Enzyme Engineering for Nonaqueous Solvents: Random Mutagenesis to Enhance Activity of Subtilisin E in Polar Organic Media

@article{Chen1991EnzymeEF,
  title={Enzyme Engineering for Nonaqueous Solvents: Random Mutagenesis to Enhance Activity of Subtilisin E in Polar Organic Media},
  author={Keqin Chen and Frances H. Arnold},
  journal={Bio/Technology},
  year={1991},
  volume={9},
  pages={1073-1077}
}
Enzyme activity is often dramatically reduced in polar organic solvents, even under conditions where the folded structures are stable. We have utilized random mutagenesis by polymerase chain reaction (PCR) techniques combined with screening for enhanced activity in the presence of dimethylformamide (DMF) to probe mechanisms by which improved enzymes for chemical synthesis in polar organic media might be obtained. Two amino acid substitutions which enhance subtilisin E activity in the presence… Expand
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References

SHOWING 1-10 OF 26 REFERENCES
Enzyme Engineering for Nonaqueous Solvents. II. Additive Effects of Mutations on the Stability and Activity of Subtilisin E in Polar Organic Media
TLDR
The double mutant Q103R+N218S is 10 times more active than the wild‐type enzyme in 20% (v/v) DMF and twice as stable in 40% DMF. Expand
Stabilization of substilisin E in organic solvents by site‐directed mutagenesis
TLDR
This study provides additional evidence that substitution of surface‐charged residues is a generally useful mechanism for stabilizing enzymes in organic media and that the stabilizing effects of such substitutions are unique to highly altered solvent environments. Expand
Engineering an enzyme by site-directed mutagenesis to be resistant to chemical oxidation.
TLDR
The feasibility of improving oxidative stability in proteins by site-directed mutagenesis is demonstrated and Mutant enzymes were expressed in Bacillus subtilis and were found to vary widely in specific activity. Expand
A search for peptide ligase: cosolvent-mediated conversion of proteases to esterases for irreversible synthesis of peptides
Serine and cysteine proteases (trypsin, chymotrypsin, papain, subtilisin) in the presence of certain concentrations of water-miscible organic solvents express no amidase activities. The esteraseExpand
Proteases of enhanced stability: Characteization of a thermostable variant of subtilisin
TLDR
The refined, 1.8‐Å crystal structures of the wild‐type and 7150 subtilisin have been compared in detail, leading to the conclusion that slight improvements in hydrogen bond parameters in the vicinity of position 218 result in the enhanced thermal stability of 7150. Expand
Engineering enzymes for non-aqueous solvents.
  • F. Arnold
  • Chemistry, Medicine
  • Trends in biotechnology
  • 1990
TLDR
The interactions that contribute to protein stability in non-aqueous solvents are discussed in the context of attempting to identify possible approaches to constructing enzymes which exhibit enhanced stability in unaqueous media. Expand
15 Subtilisin: X-Ray Structure
Publisher Summary The term subtilisin is employed to designate the group of alkaline serine proteases produced by various species of bacilli. This chapter focuses on the three-dimensional structure,Expand
Enzymatic catalysis in monophasic organic solvents
Enzymatic catalysis in nonaqueous media significantly extends conventional aqueous-based biocatalysis. Enzymes are active in organic solvents, supercritical fluids, and gaseous solvents. This paperExpand
16 Subtilisins: Primary Structure, Chemical and Physical Properties
Publisher Summary This chapter reviews the subtilisins: the diisopropylfluorophosphate (DFP)-sensitive, extracellular, alkaline proteinaees from Bacillus subtilis and other species of Bacillus.Expand
Thermitase, a thermostable subtilisin: Comparison of predicted and experimental structures and the molecular cause of thermostability
TLDR
Based on a combination of theoretical and experimental evidence, the unusually tight binding of calcium by thermitase emerges as the most likely single influence responsible for its increased thermostability. Expand
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