A single residual replacement improves the folding and stability of recombinant cassava hydroxynitrile lyase in E. coil.
Substitution of Ser113 for Gly113 in the cap domain of hydroxynitrile lyase from Manihot esculenta (MeHNL) was performed by site-directed mutagenesis to improve its self-generated folding and stability under denaturation conditions. The yield of the recombinant mutant HNL1 (mut-HNL1), which had higher specific activity than the wild type HNL0 (wt-HNL0), was increased by 2 to 3-fold. Thermostability of MeHNL was also enhanced, probably due to an increase in content of the beta-strand secondary structure according to CD analysis. Our data in this report suggest that Ser113 significantly contributes to the in vivo folding and stability of MeHNL and demonstrates an economic advantage of mut-HNL1 over the wt-HNL0.
Yan
Gonghong
G
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Zhong Guan Cun, Hai Dian, Beijing 100080, PR China.
Cheng
Shuhua
S
Zhao
Guogang
G
Wu
Sheng
S
Liu
Yanbing
Y
Sun
Wanru
W
D000097:Acetonitriles / Q000378:metabolism; D000446:Aldehyde-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D002365:Manihot / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013696:Temperature
D000097:Acetonitriles; D011994:Recombinant Proteins; C045516:mandelonitrile; D000446:Aldehyde-Lyases; C079359:hydroxymandelonitrile lyase