Improving the catalytic activity of thermostable xylanase from Thermotoga maritima via mutagenesis of non-catalytic residues at glycone subsites.
Enzyme Microb Technol
; 139: 109579, 2020 Sep.
Article
em En
| MEDLINE
| ID: mdl-32732029
ABSTRACT
Endo-ß-1,4-xylanase from Thermotoga maritima, TmxB, is an industrially attractive enzyme due to its extreme thermostability. To improve its application value, four variants were designed on the basis of multiple sequence and three-dimensional structure alignments. Wild-type TmxB (wt-TmxB) and its mutants were produced via a Pichia pastoris expression system. Among four single-site mutants, the tyrosine substitution of a threonine residue (T74Y) at putative -3/-4 subsite led to a 1.3-fold increase in specific activity at 40⯰Câ¯-â¯100⯰C and pH 5 for 5â¯min, with beechwood xylan as the substrate. T74Y had an improved catalytic efficiency (kcat/Km), being 1.6 times that of wt-TmxB. Variants DY (two amino acid insertions) and N68Q displayed a slight increase (1.2 fold) and dramatic decline (1.7 fold) in catalytic efficiency, respectively. Mutant E67Y was totally inactive under all test conditions. Structural modeling and docking simulation elucidated structural insights into the molecular mechanism of activity changes for these TmxB variants. This study helps in further understanding the roles of the non-catalytic amino acids at the glycone subsites of xylanases from glycoside hydrolase family 10.
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Base de dados:
MEDLINE
Assunto principal:
Mutagênese Sítio-Dirigida
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Thermotoga maritima
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Endo-1,4-beta-Xilanases
Idioma:
En
Revista:
Enzyme Microb Technol
Ano de publicação:
2020
Tipo de documento:
Article
País de afiliação:
China