Synthesis Of Polymer Grafted Lipase And Its Effect On Enzyme Activity

Enzyme is an important biocatalyst.Improvement of enzymatic activity and stability is always the important topic in the industrial application of enzymes.In recent years,the coupling of enzyme molecules with polymer molecules has become a promising and widely concerned technique.However,the regulating mechanism of polymer chains to enzyme activity and stability is still not well understanded so far.Lipase is one of the most widely studied and applied enzymes.Previous studies demonstrated that lipase activity was affected by the hydrophobicity of the material.In this work,four polymer-grafted Candida rugosa lipase(CRL)were synthesized via atom transfer radical polymerization by respectively grafting hydroxyethyl methacrylate(HEMA),glycidyl methacrylate(GMA),propyl methacrylate(n PMA)and butyl methacrylate(BMA)onto CRL surface using N-(bromoisobutyryloxy)succinimide as the initiator.The results of CD spectra showed that the ?-helical and ?-sheet contents of CRL increased in polymer-grafted enzyme,and exhibited a positive correlation with the length of alkyl group in monomers.Furthermore,a blue shift in fluorescence emission spectra was also observed in polymer-grafted CRL,meaning that polymer-grafted CRL had a more compact structure than wide-type CRL.Moreover,activity measurement showed that polymer-grafted CRL had 2.27-2.78 times enzymatic activity compared with CRL.The enzymatic activity increased in a train of HEMA-g-CRL = GMA-g-CRL,n PMA-g-CRL and BMA-g-CRL.With an increase of the length of alkyl group in the monomer,moreover,Michaelis parameter of CRL decreased from 0.17 mmol/L to 0.09 mmol/L whereas turnover number increased from 67 to 182 /s.As a result,catalytic efficiency of polymer-grafted CRL enhanced greatly and the maximum catalytic efficiency was reported in BMA-g-CRL.The result demonstrated that polymer grafting improved the lid opening of active site in CRL,leading to the exposure of the active site in CRL and enhancing the substrate binding.Results of CRL stability showed that polymer-grafted CRL had a greater thermal stability and wider p H tolerant range than wide-type CRL.The research proposed a novel methodology to regulate the CRL activity by polymer grafting,and exhibited the influence of alkyl groups of the monomers to CRL activity.The effect of different grafting densities on the activity of CRL enzymes was determined.The results showed that the increase of polymer grafting densities was beneficial to the increase the activity of CRL.The result in this research provides useful reference for deepening the cognition of mechanism that the side chain structure of polymer molecule how to regulate the CRL enzyme activity and rationally screening monomer molecules to synthesize polymer grafted CRL to improve the enzyme activity.