Evidence for multiple carboxymethylcellulase genes in Pseudomonas fluorescens subsp. cellulosa

Area of Science:

• * Molecular biology
• * Microbiology
• * Biochemistry

Background:

• * Pseudomonas fluorescens subsp. cellulosa is a bacterium known for its cellulolytic capabilities.
• * Carboxymethylcellulase (CMCase) is an enzyme crucial for cellulose degradation.
• * Gene cloning and expression in heterologous hosts like E. coli are vital for studying enzyme function and potential applications.

Purpose of the Study:

• * To clone and characterize genes encoding carboxymethylcellulase (CMCase) from Pseudomonas fluorescens subsp. cellulosa.
• * To determine the location and regulation of these CMCase genes within the host E. coli.
• * To investigate the enzymatic properties of the cloned CMCases.

Main Methods:

• * Construction of a genomic library of P. fluorescens subsp. cellulosa DNA in bacteriophage lambda 47.1.
• * Isolation of CMCase-expressing recombinants and subcloning into pUC18.
• * Characterization of CMCase activity, including substrate specificity and enzyme location.
• * Transposon mutagenesis and subcloning to map gene positions and identify regulatory elements.

Main Results:

• * Three distinct DNA fragments encoding CMCase activity were identified and cloned into E. coli.
• * The cloned enzymes exhibited CMCase activity and cleaved p-nitrophenyl-beta-D-glucopyranoside but lacked exoglucanase activity.
• * The CMCases were localized to the periplasmic space of E. coli.
• * CMCase genes in plasmids pJHH1 and pJHH3 were found to be under glucose repression.

Conclusions:

• * Genes encoding functional carboxymethylcellulases from P. fluorescens subsp. cellulosa have been successfully cloned and expressed in E. coli.
• * The cloned enzymes are extracellular and subject to glucose regulation, providing insights into their biological function and potential for industrial applications.
• * Further research can explore the optimization of these enzymes for enhanced cellulose degradation.