Immobilization of Lipase from <i>Pseudomonas fluorescens</i> on Porous Polyurea and Its Application in Kinetic Resolution of Racemic 1‑Phenylethanol

Abstract

A porous polyurea (PPU) was prepared through a simple protocol by reacting toluene diisocyanate with water in binary solvent of water–acetone. Its amine group was determined through spectrophotometric absorbance based on its iminization with <i>p</i>-nitrobenzaldehyde amines. PPU was then used as a novel polymer support for enzyme immobilization, through activation by glutaraldehyde followed by immobilization of an enzyme, lipase from <i>Pseudomonas fluorescens</i> (PFL), via covalent bonding with the amine groups of lipase molecules. Influences of glutaraldehyde and enzyme concentration and pH in the process were studied. The results revealed that the activity of the immobilized PFL reached a maximum at GA concentration of 0.17 mol/L and at pH 8. Immobilization rate of 60% or higher for PFL was obtained under optimized condition with an enzyme activity of 283 U/mg. The porous structure of PPU, prior to and after GA activation and PFL immobilization, was characterized. The activity of the immobilized PFL at different temperature and pH and its stability at 40 °C as well as its reusability were tested. The immobilized enzyme was finally used as enantioselective catalyst in kinetic resolution of racemic 1-phenylethanol (1-PEOH), and its performance compared with the free PFL. The results demonstrate that the enzyme activity and stability were greatly improved for the immobilized PFL, and highly pure enantiomers from racemic 1-PEOH were effectively achieved using the immobilized PFL. Noticeable deactivation of PFL in the resolution was observed by acetaldehyde in situ formed. In addition, the immobilized PFL was readily recovered from the reaction system for reuse. A total of 73% of the initial activity was retained after 5 repeated reuse cycles. This work provides a novel route to preparation of a polyurea porous material and its enzyme immobilization, leading to a novel type of immobilized enzyme for efficient kinetic resolution of racemic molecules