Substrate binding ability of chemically inactivated pectinase for the substrate pectic acid

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Pectinase, a polygalacturonase, plays a key role in polysaccharide degradation.
• Understanding enzyme-substrate interactions is crucial for enzyme engineering and application.
• Commercial pectinase preparations are widely used but their specific binding mechanisms require detailed investigation.

Purpose of the Study:

• To investigate the substrate binding properties of pectinase after chemical modification.
• To identify the specific peptide fragments responsible for pectinase-substrate interactions.
• To explore the potential of inactivated pectinase as an acidic polysaccharide recognizing protein.

Main Methods:

• Purification of pectinase from a commercial mold preparation.
• Centrifugal affinity chromatography using immobilized pectic acid to measure substrate binding.
• Chemical modification of pectinase using EDC and DEP.
• Enzyme digestion with lysyl endopeptidase and peptide recovery via affinity chromatography.
• Peptide sequencing to identify binding fragments.

Main Results:

• Pectinase exhibited substrate binding with dissociation constants (Kd) of 5.3 mg/ml for pectin and 8.5 mg/ml for pectic acid.
• Chemical modification with EDC and DEP inactivated the enzyme but did not affect substrate binding.
• Three peptide fragments were identified, corresponding to sequences within the Aspergillus niger pectinase gene, indicating their role in substrate recognition.

Conclusions:

• Inactivated pectinase retains significant substrate binding ability, highlighting the independence of binding and catalytic activity.
• Specific peptide sequences (Asp101-Ser117 and Asn152-Asp169) are crucial for pectinase's interaction with pectic acid.
• Modified pectinase holds potential as a tool for recognizing and interacting with acidic polysaccharides.