Activation and stabilization of lipase by grafting copolymer of hydrophobic and zwitterionic monomers onto the enzyme

We report herein new enzyme-polymer conjugates synthesized by grafting polymerization onto Candida rugosa lipase (CRL) with zwitterionic carboxyl betaine methacrylate (CBMA), hydrophobic tert-butyl methacrylate (TBMA) and their equimolar mixture, which respectively created CRL-pCBMA, CRL-pTBMA, and CRL-p(T50-C50). All the enzyme conjugates presented improved catalytic activity, thermostability and pH tolerance, and the kinetic studies indicated that the polymer-grafting resulted in significantly higher enzymatic reaction efficiency and enzyme-substrate affinity. CRL-pTBMA increased the catalytic activity by 2.26-fold and half-life by 43.1-fold at 50 degrees C as compared with the free enzyme due to the interfacial activation effect of TBMA. However, despite the lower activity of the copolymer conjugate (1.56 times of free enzyme), CRL-p(T50-050), its half-life was 2.2-fold higher than CRL-pTBMA. This was due to the stabilization effect of the zwitterionic monomer component (CBMA) in the copolymer. This is evident from the similar stability of CRL-p(T50-050) with CRL-pCBMA, but the latter showed lower activity than the former due to the lack of the interfacial activation effect of TBMA. Thus, a zwitterionic/hydrophobic balance is of vital importance to enhance the enzymatic performance. Spectroscopic characterizations revealed the changes in the microenvironment and secondary structures of the enzyme in CRL-polymer conjugates. Therefore, modification with zwitterionic-hydrophobic copolymer was found more effective in improving the catalytic performance of lipase.