Thiol-Ene Photoimmobilization Of Chymotrypsin On Polysiloxane Gels For Enzymatic Peptide Synthesis

Enzymes are regarded as highly efficient and multi-functional biocatalysts,which can be widely applicable and useful tools for organic chemical reactions,particularly in chemo-,regio-,and enantio-selective processes.Nowadays,due to the large and growing interest of peptides in the market-oriented applications for food and pharmaceutical industry,enzymatic synthesis of peptides has become a typical bioprocess with many advantages,including high efficiency,high selectivity,high product yields,mild reaction conditions,and minimal side chain protection requirements.However,the application of enzymes in their native forms for biotransformation in organic media is plagued by several drawbacks,such as autolysis,structural denaturation,low operational stability and poor recyclability,etc.In order to overcome these limitations,it is necessary to adopt proper strategies for enzyme stabilization in organic solvents,for example,by physisorption,covalent immobilization,entrapment,or encapsulation of enzymes in various solid supports.Among various support media and enzyme immobilization techniques,chemically bonding of enzymes onto polymeric materials offers several advantages due to its good resistance to organic solvents,excellent processability,biocompatibility,and inertness to microbial attack.In particular,cross-linked polysiloxane gels as typical super-hydrophobic supports,have garnered so much excitement for their advantageous properties,such as excellent chemical resistance,good thermal stability,and extraordinary permeability in organic solvents,which render it a good candidate for supporting enzymes in organic media to efficiently catalyze peptide synthesis.The light-stimulated thiol-enephotoimmobilization provides an alternative approach as a mild,efficient and green method for surface immobilization purposes.Accordingly,olefin-functionalized chymotrypsin is photoimmobilized onto cross-linked polysiloxane gels via facile and fast UV-initiated thiol-ene click chemistry.We also explore the properties of polysiloxane-gel-bonded chymotrypsin for peptide synthesis in organic solvents.The main research contents are divided into the following two parts:1.Native chymotrypsin was firstly functionalized with terminal olefin groups by a“grafting to”strategy in aqueous solution.The vinyl-decorated chymotrypsin was then immobilized onto PMMS gels by UV-initiated thiol-ene click chemistry reaction,to provide the desired polysiloxane-gel-bonded chymotrypsin?PMMS-CT?.Consequently,the photo-immobilization processes are determined by the change of C=C-H stretching vibration in FT-IR spectra of the native chymotrypsin,vinyl-modified chymotrypsin and PMMS-CT samples.The absorption peak at 3070 cm^-1 of the vinyl-modified chymotrypsin ascribed to the C=C-H stretching vibration disappeared in PMMS-CT sample,which indicated that the C=C groups of the vinyl-modified chymotrypsin have been completely consumed by the mercapto groups of PMMS and chymotrypsin were successfully imobilized.2.Dipeptide synthesis of L-phenylalaninamide?Phe-NH₂?and N-benzoyl-L-tyrosine ethyl ester?Bz-Tyr-OEt?providing N-benzoyl-tyrosyl phenylalaninamide?Bz-Tyr-Phe-NH₂?catalysed by PMMS-CT is chosen as a modal reaction for peptide synthesis in this thesis.In enzymatic peptide synthesis process,hydrophobic polysiloxane matrice could prevent water from attacking the acyl-enzyme intermediate,which was beneficial for the shift in equilibrium to peptide formation.The results show that the prepared polysiloxane-immobilized chymotrypsin could efficiently catalyze peptide synthesis in low water organic medium.Compared with the native chymotrypsin,polysiloxane-immobilized chymotrypsin showed advantageous catalytic activity,higher thermal stability and superior recyclability.