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A C-terminal proline-rich sequence simultaneously broadens the optimal temperature and pH ranges and improves the catalytic efficiency of glycosyl hydrolase family 10 ruminal xylanases
- Source :
- Applied and environmental microbiology. 80(11)
- Publication Year :
- 2014
-
Abstract
- Efficient degradation of plant polysaccharides in rumen requires xylanolytic enzymes with a high catalytic capacity. In this study, a full-length xylanase gene ( xynA ) was retrieved from the sheep rumen. The deduced XynA sequence contains a putative signal peptide, a catalytic motif of glycoside hydrolase family 10 (GH10), and an extra C-terminal proline-rich sequence without a homolog. To determine its function, both mature XynA and its C terminus-truncated mutant, XynA-Tr, were expressed in Escherichia coli . The C-terminal oligopeptide had significant effects on the function and structure of XynA. Compared with XynA-Tr, XynA exhibited improved specific activity (12-fold) and catalytic efficiency (14-fold), a higher temperature optimum (50°C versus 45°C), and broader ranges of temperature and pH optima (pH 5.0 to 7.5 and 40 to 60°C versus pH 5.5 to 6.5 and 40 to 50°C). Moreover, XynA released more xylose than XynA-Tr when using beech wood xylan and wheat arabinoxylan as the substrate. The underlying mechanisms responsible for these changes were analyzed by substrate binding assay, circular dichroism (CD) spectroscopy, isothermal titration calorimetry (ITC), and xylooligosaccharide hydrolysis. XynA had no ability to bind to any of the tested soluble and insoluble polysaccharides. However, it contained more α helices and had a greater affinity and catalytic efficiency toward xylooligosaccharides, which benefited complete substrate degradation. Similar results were obtained when the C-terminal sequence was fused to another GH10 xylanase from sheep rumen. This study reveals an engineering strategy to improve the catalytic performance of enzymes.
- Subjects :
- Rumen
Protein Conformation
Gene Expression
Biology
Xylose
Calorimetry
Protein Sorting Signals
Applied Microbiology and Biotechnology
chemistry.chemical_compound
Hydrolysis
Catalytic Domain
Glycoside hydrolase family 10
Hydrolase
Enzyme Stability
Escherichia coli
Fagus
Animals
Glycosyl
Cloning, Molecular
Enzymology and Protein Engineering
Triticum
Sheep
Ecology
Circular Dichroism
Temperature
Isothermal titration calorimetry
Sequence Analysis, DNA
Hydrogen-Ion Concentration
Protein Structure, Tertiary
Xylosidases
Biochemistry
chemistry
Xylanase
Xylans
Xylooligosaccharide
Food Science
Biotechnology
Protein Binding
Subjects
Details
- ISSN :
- 10985336
- Volume :
- 80
- Issue :
- 11
- Database :
- OpenAIRE
- Journal :
- Applied and environmental microbiology
- Accession number :
- edsair.doi.dedup.....1ef8dca9923acc1e447224c4a38bf76d