Enantioselectivity and catalysis improvements of Pseudomonas cepacia lipase with Tyr and Asp modification

Abstract

A concise strategy to improve the p-NPP (p-nitrophenyl palmitate) catalytic activity and enantioselectivity towards secondary alcohols of Pseudomonas cepacia lipase (PcL) has been described. The PcL was modified by I₃⁻, N-acetyl imidazole (NAI), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and ethylenediamine (EDA) in the absence or presence of n-hexane, respectively. After being modified by the four modification reagents, the enantioselectivity (E value) of the PcL towards secondary alcohols was enhanced by 2- to 4-fold. The catalytic activity of EDA-PcL was increased by about 6-fold. The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of modified PcL showed that Tyr⁴, Tyr²⁹, Tyr⁴⁵, Tyr⁹⁵, Asp³⁶ and Asp⁵⁵ were the modified sites. When Tyr²⁹ was modified, the E value of PcL towards secondary alcohols was largely improved. MALDI-TOF-MS characterization and molecular dynamics simulation of the lipase indicated that Tyr²⁹ located inside the catalytic cavity had a significant impact on the E value. The strong steric hindrance of acetyl and iodine ion to the groups on the chiral center of the substrates is responsible for the improvement. In addition, the enhancement of hydrophobicity on the surface of the lipase due to the sidechain replacement of Asp with uncharged hydrophobic groups also improved the E value.