine .gamma.-lyase (EC 4.4.1.11) is an enzyme which requires pyridoxal phosphate as a co-enzyme and catalyzes .alpha., .gamma.-elimination and .gamma.-replacement of L-methionine or its derivatives and also .alpha., .beta.-elimination and .beta.-replacement of S-substituted L-cysteine or its derivatives Tanaka, H. et al., Biochemistry, 16, 100-106 (1977)!. This enzyme has been isolated and purified mainly from Pseudomonas putida and its physicochemical and enzymological properties have already been shown Nakayama, T. et al., Anal. Biochem., 138, 421-424 (1984)!. There have been some researches reporting the mechanism of enzymatic reaction catalyzed by L-methionine .gamma.-lyase Esaki, N. et al., FEBS Lett., 84, 309-312 (1977); Nakayama, T. et al., Biochemistry, 27, 1587-1591 (1988), and the like!. The references which have been provided so far are all related to L-methionine .gamma.-lyases purified from natural source such as bacteria belonging to genus Pseudomonas. Thus, there are no references describing or suggesting the preparation of a recombinant enzyme by means of genetic engineering.
Recently, it has been suggested that naturally occurring L-methionine .gamma.-lyase purified from a culture of P. putida may have an anti-tumor activity WO94/11535, Publication date, May 26, 1994).
To develop a therapeutically applicable and useful drug, a substance to be used as an active ingredient should be pure or essentially free from undesirable contaminants, for example, proteins or the like by which the substance is naturally accompanied, and can be produced on a large scale so as to meet the demands. Accordingly, the establishment of a method of preparing L-methionine .gamma.-lyase or its variants with essentially the same enzymatic activity by means of recombinant DNA technique must contribute to the development of useful anti-tumor agents.
WO95/17908 has revealed production of L-methionine .gamma.-lyase using gene recombinant technique, and describes the DNA sequence and the deduced amino acid sequence L-methionine .gamma.-lyase. However, the document shows neither any primers for isolating the DNA encoding L-methionine .gamma.-lyase nor any source from which the DNA comes.