Altering lipase activity and enantioselectivity in organic media using organo-soluble bases: Implication for rate-limiting proton transfer in acylation step.
With the hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl esters via a partially purified papaya lipase (PCPL) in water-saturated isooctane as the model system, the enzyme activity, and enantioselectivty is altered by adding a variety of organo-soluble bases that act as either enzyme activators (i.e., TEA, MP, TOA, DPA, PY, and DMA) or enzyme inhibitors (i.e., PDP, DMAP, and PP). Triethylamine (TEA) is selected as the best enzyme activator as 2.24-fold increase of the initial rate for the (S)-ester is obtained when adding 120 mM of the base. By using an expanded Michaelis-Menten mechanism for the acylation step, the kinetic analysis indicates that the proton transfer for the breakdown of tetrahedral intermediates to acyl-enzyme intermediates is the rate-limiting step, or more sensitive than that for the formation of tetrahedral intermediates when the enzyme activators of different pKa are added. However, no correlation for the proton transfers in the acylation step is found when adding the bases acting as enzyme deactivators.
Chen
Chun-Chi
CC
Department of Chemical Engineering, National Cheng Kung University, Tainan 70101, Taiwan.
Chen
Teh-Liang
TL
Tsai
Shau-Wei
SW
D000215:Acylation; D000438:Alcohols / Q000737:chemistry; D001665:Binding Sites; D029441:Carica / Q000201:enzymology*; D004952:Esters; D006868:Hydrolysis; D007700:Kinetics; D008049:Lipase / Q000737:chemistry* / Q000378:metabolism*; D008968:Molecular Conformation; D009288:Naproxen / Q000737:chemistry*; D009795:Octanes / Q000737:chemistry; D009930:Organic Chemicals / Q000737:chemistry* / Q000302:isolation & purification; D011522:Protons; D013237:Stereoisomerism; D014867:Water / Q000737:chemistry
D000438:Alcohols; D004952:Esters; D009795:Octanes; D009930:Organic Chemicals; D011522:Protons; D014867:Water; D009288:Naproxen; D008049:Lipase; C045798:2,2,4-trimethylpentane