Purification and characterization of a lysine aminopeptidase from Kluyveromyces marxianus

• Published in: FEMS microbiology letters 2004-06, Vol.235 (2), p.369-375
• Main Author: RAMIREZZAVALA, B
• Format: Article
• Language: English
• Subjects: Aminopeptidase; Aminopeptidases - antagonists & inhibitors; Aminopeptidases - chemistry; Aminopeptidases - isolation & purification; Aminopeptidases - metabolism; Biological and medical sciences; Biotechnology - methods; Enzyme Stability; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Kinetics; Kluyveromyces - enzymology; Kluyveromyces marxianus; Microbiology; Miscellaneous; Mycology; plant biochemistry; plant physiology; Protease; Purification; Substrate Specificity; Temperature
• ISSN: 0378-1097; 1574-6968
• DOI: 10.1016/j.femsle.2004.05.009

A lysine aminopeptidase was purified from the yeast Kluyveromyces marxianus. This enzyme was purified 100-fold from a soluble extract obtained at 100,000 g. The purification procedure consisted in fractionated precipitation with ammonium sulfate and five chromatography steps. The native enzyme had a molecular mass of 46 kDa assessed through gel filtration. This aminopeptidase depicted an optimal pH of 7.0 and was stable at a pH range of 4–8, its optimal temperature was 45 °C and the enzyme became unstable at temperatures above 55 °C. The isoelectric point of the purified enzyme was 4.4. Michaelis constant and V max for l-lysine– p-nitroanilide were 0.33 mM and 2.2 mM min −1 per milligram of protein, respectively. The enzyme was strongly inhibited by bestatin, o-phenanthroline and, to a lesser extent, by EDTA, suggesting that this enzyme is a metalloprotease. Our results suggest that the lysine aminopeptidase from Kluyveromyces marxianus might be of biotechnological relevance.