Purification of subtilisin by single-step affinity chromatography.

Area of Science:

• Biochemistry
• Protein Purification
• Affinity Chromatography

Background:

• Subtilisin is a widely used serine protease with significant industrial applications.
• Efficient purification methods are crucial for obtaining homogeneous subtilisin for research and industrial use.
• Existing purification techniques can be complex and time-consuming.

Purpose of the Study:

• To develop and evaluate a novel affinity matrix for the efficient purification of subtilisin.
• To assess the effectiveness of the affinity matrix in terms of purification fold and enzyme activity recovery.
• To demonstrate the homogeneity of the purified subtilisin.

Main Methods:

• Coupling of 4-(4-aminophenylazo)phenylarsonic acid dye to activated CH-Sepharose 4B to create an affinity matrix.
• Purification of crystalline subtilisin using the developed affinity matrix.
• Purification of subtilisin from a crude enzyme preparation using the same affinity matrix.
• Analysis of enzyme homogeneity using polyacrylamide gel electrophoresis (PAGE).

Main Results:

• The affinity matrix demonstrated high efficiency for subtilisin purification.
• Crystalline subtilisin was purified to homogeneity with a 1.4-fold increase and 98% activity yield.
• Subtilisin from crude preparations was purified 211-fold in a single step with 94% activity recovery.
• Polyacrylamide gel electrophoresis confirmed the homogeneity of the purified subtilisin.

Conclusions:

• The developed affinity matrix is highly effective for the single-step purification of subtilisin.
• This method provides a high yield and purity of subtilisin from various sources.
• The affinity chromatography procedure offers a valuable tool for efficient subtilisin isolation.