The unique stability of Vibrio proteolyticus neutral protease under alkaline conditions affords a selective step for purification and use in amino acid-coupling reactions.
A procedure is described for the purification of a neutral protease from fermentation broths of Vibrio proteolyticus. The key feature of the purification scheme is the selective, irreversible inactivation of a contaminating exoenzyme, aminopeptidase, by alkali treatment, rather than removal of this enzyme by conventional chromatographic methods. Fermentation broths or concentrates were brought to pH 11.5 to 11.7 by Na2CO3-NaOH addition and incubated at 25 degrees C until aminopeptidase activity was diminished. The alkali treatment resulted in greater than 99% reduction of aminopeptidase activity with minimal loss of neutral protease activity. The neutral protease could be further purified to apparent homogeneity by QA-52 cellulose chromatography. The alkali treatment of fermentation concentrates was also useful for preparation of V. proteolyticus neutral protease to effect the coupling of N-protected aspartic acid and phenylalanine methyl ester for the production of N-aspartylphenylalanine methyl ester, a precursor for the sweetener aspartame.
Durham
D R
DR
Biotechnology Research Department, W.R. Grace & Co., Columbia, Maryland 21044.
D000596:Amino Acids; D000626:Aminopeptidases / Q000037:antagonists & inhibitors; D001709:Biotechnology; D004151:Dipeptides / Q000096:biosynthesis; D004355:Drug Stability; D005285:Fermentation; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000302:isolation & purification*; D014733:Vibrio / Q000201:enzymology*
D000596:Amino Acids; D004151:Dipeptides; D000626:Aminopeptidases; D008666:Metalloendopeptidases; C044194:microbial metalloproteinases