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J Agric Food Chem ; 66(16): 4182-4188, 2018 Apr 25.
Article in English | MEDLINE | ID: mdl-29633613

ABSTRACT

To simplify purification and improve heat tolerance of a thermostable ß-xylosidase (ThXylC), a short ELK16 peptide was attached to its C-terminus, which is designated as ThXylC-ELK. Wild-type ThXylC was normally expressed in soluble form. However, ThXylC-ELK assembled into aggregates with 98.6% of total ß-xylosidase activity. After simple centrifugation and buffer washing, the ThXylC-ELK particles were collected with 92.57% activity recovery and 95% purity, respectively. Meanwhile, the wild-type ThXylC recovery yield was less than 55% after heat inactivation, affinity and desalting chromatography followed by HRV 3C protease cleavage purification. Catalytic efficiency ( Kcat/ Km) was increased from 21.31 mM-1 s-1 for ThXylC to 32.19 mM-1 s-1 for ThXylC-ELK accompanied by a small increase in Km value. Heat tolerance of ThXylC-ELK at high temperatures was also increased. The ELK16 peptide attachment resulted in 6.2-fold increase of half-life at 65 °C. Released reducing sugars were raised 1.3-fold during sugar cane bagasse hydrolysis when ThXylC-ELK was supplemented into the combination of XynAΔSLH and Cellic CTec2.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Peptides/chemistry , Thermoanaerobacterium/enzymology , Xylosidases/chemistry , Xylosidases/isolation & purification , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Enzyme Stability , Hot Temperature , Hydrogen-Ion Concentration , Hydrolysis , Kinetics , Peptides/metabolism , Thermoanaerobacterium/chemistry , Thermoanaerobacterium/genetics , Xylosidases/genetics , Xylosidases/metabolism
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