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

  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},
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
Random mutagenesis to enhance the activity of subtilisin in organic solvents: Characterization of Q103R subtilisin E
Kinetic experiments performed at high salt concentration reveal that the mechanism behind the Q103R variant's enhanced activity toward succinyl‐Ala‐ Ala‐Pro‐Phe‐p‐nitroanilide is both electrostatic and nonelectrostatic in origin. Expand
Enhancement of stability and activity of phospholipase A(1) in organic solvents by directed evolution.
An enzyme variant having superior catalytic efficiency in most of the organic solvents could be obtained by using any solvent suitable for designing the efficient screening system, regardless of the properties of the particular solvent. Expand
Protein Engineering of a Metalloprotease in Order to Improve Organic Solvents Stability and Activity
Abstract Recently, improve the protease activity in the presence of organic solvents has been appreciated for the researchers. In the current study, we have tried to increase the organic solventExpand
Directed evolution of a para-nitrobenzyl esterase for aqueous-organic solvents
Although the contributions of individual effective amino acid substitutions to enhanced activity are small (<2-fold increases), the accumulation of multiple mutations by directed evolution allows significant improvement of the biocatalyst for reactions on substrates and under conditions not already optimized in nature. Expand
Characterization and molecular modelling of an engineered organic solvent tolerant, thermostable lipase with enhanced enzyme activity
Abstract To evolve a variant with improved enzymatic properties i.e. thermostability, catalytic efficiency, by error prone mutagenesis, lipR1 was used as template. Using two tier screening protocol,Expand
Improving the carboligase activity of benzoylformate decarboxylase from Pseudomonas putida by a combination of directed evolution and site-directed mutagenesis.
Surprisingly, all L476 mutants catalyze the formation of 2-hydroxy-1-phenyl-propanone with significantly higher enantioselectivity than the wild-type enzyme although enantiOSElectivity was not a selection parameter. Expand
Proteins designed for challenging environments and catalysis in organic solvents
Abstract The discovery and application of protein design guidelines continue to enhance successfully the stability of proteins in hars environments such as elevated temperatures, extreme alkaline pH,Expand
Engineering the loops in a lipase for stability in DMSO.
Six mutations that improve the stability and activity of lipase in dimethyl sulfoxide (DMSO) are identified based on a novel screening system and the melting temperature of 6SR is lower than the wild type and binds the least to hydrophobic fluorescent probes, indicating that the surface has become more polar in nature. Expand
Molecular mechanism of enzyme tolerance against organic solvents: Insights from molecular dynamics simulation.
It was obtained that low concentrations of hexane may impose more instability on the enzyme conformation than higher percentages, and results showed that presence of water is determining factor in the enzyme stability at high concentrations ofhexane. Expand
Improve Salinivibrio zinc-metalloprotease function in less polar organic solvents by increasing surface hydrophobicity.
There was a correlation between surface hydrophobicity of SVP and hydrophobic strength of organic solvents and D24A is found to be the most efficient mutant for its remarkable decrease in ki value in the presence of isopropanol and n-propanol. Expand


Enzyme Engineering for Nonaqueous Solvents. II. Additive Effects of Mutations on the Stability and Activity of Subtilisin E in Polar Organic Media
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
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.
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
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
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
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