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Purification of an acid protease from Mucor rouxii that inactivates chitin synthetase
1Departamento de Genética y Biología Molecular, Centro de Investigación y Estudios Avanzados, I.P.N., Guanajuato, Mexico.
Abstract:
An acid protease was purified from the mycelial form of Mucor rouxii by a method which involved salt and acid precipitation, gel filtration and anion-exchange chromatography. The enzyme had a molecular mass of 16,000 Da. Its optimum pH was 4.0, maximal activity was obtained at 50 degrees C, and it was inactivated at 70 degrees C. It was not affected by leupeptin or N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) but diazoacetyl-DL-norleucine methyl ester (DNME) in the presence of Cu2+ and more noticeably pepstatin A, strongly inhibited the activity. This acid protease did not activate "zymogenic" chitin synthetase from the fungus, but brought about its inactivation even at low concentrations and after short periods of incubation time.
Insights
Researchers purified an acid protease from Mucor rouxii, finding it optimally active at pH 4.0 and 50°C. This enzyme inhibited chitin synthetase activity, suggesting a role in fungal cell wall regulation.
Area of Science:
- Biochemistry
- Mycology
Background:
- Mucor rouxii is a fungus with a complex life cycle.
- Acid proteases play various roles in fungal physiology.
Purpose of the Study:
- To purify and characterize an acid protease from the mycelial form of Mucor rouxii.
- To investigate the enzyme's effect on chitin synthetase activity.
Main Methods:
- Purification involved salt and acid precipitation, gel filtration, and anion-exchange chromatography.
- Enzyme activity was assessed at different pH and temperature conditions.
- Inhibition studies were performed using specific protease inhibitors.
- Effects on zymogenic chitin synthetase were evaluated.
Main Results:
- An acid protease with a molecular mass of 16,000 Da was successfully purified.
- Optimal activity was observed at pH 4.0 and 50°C; the enzyme was inactivated at 70°C.
- Pepstatin A and diazoacetyl-DL-norleucine methyl ester (DNME) with Cu2+ strongly inhibited the protease.
- The purified acid protease inactivated, rather than activated, zymogenic chitin synthetase.
Conclusions:
- The study characterized a novel acid protease from Mucor rouxii.
- This protease demonstrates inhibitory effects on fungal chitin synthetase, indicating a potential regulatory role in cell wall biosynthesis.