Expression, purification and characterization of a functional carbohydrate-binding module from Streptomyces sp....

Area of Science:

• Biochemistry
• Microbiology
• Biotechnology

Background:

• Streptomyces sp. SirexAA-E is a potent cellulolytic bacterium.
• Carbohydrate-active enzymes (CAZymes) in SirexAA-E often feature carbohydrate-binding module family 2a (CBM2a).
• CBM2a plays a role in the deconstruction of lignocellulosic biomass.

Purpose of the Study:

• To characterize the CBM2a from the most abundant cellulase in SirexAA-E.
• To investigate the binding properties of purified CBM2a to cellulose.

Main Methods:

• Gene cloning and expression of CBM2a as a GFP fusion protein in Escherichia coli.
• Purification of the fusion protein from inclusion bodies and subsequent refolding.
• Size-exclusion chromatography to separate monomers and multimers.
• Affinity measurements using cellulose binding assays.

Main Results:

• Monomeric CBM2a, but not multimers, exhibited high affinity for cellulose (partition coefficient of 0.62±0.04 L/g).
• The binding of monomeric CBM2a to cellulose was found to be fully reversible.
• The CBM2a was derived from gene locus SACTE_0237, the most expressed cellulase.

Conclusions:

• Monomeric CBM2a from Streptomyces sp. SirexAA-E is a high-affinity cellulose-binding protein.
• This CBM2a demonstrates reversible binding, suggesting potential applications in biomass processing.
• The findings contribute to understanding the enzymatic machinery for lignocellulosic biomass deconstruction.