Alcalase rapeseed inhibitors: purification and partial characterization

Area of Science:

• Biochemistry
• Food Science
• Proteomics

Background:

• Enzymatic hydrolysis of rapeseed protein yields peptides with potential biological activities.
• Protease inhibitors are crucial in regulating enzymatic processes and have various applications.
• Understanding the characteristics of rapeseed-derived protease inhibitors is essential for food processing and biotechnology.

Purpose of the Study:

• To investigate the inhibitory activity of rapeseed protein hydrolysates against Alcalase.
• To purify and characterize the Alcalase inhibitors present in rapeseed hydrolysates.
• To assess the stability of these inhibitors under different heat treatments.

Main Methods:

• Sequential enzymatic hydrolysis of rapeseed protein using Alcalase and Flavourzyme.
• Purification of inhibitors via gel filtration and ion exchange chromatography.
• Analysis of inhibitor composition and molecular weight using SDS-PAGE.
• Heat stability assays at varying temperatures and durations.

Main Results:

• Rapeseed protein hydrolysates exhibited inhibitory activity towards Alcalase, which diminished with prolonged hydrolysis.
• Purified inhibitors consisted of 8.4 and 6.1 kDa peptides linked by disulfide bonds, with a native molecular weight of 18 kDa.
• The inhibitors were rich in methionine and cysteine and retained over 50% activity after heating at 70°C for 45 minutes, but rapidly lost activity at 90°C for 5 minutes.

Conclusions:

• Rapeseed protein contains Alcalase inhibitors that are peptides with disulfide linkages.
• These inhibitors possess partial heat stability, suggesting potential applications in controlled enzymatic processes.
• The study discusses a possible link between these protease inhibitors and 2S albumin storage proteins in rapeseed.