Alkaline protease from Neurospora crassa. Purification and partial characterization

Area of Science:

• Biochemistry
• Enzymology
• Microbial Biotechnology

Background:

• Extracellular alkaline proteases are valuable industrial enzymes.
• Efficient purification methods are crucial for their application.
• Neurospora crassa protease offers potential for various biotechnological uses.

Purpose of the Study:

• To develop a simple and efficient purification procedure for extracellular alkaline protease from Neurospora crassa.
• To characterize the purified enzyme's properties and molecular weight.

Main Methods:

• Utilized gelatin as a low-concentration protein inducer.
• Employed heat treatment to digest the inducer.
• Incorporated a concentration step to remove digested inducer and avoid precipitation.
• Applied ion exchange chromatography and gel filtration for purification.
• Assessed homogeneity using gel electrophoresis and ultracentrifugal analyses.

Main Results:

• Developed a straightforward purification protocol for Neurospora crassa alkaline protease.
• Achieved a homogeneous enzyme preparation.
• Determined a molecular weight of approximately 30,500 Da.
• Established optimal activity between pH 6.0 and 10.0.
• Identified heat lability above 45°C and susceptibility to autodigestion.
• Observed preferential cleavage of insulin beta chain at neutral and aromatic amino acid carboxyl groups.

Conclusions:

• The developed purification method is effective and simple for obtaining homogeneous Neurospora crassa alkaline protease.
• The enzyme exhibits robust activity over a wide alkaline pH range.
• Characterization provides essential data for potential industrial applications of this protease.