Stabilization of Candida antarctica lipase B in hydrophilic organic solvent by rational design of hydrogen bond

Exploring the full natural diversity of single amino acid exchange reveals that 40–60% of BSLA positions improve organic solvents resistance

“…A role of substitutions in loop regions to improve cosolvent resistance has previously been reported (Wintrode and Arnold 2001). Substitutions of surface located residues were in many cases reported to contribute to organic solvent resistance of enzymes (Park et al 2012(Park et al , 2013Kawata and Ogino 2009;Martinez and Arnold 1991;Martinez et al 1992). Ogino et al (2007) speculated that charged substitutions on the surface prevent organic solvents molecules to diffuse into the protease from Pseudomonas aeruginosa PST-01.…”

“…Both charged and polar amino acids have been reported to contribute to improved organic solvents resistance of enzymes (Park et al 2012, 2013; Chen and Arnold 1991;Song and Rhee 2001;Takahashi et al 2005;Zumarraga et al 2007), including methanol, ethanol, DMF, 2-propanol, Acetonitrile and DMSO, etc. Postulated reasons were that charged and polar residues could enable the formation of hydrogen bonds between amino acid residues or between surface residues and water molecules which help in maintaining the enzyme structure intact (Park et al 2012). Furthermore, substitution of aromatic, polar, charged, and aliphatic amino acid residues with chemically different ones yielded for all three organic solvents more improved variants (68-95%) than substitutions to chemically similar amino acids.…”

“…Hydrophilic organic solvents (e.g. EtOH, MeOH, and DMSO) with a polarity similar to water are fully miscible (homogeneous solution) (Park et al 2012) and organic molecules are in close contact with enzymes. The latter can result in a competition of solvent molecules with enzyme-bound water molecules and results in structural changes or denaturation (Ogino and Ishikawa 2001;Yang et al 2004).…”

“…For instance, it was found that substitutions of two charged amino acid residues on the surface of α-lytic protease to hydrophobic residues improved protease stability in presence of hydrophilic solvents (Martinez and Arnold 1991). Interestingly, it was reported that some beneficial substitutions were surface exposed (Park et al 2012(Park et al , 2013Kawata and Ogino 2009;Martinez et al 1992). Furthermore, the formation of salt bridges and hydrogen bonds led to improved organic solvent stability of the lipase (Pseudomonas aeruginosa LST-03) in presence of organic solvents (Kawata and Ogino 2010).…”

Protein Engineering by Random Mutagenesis and Structure-Guided Consensus of Geobacillus stearothermophilus Lipase T6 for Enhanced Stability in Methanol

“…Enzyme stability in organic solvents can be successfully enhanced by a rational design approach (28)(29)(30). However, the main drawbacks of this approach are the lack of structural data, in many cases, together with a limited understanding of the enzyme-solvent interactions, and also the limited ability to predict how amino acid alternations influence the enzyme activity and stability.…”