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Pcal_0842, a highly thermostable glycosidase from Pyrobaculum calidifontis displays both α-1,4- and β-1,4-glycosidic cleavage activities
- Source :
- International Journal of Biological Macromolecules. 165:1745-1754
- Publication Year :
- 2020
- Publisher :
- Elsevier BV, 2020.
-
Abstract
- The gene encoding Pcal_0842, annotated as a cellulase (accession no. ABO08268), was cloned and expressed in Escherichia coli. The gene product was produced in insoluble form in E. coli in high amounts even without addition of the inducer isopropyl β-D-1-thiogalactopyranoside. The recombinant protein was solubilized in 8 M urea and refolded by gradual removal of urea. The refolded protein exhibited both α-1,4- and β-1,4-glycosidic cleavage activities. The enzyme activity increased with the increase in temperature till 120 °C. Apart from very high optimal temperature, recombinant Pcal_0842 was extremely thermostable. There was no significant loss in activity even after heating for 100 h at 100 °C. The half-lives of Pcal_0842 were 6 and 2.5 h at 110 and 120 °C, respectively. To the best of our knowledge, Pcal_0842 is the most thermostable glycosidase characterized to date and this is the first report on cloning and characterization of an enzyme from archaea that displays both α-1,4- and β-1,4-glycosidic cleavage activities.
- Subjects :
- Models, Molecular
Glycoside Hydrolases
Archaeal Proteins
Gene Expression
02 engineering and technology
Cellulase
medicine.disease_cause
Cleavage (embryo)
Biochemistry
Substrate Specificity
law.invention
Gene product
03 medical and health sciences
Structural Biology
law
Enzyme Stability
medicine
Glycoside hydrolase
Amino Acid Sequence
Glycosides
Molecular Biology
Escherichia coli
030304 developmental biology
chemistry.chemical_classification
0303 health sciences
biology
Circular Dichroism
Hydrolysis
Temperature
General Medicine
Hydrogen-Ion Concentration
021001 nanoscience & nanotechnology
Recombinant Proteins
Enzyme assay
Kinetics
Enzyme
Solubility
chemistry
Pyrobaculum
biology.protein
Recombinant DNA
0210 nano-technology
Subjects
Details
- ISSN :
- 01418130
- Volume :
- 165
- Database :
- OpenAIRE
- Journal :
- International Journal of Biological Macromolecules
- Accession number :
- edsair.doi.dedup.....e5f191a9903edc0fc0df5932249a121f