Chemical profiling of the deacetylase activity of acetyl xylan esterase A (AxeA) variants on chitooligosaccharides using hydrophilic interaction chromatography-mass spectrometry.
J Biotechnol
; 155(2): 257-65, 2011 Sep 10.
Article
em En
| MEDLINE
| ID: mdl-21767585
ABSTRACT
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 24h 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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Oligossacarídeos
/
Acetilesterase
/
Engenharia de Proteínas
/
Streptomyces lividans
/
Quitosana
Idioma:
En
Revista:
J Biotechnol
Assunto da revista:
BIOTECNOLOGIA
Ano de publicação:
2011
Tipo de documento:
Article
País de afiliação:
Canadá
País de publicação:
HOLANDA
/
HOLLAND
/
NETHERLANDS
/
NL
/
PAISES BAJOS
/
THE NETHERLANDS