Three amino acid changes contribute markedly to the thermostability of β-glucosidase BglC from Thermobifida fusca.
Thermostability of β-glucosidase was enhanced by family shuffling, site saturation mutagenesis, and site-directed mutagenesis. Family shuffling was carried out based on β-glucosidase BglC from Thermobifida fusca and β-glucosidase BglB from Paebibacillus polymxyxa with the help of synthetic primers. High-throughput screening revealed mutants with higher thermostability than both parental enzymes. The most thermostable mutant VM2 containing three key amino acid changes in L444Y/G447S/A433V had a 144-fold increase in half-life of inactivation as compared to the parental enzyme BglC. The mutant VM2 showed 28% and 94% increase in k(cat) towards p-nitrophenyl-β-D-glucopyranoside (pNPG) and cellobiose, respectively. The mutant with enhanced stability would facilitate the recycle of β-glucosidase in the bioconversion of cellulosic biomass.
Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, Sichuan, China.
Xiao-Qiong
XQ
Pei
Zhuo-Lin
ZL
Yi
Chuan-Gen
CG
Tang
Zhong-Liu
ZL
Wu
D000192:Actinomycetales / Q000737:chemistry* / Q000201:enzymology* / Q000235:genetics; D019943:Amino Acid Substitution; D004795:Enzyme Stability; D006358:Hot Temperature; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D015202:Protein Engineering / Q000379:methods*; D013329:Structure-Activity Relationship; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D001617:beta-Glucosidase