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A modular esterase from Pseudomonas fluorescens subsp. cellulosa contains a non-catalytic cellulose-binding domain
L M Ferreira1, T M Wood, G Williamson
1Department of Biological and Nutritional Sciences, University of Newcastle upon Tyne, U.K.
The Biochemical Journal
|September 1, 1993
Summary
Researchers identified a new esterase, XYLD, from Pseudomonas fluorescens. This enzyme has a modular structure with separate domains for cellulose binding and ester hydrolysis, aiding in biomass breakdown.
Area of Science:
- Microbiology
- Enzymology
- Biochemistry
Background:
- The Pseudomonas fluorescens subsp. cellulosa genome contains reiterated 5' regions of xynB and xynC genes.
- These genes encode xylanase and arabinofuranosidase enzymes, respectively, suggesting potential for complex carbohydrate degradation.
Purpose of the Study:
- To isolate and characterize novel enzymes involved in biomass degradation from Ps. fluorescens subsp. cellulosa.
- To elucidate the functional and structural properties of a newly identified esterase, XYLD.
Main Methods:
- Screening a genomic library of Ps. fluorescens subsp. cellulosa DNA using a probe from the conserved xynB region.
- Subcloning a positive phage isolate into pMTL22p to create recombinant plasmid pFG1.
- Expressing and characterizing the encoded protein (XYLD) in Escherichia coli, including enzymatic activity assays and nucleotide sequencing of the xynD gene.
Main Results:
- A novel esterase, XYLD, was expressed, exhibiting cellulose binding but not xylan binding capabilities.
- XYLD demonstrated hydrolysis of aryl esters, acetylxylan, and liberation of ferulic acid from wheat bran.
- Nucleotide sequencing revealed XYLD's gene (xynD) shares significant homology with xynB and xynC, indicating a modular structure with N-terminal cellulose-binding and C-terminal catalytic domains.
Conclusions:
- XYLD possesses a modular enzymatic structure, critical for its function in biomass degradation.
- The N-terminal domain is responsible for cellulose binding, while the C-terminal domain carries the catalytic activity for ester hydrolysis.