Structure-based engineering of a pectate lyase with improved specific activity for ramie degumming

Area of Science:

• Biotechnology
• Structural Biology
• Enzymology

Background:

• Microbial pectate lyases (Pels) are eco-friendly alternatives to chemical degumming in plant fiber processing.
• Understanding the molecular structure of Pels is crucial for optimizing their industrial applications.

Purpose of the Study:

• To determine the high-resolution crystal structure of pectate lyase (pelN) from Paenibacillus sp. 0602.
• To engineer a novel pelN mutant with enhanced enzymatic properties for industrial use.

Main Methods:

• High-resolution (1.45 Å) X-ray crystallography was used to determine the apo structure of pelN.
• Sequence alignment and structural superposition with other polysaccharide lyase family 1 (PL1) members were performed.
• Rational design based on structural insights was employed to engineer a pelN mutant.

Main Results:

• The crystal structure revealed pelN shares the characteristic right-handed β-helix fold of PL1 family members, with distinct catalytic and substrate-binding residues.
• The engineered pelN mutant exhibited an optimal temperature shift for enzymatic activity from 67.5°C to 60°C.
• The mutant displayed significantly higher specific activity and improved ramie fiber degumming ability compared to the wild-type enzyme.

Conclusions:

• The high-resolution structure of pelN provides a foundation for structure-based enzyme engineering.
• Rational design of pelN resulted in a mutant with enhanced industrial applicability.
• This study demonstrates the potential of structure-guided engineering for developing improved microbial enzymes for plant fiber processing.