Insight into subtilisin E-S7 cleavage pattern based on crystal structure and hydrolysates peptide analysis

Area of Science:

• Enzymology
• Structural Biology
• Biochemistry

Background:

• Subtilisin E is a well-studied serine protease.
• Understanding protease structure-function relationships is crucial for enzyme engineering.

Purpose of the Study:

• Determine the X-ray crystallographic structure of mature subtilisin E-S7 (SES7).
• Investigate the substrate selectivity mechanism of SES7.
• Assess the industrial application potential of SES7 hydrolysates.

Main Methods:

• X-ray crystallography at 1.90 Å resolution.
• Hydrolysis of skim milk by SES7.
• Liquid chromatography-mass spectrometry (LC-MS) for peptide identification.
• Determination of ACE-inhibitor and NLN-inhibitor activity.

Main Results:

• The crystal structure of mature SES7 was determined.
• Structural comparison with the propeptide-subtilisin E complex revealed active site adjustments.
• Cleavage pattern analysis indicated a preference for proline at the P2 position.
• Hydrolysates exhibited ACE-inhibitor (IC50 = 67 ± 0.92 μg/mL) and NLN-inhibitor (IC50 = 263 ± 13 μg/mL) activity.

Conclusions:

• Structural and substrate analysis provide insights into SES7's catalytic mechanism and specificity.
• SES7 demonstrates potential for protein engineering and industrial applications, particularly in producing bioactive peptides.