Purified enzymatic preparation derived from chromobacterium violaceum, method for preparing it and its use in the production of .alpha., .beta.-dehydrotryptophanyl-peptides

A purified enzymatic preparation derived from Chromobacterium violaceum having:

• (i) L-tryptophan dehydrogenase activity,
• (ii) a specific activity of between 35 and 45 units for N-acetyl-L-tryptophanamide (NATA), wherein said units represents a mole of product formed per second per mole of heme,
• (iii) an apparent molecular weight of approximately 680 kDa,
• (iv) an isoelectric point of about 4, and
• (v) a temperature optimum for activity greater than 80.degree. C.;
• wherein said preparation catalyzes the conversion of tryptophanyl residues to dehydrotryptophanyl residues at a pH of from 3 to 8.

A purified enzymatic preparation obtained from Chromobacterium violaceum by:

• (i) preparing a crude enzymatic fraction by:
• (a) extracting total proteins from Chromobacterium violaceum cells by
• (i) grinding said cells in the presence of an inhibitor of intrinsic proteolytic activity,
• (ii) ultracentrifugating, and
• (iii) separating proteins by fractional precipitation in a buffer, and
• (b) subsequently fractionating said proteins to obtain an enzymatic fraction Q1 by a succession of chromatographic steps, and
• (ii) subsequently purifiying said enzymatic fraction Q1 by
• (c) incubating said enzymatic fraction in a denaturing medium comprising (i) 0.1 to 2% w/v of a denaturing agent selected from the group consisting of 3-4M urea and sodium dodecyl sulphate and (ii) 1 to 5% v/v of a reducing agent selected from the group consisting of >50 mM dithiothreitol and 2-mercaptoethanol, and
• (d) separating, by gel permeation, said incubated active fraction.