Immobilization Of Phenylalanine Ammonia Lyase Based On Calcium Carbonate Templates And Its Catalytic Performance

Phenylalanine ammonia lyase(PAL)is the key enzyme for production of L-phenylalanine(L-Phe)and treatment of phenylketonuria.However,free PAL exhibits low catalytic efficiency,poor stability and poor reusability in industrial applications,which seriously affecting the efficiency of the PAL.In this study,PAL protein particles and protein particles with biosilica shell were prepared by using calcium carbonate as a template,respectively.Furthermore,the properties of the immobilized enzymes were also investigated.1)Preparation of different morphology of calcium carbonate template and the immobilization of enzymeIn this section,different morphology of calcium carbonate template was prepared,and PAL was immobilized by these calcium carbonate template,respectively.The results showed that spherical PAL protein particles with high enzyme activity was obtained when spherical calcium carbonate was used as a template.The optimized conditions for preparing template were as follow: stirring time for 2 h,stirring speed at 700 rpm,and 0.7 M salt solution with solvent(ethylene glycol:water=3:1).Therefore,spherical calcium carbonate particles with narrow size distribution and large surface area were obtained under the optimized conditions.2)Preparation and catalytic properties of porous PAL protein particlesPorous spherical protein particles were prepared by the covalent cross-linked technology.The concentration of glutaraldehyde and crosslinking time were optimized.The optimized conditions for preparing spherical protein particles were glutaraldehyde concentration at 0.4% and cross-linking time for 1 h.We prepared successfully spherical protein particles with uniform size under optimized conditions.the optimum catalytic temperature and p H of spherical PAL protein particles was 50 ?and 7.0,respectively.Furthermore,thermostability,p H stability,resistance to denaturants,trypsin tolerance,storage stability,and reusability of the immobilized enzyme was better than that of the free enzyme.Kinetic parameters showed that Vmax of the immobilized enzyme was lower than that of the free enzyme.however,Km of the immobilized enzyme was higher than that of the free enzyme.The results indicated that the mass transfer resistance of the protein particles contacting with the substrate was bigger than that of the free enzyme,and the protein particles had a low affinity with substrate.3)Preparation and catalytic properties of protein particles with biosilica shellThe spherical PAL protein particles with biosilica shell was prepared by biomimetic silicification.The spherical PAL protein particles was encapsulated into biomimetic silicification and hollow biosilica shell,respectively.The conditions for the preparation of protein particles with biosilica shell were optimized.The optimized conditions were as follows: 2.2 m L Tris-HCl buffer(containing the protein particles),then 300 ?L dodecyl trimethyl ammonium bromide(CTAB),and 20 ?L tetramethoxysilane(TMOS).The optimum catalytic temperature of PAL protein particles with biosilica shell and PAL protein particles hollow biosilica shell were 70 ? and 60 ?,respectively.Moreover,the optimum catalytic p H of PAL protein particles with biosilica shell and PAL protein particles hollow biosilica shell were 8.0 and 9.0,respectively.Compared to the free enzyme,the spherical PAL protein particles with biosilica shell exhibited superior thermostability,p H stability,the denaturant tolerance,trypsin tolerance and storage stability.