Design And Fabrication Of Porous Microspheres For Enzyme Immobilization Based On Natural Polymers

Mild fabrication,rapid transfer of substrate/product and easy to recycle were the three crucial issues in enzyme immobilization.Based on the three crucial issues,porous microspheres were fabricated by choosing natural polymers as the raw materials.These porous microspheres were utilized as the carriers for yeast alcohol dehydrogenase immobilization.The porous structure promoted the transfer of substrate/product,enhancing the activity of the enzyme and the biocompatibility of the carriers enhanced the stabilities of the enzyme.The details were summarized as follows:Firstly,an inverse replication method based on porous CaCO₃ templates was developed to fabricate porous magnetic polymer microspheres（PMMSs）composed of biocompatible polydopamine and magnetic Fe₃O₄ nanoparticles.The preparation procedure involved the synthesis of Fe₃O₄@CaCO₃ templates,the infiltration and spontaneous polymerization of dopamine in template pores and finally the mild removal of templates.The particle size,surface morphology and pore structure（e.g.,average pore size,pore volume,surface area,etc.）of porous PMMSs were facilely tailored by altering the templates.The as-prepared polydopamine microspheres PMMSs were applied to covalently immobilize YADH for catalyzing the conversion of formaldehyde to methanol.In comparison to the enzyme-conjugated PDA-coated Fe₃O₄ nanoparticles（PMNPs）,the immobilized enzyme upon porous PMMSs exhibited remarkably enhanced activity（specific activity: 162.3 U·mg-1 enzyme vs.97.6 U·mg-1 enzyme;methanol yield: 95.5% vs.57.1%;initial reaction rate: 0.15% s-1 vs.0.08% s-1）,and desirable thermal/pH/recycling/storage stabilities,and particularly,easy separation from the bulk solution by an external magnetic field.Secondly,a facile coordination-enabled self-assembly of tannic acid（TA）and titanium（IV）bis（ammonium lactate）dihydroxide（Ti-BALDH）was adopted to confer natural-derived porous chitin microspheres with surface multifunctionality.Porous chitin microspheres with an average diameter of 111.5 μm and a porous architecture were fabricated through thermally induced phase separation method.Then,the porous chitin microspheres were conferred with surface multifunctionality through facile coordination-enabled self-assembly of tannic acid（TA）and titanium（TiIV）bis（ammonium lactate）dihydroxide（Ti-BALDH）.The multipoint hydrogen bonds between TA and chitin microspheres conferred the TA-TiIV coating with high adhesion capability to firmly adhere to the surface of the chitin microspheres.In view of the biocompatibility,porosity and surface activity,the multifunctional chitin microspheres were used as carriers for enzyme immobilization.The enzyme-conjugated multifunctional porous microspheres exhibited high catalytic performance（102.8 U·mg-1 yeast alcohol dehydrogenase）.Besides,the multifunctional chitin microspheres also found potential applications in the catalytic reduction（e.g.,reduction of silver ions to silver nanoparticles）and efficient adsorption of heavy metal ions（e.g.,Pb2+）taking advantages of their porosity,reducing capability and chelation property.Thirdly,one-pot method was developed to fabricate the porous composite microspheres composed of biocompatible chitin and shellac.Chitin and shellac were dissolved at specific condition.Then the porous composite microspheres were fabricated by the thermally induced phase separation.The effect of the content of shellac on the formation and porosity of the porous composite microspheres were studied.The activities and stabilities of immobilized enzyme were markedly enhanced.Specially,compared to YADH immobilized on chitin microspheres（54.5 U·mg-1 yeast alcohol dehydrogenase）,YADH immobilized on the porous composite microspheres showed enhanced specific activity（128.2 U·mg-1 yeast alcohol dehydrogenase）.