The Improved Cross-linked Enzyme Aggregates And Its Application In Biocatalysis

In this thesis, cross-linked enzyme aggregates (CLEAs) as a carrier-free enzymeimmobilization approach was considered as the core of the research. CLEAstechnology was applied to penicillin G acylase (PGA) and-mannanase.PGA-CLEAs has been prepared and optimized. Meanwhile, a new encapsulatedCLEAs method was put forward to expand the industrial application of CLEAs. Thepreparing method of the encapsulated CLEAs has been designed and evaluated, andthe encapsulated PGA-CLEAs were applied to the synthesis of amoxicillin. Aspreparing of-mannanase CLEAs, macromolecular cross-linker was used in thecross-linking step instead of traditional glutaraldehyde to improve the activity ofCLEAs toward macromolecular substrates. The conclusions can be summarized asfollows:Preparation of PGA-CLEAs: PGA-CLEAs have been prepared. The optimumpreparation conditions were summarized as: precipitant (NH4)2SO4, volume ratio ofprecipitant to enzyme2.3:1, cross-linker glutaraldehyde, cross-linker concentration0.35%and reaction time2h. The reservation of activity was30.1%. The optimumtemperature and pH of CLEAs were both improved compared with free PGA.Kinetics of thermal deactivation of PGA-CLEAs: The deactivation model ofPGA-CLEAs was established as the series model whereas the free PGA was the onestep model. The decrease of Eiof PGA-CLEAs explained the excellent thermalstability of PGA-CLEAs compared with free enzyme.Preparation of encapsulated PGA-CLEAs: Four methods were evaluated inorder to prepare encapsulated CLEAs. Calcium alginate microsphere has beenprepared.10mg/mL CaCl2was used as hardening agent and the optimum hardeningtime was30min. ALG-SiO2microsphere has been hardened by15mg/mL CaCl2for40min. Moreover, sodium alginate-chitosan microsphere and chitosan microspherehave been prepared. After inspecting the properties of four microspheres, ALG-SiO2microsphere was selected as the optimum carrier for PGA-CLEAs.Synthesis of amoxicillin by encapsulated PGA-CLEAs: Amoxicillin wassuccessfully synthesized by using PGA-CLEAs, which were encapsulated inALG-SiO2microspheres. The optimum reaction conditions can be summarized as:reaction temperature4℃, pH of substrates6.5, mole ratio of substrates HPGM： 6-APA=3:1, and reaction time75h. The final yield of amoxicillin was55.2%and evenif reused by four times, the yield was85%of the starting value.Preparation of-mannanase CLEAs:-mannanase CLEAs has been preparedby precipitation and subsequent cross-linking. Isopropanol was the ideal precipitantand the optimum volume was5:1compared with enzyme solution. Dextranpolyaldehyde (2000kDa) was selected as the ideal cross-linker and the optimumconcentration was0.6%. The optimum cross-linking time was determined as16h, andthe final activity of β-mannanase CLEAs was2.67U/mg (64.66%), which was about16times higher than CLEAs prepared with glutaraldehyde (4.68%).