Novel Properties for Endoglucanase Acquired by Cell-Surface Display Technique

Area of Science:

• Biotechnology
• Enzyme Engineering
• Microbial Cell Surface Display

Background:

• Endoglucanase enzymes often exhibit limited stability under industrial process conditions (thermal and acidic).
• Cell surface display is a promising strategy to immobilize enzymes and potentially enhance their properties.
• Saccharomyces cerevisiae PIR gene encodes a cell wall protein useful for enzyme display.

Purpose of the Study:

• To improve the thermal and acidic stability of endoglucanase.
• To display endoglucanase on the surface of Pichia pastoris.
• To evaluate the catalytic efficiency and reusability of the displayed endoglucanase.

Main Methods:

• Genetic fusion of endoglucanase gene to the Saccharomyces cerevisiae PIR gene.
• Transformation and expression of the fusion gene in Pichia pastoris GS115.
• Selection of high-expression strains and characterization of displayed endoglucanase (DEG) activity, stability, and reusability via SDS-PAGE, enzyme assays, and stability tests.

Main Results:

• Successfully displayed endoglucanase (DEG) on Pichia pastoris cell surface, confirmed by SDS-PAGE.
• DEG demonstrated significantly enhanced stability across a broad pH range (2.5-8.5) and higher thermal tolerance (up to 70°C) compared to free endoglucanase (FEG).
• DEG exhibited superior operational stability, retaining 91.1% activity at 65°C for 120 min and a longer half-life (270 min vs. 150 min for FEG), with good reusability (at least three cycles).

Conclusions:

• Cell surface display of endoglucanase using the PIR gene fusion in Pichia pastoris effectively enhances enzyme stability and reusability.
• The displayed endoglucanase (DEG) possesses high catalytic efficiency and remarkable acid-thermal stability, making it suitable for industrial applications.
• DEG represents a robust whole-cell biocatalyst with broad application potential as a yeast biocatalyst.