Chemical profiling of the deacetylase activity of acetyl xylan esterase A (AxeA) variants on chitooligosaccharides using hydrophilic interaction chromatography-mass spectrometry

Autor: Joelle N. Pelletier, Karen C. Waldron, Claude Dupont, Marie-Christine Tang, Audrey Nisole
Přispěvatelé: Université de Montréal (UdeM), Institut Armand Frappier (INRS-IAF), Institut National de la Recherche Scientifique [Québec] (INRS)-Réseau International des Instituts Pasteur (RIIP), Funding for this work was provided by The Natural Sciences and Engineering Research Council of Canada Strategic Grant No. STPGP-257885.
Rok vydání: 2011
Předmět:
MESH: Sequence Analysis
DNA
MESH: Acetylesterase
MESH: Streptomyces lividans
Oligosaccharides
Bioengineering
Protein Engineering
Applied Microbiology and Biotechnology
Mass Spectrometry
Hydrolysis
MESH: Directed Molecular Evolution
MESH: Gene Library
MESH: Mutagenesis
Gene Library
MESH: Mass Spectrometry
chemistry.chemical_classification
Chitosan
Chromatography
Hydrophilic interaction chromatography
MESH: Hydrophobic and Hydrophilic Interactions
Biological activity
Acetylation
General Medicine
Sequence Analysis
DNA
Directed evolution
HEXA
MESH: Protein Engineering
carbohydrates (lipids)
MESH: Chitosan
Enzyme
chemistry
Biochemistry
Mutagenesis
[SDV.TOX]Life Sciences [q-bio]/Toxicology
Acetylesterase
Streptomyces lividans
Directed Molecular Evolution
MESH: Chromatography
Liquid
MESH: Oligosaccharides
Hydrophobic and Hydrophilic Interactions
MESH: Acetylation
Biotechnology
Deacetylase activity
Chromatography
Liquid
Zdroj: Journal of Biotechnology
Journal of Biotechnology, Elsevier, 2011, 155 (2), pp.257-65. ⟨10.1016/j.jbiotec.2011.06.041⟩
ISSN: 1873-4863
0168-1656
Popis: Chitosan oligosaccharides (oligomers of (GlcNAc) x (GlcN) y ) are used in the pharmaceutical, cosmetic and food industries and are reported to have therapeutic benefits. However, it is unknown whether their biological activity depends on the degree of deacetylation or the sequence of residues within the oligomer. We report here the development of a random mutagenesis method for directed evolution of Streptomyces lividans acetyl xylan esterase (AxeA), which we previously showed is able to deacetylate chitinous substrate, in order to obtain chitooligosaccharides with well-defined structural properties. A colorimetric assay was used to pre-screen libraries for p -nitrophenol acetate hydrolysis activity and an HPLC-UV absorbance assay was optimized to subsequently screen for deacetylase activity toward hexa- N -acetyl-glucosamine substrate (GlcNAc) 6 . Native AxeA and two variants displaying > 50% deacetylation of the oligohexamer substrate after reaction at 50 °C for 24 h in diluted culture supernatant were then selected for detailed analysis of the enzymatic products. A HILIC (hydrophilic interaction chromatography)-mode LC method was developed for profiling the deacetylated chitooligosaccharide products and HILIC-MS/MS sequencing revealed that ca. 30 different deacetylation products ranging from (GlcNAc) 5 (GlcN) 1 to (GlcNAc) 1 (GlcN) 5 and isomers thereof were produced. The AxeA variants produced, on average, 26% more unique products than the native enzyme; however, none were able to fully deacetylate the substrate to make (GlcN) 6 . The long term goal of this multidisciplinary approach is to improve the activity of chitosan oligosaccharides to an industrially applicable level.
Databáze: OpenAIRE
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