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Purification and characterization of a pineapple crown leaf thiol protease
L Rupachandra Singh1, Th Premila Devi, S Kunjeshwori Devi
1Department of Biochemistry, Manipur University, Canchipur, Imphal, India. rslaishram@yahoo.com
Preparative Biochemistry & Biotechnology
|March 30, 2004
Summary
A novel thiol protease was purified from pineapple crown leaves. Its properties closely resemble those of stem bromelain, suggesting a close relationship between these pineapple enzymes.
Area of Science:
- Biochemistry
- Enzymology
- Plant Science
Background:
- Pineapple (Ananas comosus) contains various proteases, with stem bromelain being well-characterized.
- Crown leaf tissue is a potential source of novel proteolytic enzymes.
Purpose of the Study:
- To isolate and purify a thiol protease from pineapple crown leaves.
- To characterize the physico-chemical and kinetic properties of the purified enzyme.
- To compare the enzyme's properties with known bromelains.
Main Methods:
- Immunoaffinity purification of thiol protease from pineapple crown leaf extract.
- Electrophoretic homogeneity confirmation.
- Characterization of molecular weight, kinetic parameters (Km, kcat), thiol activator concentration, optimal pH, thermal stability, and SH-group content.
Main Results:
- A thiol protease was successfully purified to electrophoretic homogeneity.
- Key properties determined include molecular weight (22.4-22.9 kDa), Km (97 µM), kcat (8.8 s⁻¹), L-cysteine activation constant (A0.5 = 9.9 µM), optimal pH (6.5), thermal inactivation T½ (60°C), and SH-group content (0.98 mol/mol enzyme).
- These properties are highly similar to those of stem bromelain (EC 3.4.22.32).
Conclusions:
- The immunoaffinity purified crown leaf protease is closely related to stem bromelain.
- This finding contributes to understanding the diversity and relationships of proteases within the pineapple plant.
- The characterized enzyme represents a potential source for further biochemical and biotechnological applications.