Multifunctional cellulase catalysis targeted by fusion to different carbohydrate-binding modules.

Area of Science:

• Biochemistry
• Biotechnology
• Enzymology

Background:

• Carbohydrate-binding modules (CBMs) target glycoside hydrolases to polysaccharide substrates.
• Ruminoclostridium thermocellum's CelE enzyme (a GH5 catalytic domain) hydrolyzes various polysaccharides.
• No previous fusions of CBMs to CelE existed.

Purpose of the Study:

• To investigate how CBMs improve cellulolytic enzyme reactivity.
• To create and test novel fusions of CBMs with the multifunctional CelE catalytic domain.

Main Methods:

• Fusing representatives from 18 CBM families to the CelE catalytic domain.
• Assaying hydrolytic activity on cellulose, lichenan, xylan, and mannan.
• Conducting binding studies and quantitative catalysis using nanostructure-initiator mass spectrometry (NIMS).

Main Results:

• Several CelEcc-CBM fusions exhibited enhanced hydrolytic activity compared to controls.
• Enzyme reactivity correlated with moderate CBM binding affinity.
• CelEcc-CBM44 demonstrated the fastest hydrolysis rates for both hexose and pentose fractions of pretreated switchgrass.

Conclusions:

• Fusion of CBMs to a single catalytic domain increases reaction rates with polysaccharides and pretreated biomass.
• This fusion approach offers a novel strategy to enhance enzyme combinations for biomass conversion.