The cellodextrinase from Pseudomonas fluorescens subsp. cellulosa consists of multiple functional domains

Area of Science:

• Microbiology
• Enzymology
• Molecular Biology

Background:

• Pseudomonas fluorescens subsp. cellulosa is known for its ability to degrade plant biomass.
• Understanding the specific enzymes involved in cellulose degradation is crucial for biotechnological applications.

Purpose of the Study:

• To isolate and characterize a novel enzyme involved in cellodextrin hydrolysis from Pseudomonas fluorescens subsp. cellulosa.
• To elucidate the structure and function of the identified enzyme, including its substrate specificity and cellulose-binding properties.

Main Methods:

• Construction of a genomic library of Pseudomonas fluorescens subsp. cellulosa DNA in Escherichia coli.
• Screening for recombinants expressing 4-methylumbelliferyl beta-D-cellobioside-hydrolysing activity.
• Enzyme activity assays using various cello-oligosaccharides and polysaccharides.
• Nucleotide sequencing of the gene encoding the enzyme.
• Analysis of protein homology and cellulose-binding capabilities of full-length and truncated enzyme forms.

Main Results:

• Isolation and identification of an endo-acting cellodextrinase, designated cellodextrinase C (CELC).
• CELC specifically cleaves cellopentaose and cellohexaose, producing cellobiose and cellotriose, with no activity on shorter cello-oligosaccharides or CM-cellulose.
• Nucleotide sequencing revealed a 2153 bp open reading frame encoding an 80,189 Mr protein.
• The N-terminal region of CELC contains a non-catalytic cellulose-binding domain, distinct from the catalytic domain, which mediates strong binding to crystalline cellulose.

Conclusions:

• Cellodextrinase C (CELC) is a novel endo-acting enzyme from Pseudomonas fluorescens subsp. cellulosa with specific activity on longer cellodextrins.
• The enzyme possesses a distinct cellulose-binding domain essential for its interaction with crystalline cellulose.
• The findings contribute to the understanding of cellulolytic enzyme systems and their potential applications in biomass degradation.