ISSN:
1476-5535
Keywords:
leucine aminopeptidase
;
Penicilium citrinum
;
methionine removal
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Process Engineering, Biotechnology, Nutrition Technology
Notes:
Abstract An intracellular leucine aminopeptidase (LAP) fromPenicillium citrinum (IFO 6352) was purified to homogeneity using three successive purification steps. The enzyme has a native molecular mass of 63 kDa using HPLC gel filtration analysis and a molecular mass of 65 kDa when using SDS-polyacrylamide gel electrophoresis. This monomeric aminopeptidase showed maximum enzyme activity at pH 8.5. An optimum temperature was 45–50°C whenl-Leu-p-nitroanilide (pNA) was the substrate, and enzyme activity drastically decreased above 60°C. The Michaelis-Menten constants forl-Leu-pNA andl-Met-pNA were 2.7 mM and 1.8 mM, respectively. When the enzyme reacted with biosynthetic methionyl human growth hormone, it showed high specificity for N-terminal methionine residue and recognized a stop sequence (Xaa-Pro). The aminopeptidase was inactivated by EDTA or 1,10-phenanthroline, indicating that it is a metallo-exoprotease. Enzyme activity was restored to 90% of maximal activity by addition of Co2+ ions. The activity of EDTA-treated enzyme was restored by addition of Zn2+, but reconstitution with Ca2+, Mg2+ or Mn2+ restored some enzyme activity. It is likely that Co2+ ions play an important role in the catalysis or stability of thePenicillium citrinum aminopeptidase, as zinc plays a similar function in other leucine aminopeptidases.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF01570145
