|
KMID : 1094720160210060704
|
|
Biotechnology and Bioprocess Engineering
2016 Volume.21 No. 6 p.704 ~ p.711
|
|
|
Improved temperature characteristics of an Aspergillus oryzae GHF11 xylanase, by in silico design and site-directed mutagenesis
|
|
Hu Die
Li Jianfang
Wu Qin
Zang Jia
Cheng Jianqing
Wu Minchen
|
|
|
Abstract
|
|
|
|
To improve the temperature characteristics of a mesophilic glycoside hydrolase family (GHF) 11 xylanase AoXyn11A from Aspergillus oryzae, both introduction of a disulfide bridge and the substitution of a specific amino acid were carried out by in silico design and site-directed mutagenesis. Based on the analysis of a known crystal structure of thermophilic xylanase TlXynA from Thermomyces lanuginosus, and the alignment of primary structures between AoXyn11A and TlXynA, one mutant AoXyn11AM with a disulfide bridge (Cys108?Cys152) was designed by replacing the Ser108 and Asn152 of AoXyn11A with Cys residues, respectively. Additionally, based on the analysis of amino acid B-factor values, another mutant AoXyn11AM-G22A was predicted by substituting Gly22 of AoXyn11AM (having the maximum B-factor value of 69.25 A, with the corresponding Ala23 of TlXynA. Thereafter, two mutant xylanase-encoding genes, Aoxyn11A M and Aoxyn11A M-G22A, were constructed by site-directed mutagenesis. Aoxyn11A and two mutant genes were expressed in E. coli BL21(DE3) respectively, and three expressed recombinant xylanases, reAoXyn11A, reAoXyn11AM and reAoXyn11AM-G22A, were purified to homogeneity. The temperature optima of reAoXyn11AM and reAoXyn11AM-G22A were 60 and 65°C, respectively, being 5 and 10°C higher than that of reAoXyn11A. Their thermal inactivation half-lives at 50°C were 1.8- and 8.4-folds longer than that of reAoXyn11A. There were no obvious alterations after mutations in specific activity and enzymatic properties, except for the temperature characteristics.
|
|
|
KEYWORD
|
|
|
GHF11 xylanase, temperature characteristics, in silico design, site-directed mutagenesis, molecular dynamics simulation
|
|
|
FullTexts / Linksout information
|
|
|
|
|
|
Listed journal information
|
|
 
|
|