Development of an effective method for purifying trypsin using a recombinant inhibitor

Area of Science:

• Biochemistry
• Affinity Chromatography
• Enzyme Purification

Background:

• Trypsin is a crucial serine protease with diverse applications.
• Efficient purification methods are needed to obtain high-purity trypsin.
• Immobilized enzyme inhibitors offer a promising approach for affinity chromatography.

Purpose of the Study:

• To develop and characterize a novel trypsin affinity material.
• To assess the material's efficiency in purifying trypsin from locust homogenate.
• To determine the properties of purified locust trypsin.

Main Methods:

• Covalent immobilization of buckwheat trypsin inhibitor (BTI) onto epichlorohydrin-activated cross-linked agarose gel (Sephinose CL 6B).
• Optimization of activation and immobilization conditions (epichlorohydrin concentration, NaOH concentration, temperature, pH).
• One-step affinity chromatography for trypsin purification, followed by characterization of the enzyme's molecular mass, kinetic parameters, and stability.

Main Results:

• Optimal conditions for material activation and BTI immobilization were established.
• The BTI-Sephinose CL 6B material exhibited a maximum adsorption capacity of 2.25 mg trypsin/g support and >90% reusability after 30 cycles.
• High-purity locust trypsin (27 kDa, Km = 0.241 mM) was obtained, with optimal activity at pH 9.0 and 55°C, and good stability between pH 4.0-10.0 and 30-50°C.

Conclusions:

• The developed BTI-Sephinose CL 6B affinity material is effective for high-purity trypsin preparation.
• The material demonstrates excellent adsorption capacity and reusability.
• This approach holds potential for purifying trypsin from various biological sources and discovering novel trypsin variants.