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Development of an improved variant of GH51 α-l-arabinofuranosidase from Pleurotus ostreatus by directed evolution
- Source :
- New Biotechnology, New Biotechnology, Elsevier, 2014, 31 (3), pp.230-236. ⟨10.1016/j.nbt.2014.02.004⟩, New Biotechnology, 2014, 31 (3), pp.230-236. ⟨10.1016/j.nbt.2014.02.004⟩
- Publication Year :
- 2014
- Publisher :
- HAL CCSD, 2014.
-
Abstract
- In this study, the α- l -arabinofuranosidase from Pleurotus ostreatus was subjected to directed evolution by expressing a library of around 7000 randomly mutated variants by error prone Polymerase Chain Reaction. High-throughput screening of the library for the most active variants was performed by assaying activity towards p-nitrophenyl α- l -arabinofuranoside, and a variant with higher activity than the wild type was selected, purified and characterised. It exhibited a kcat of 7.3 × 103 ± 0.3 min−1, around 3-fold higher than that of the wild type (2.2 × 103 ± 0.2 min−1), and a KM (0.54 ± 0.06 mM) 30% lower than that of the wild type (0.70 ± 0.05 mM) towards this substrate. The mutant also showed improved catalytic properties towards pNP-β- d -glucopyranoside (kcat of 50.85 ± 0.21 min−1 versus 11.0 ± 0.6 min−1) and it was shown able to hydrolyse larch arabinogalactan which is not recognised by the wild type. The mutant was also more active than the wild type towards arabinoxylan and was able to hydrolyse arabinan, which was not transformed by the wild type. The ability of rPoAbf F435Y/Y446F to hydrolyse these insoluble substrates expands its potential for application also to hemicelluloses, which in some types of pretreatment are recovered in solid fractions.
- Subjects :
- 0106 biological sciences
Models, Molecular
Glycoside Hydrolases
Mutant
Bioengineering
Pleurotus
01 natural sciences
03 medical and health sciences
chemistry.chemical_compound
Arabinogalactan
010608 biotechnology
Arabinoxylan
[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology
Enzyme kinetics
Molecular Biology
ComputingMilieux_MISCELLANEOUS
030304 developmental biology
Gene Library
0303 health sciences
biology
[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM]
Wild type
Temperature
Substrate (chemistry)
[SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology
General Medicine
Hydrogen-Ion Concentration
biology.organism_classification
Directed evolution
Arabinose
[SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics
Biochemistry
chemistry
Mutation
Electrophoresis, Polyacrylamide Gel
Mutant Proteins
Pleurotus ostreatus
Directed Molecular Evolution
Biotechnology
Subjects
Details
- Language :
- English
- ISSN :
- 18716784 and 18764347
- Database :
- OpenAIRE
- Journal :
- New Biotechnology, New Biotechnology, Elsevier, 2014, 31 (3), pp.230-236. ⟨10.1016/j.nbt.2014.02.004⟩, New Biotechnology, 2014, 31 (3), pp.230-236. ⟨10.1016/j.nbt.2014.02.004⟩
- Accession number :
- edsair.doi.dedup.....e30d68506472aa4aa9bfa1d694472666