Surface Modification Of Halloysite Nanotubes For Immobilizing Laccase

Halloysite nanotubes(HNTs)have a special tubular structure,good biocompatibility and stable physical and chemical properties,so there is considerable potential in the field of biological enzyme immobilization.HNTs have been studied in terms of dye adsorption,phase change materials,biosensing,drug release and preparation of composite materials,but the systematic research in the field of enzyme immobilization is not perfect.Researchers at home and abroad generally pay attention to the adsorption of immobilized enzyme in Lumen of HNTs,but the space of Lumen is limited and HNTs are prone to agglomeration,leading to the increase of mass transfer resistance and the low amount of immobilized enzyme.In view of this,this paper focuses on improving the dispersibility of nanotubes and improving the loading of the immobilized enzyme.In this paper,the surface modification of HNTs was carried out by two methods.Roughed HNTs with defects(RHNTs)and positive charged HNTs(PHNTs)were prepared and then study the immobilized enzyme properties of these carriers.In order to broaden the industrial application of biological enzymes,improve the recovery of fixed enzymes and batch use,this paper prepared micron-sized porous alanite composite microspheres and millimeter-grade porous alanite composite particles.Both the lumen of HNTs and the polymer network structure of the sample,providing the mass transfer channel and binding site for enzyme,and providing good solid buffer protection for the subsequent enzymatic reaction.This paper selected laccase as a research object.The main contents are as follows:(1)Preparation of RHNTs and study on the performance of immobilized enzymes:HNTs have a smooth outer surface with weak adhesion and less crystal defects,which make it hard to bind with the amino residues.Activated by high temperature activation and selective corrosion,surface modification of nanotubes was investigated for the first time by NaNO3/Na2CO3 molten salt system.Na2CO3 in the molten state corrodes the silica of Halloysite.So,the controllable corrosion modification of the outer surface of the HNTs can be achieved by regulating the ratio of HNTs: NaNO3:Na2CO3.RHNTs were successfully prepared with reduced organic content,surface roughness and lattice defects in the outer surface.The results showed that RHNTs maintain a complete tubular structure with a rough wall and a significant improvement in dispersibility.This showed that the surface groups and lattice defects have increased in varying degrees.The amount of laccase immobilized on RHNTs was 37.55 mg/g,which was higher than that of HNTs(21.46 mg/g).Comparison of free laccase,pH tolerance,temperature tolerance and thermal stability of immobilized enzyme on RHNTs are slightly improved.The results indicated that the change of HNTs is conducive to improving the amount of enzyme loading and the stability of immobilized enzyme.(2)Preparation of PHNTs and study on the performance of immobilized enzymes:Polyallyl dimethyldimethylammonium chloride is an environmentally friendly cationic polyelectrolyte that can attract HNTs electrostatically.PHNTs with positive charge and increased charge density were prepared by changing the PDDA concentration and reaction time.The results showed that PDDA has successfully covered the surface of HNTs by electrostatic attraction to form uniformly deposited PDDA nanofilm.Compared with free enzyme,the immobilized laccase exhibited enhanced pH and temperature tolerance and thermal stability.Besides,after tenth cycles of continuous use,the activity of immobilized laccase remained above 50 %.Also,immobilized laccase was used to remove 2,4-DCP,resulting in 81.66 %removal efficiency in 6 h by adding redox mediator ABTS.It is expected that PHNTs could be of great potential as a promising support material for biomacromolecule immobilization.The results show that the positive charged PHNTs with higher charge density and increased active groups provided enough binding sites for enzyme loading.The immobilized enzyme on PHNTs also has a good performance in terms of reusability.(3)Preparation of dopamine biomimetic composite microspheres and study on their immobilized enzyme properties: The composite microspheres with 3D architecture were prepared by using HNTs and chitosan as raw materials and oleic acid droplets as the soft template.Then,washed the soft template with ethanol,and strengthened the microsphere structure by dopamine.The thermal stability of themicrospheres was also improved by the self-polymerization of dopamine.The results showed that the composite microspheres had a large specific surface area(114.6m2/g),and the surface was successfully grafted with o-quinone,catechol and amino groups.The activity of the immobilized enzyme was higher and free enzyme loading on the microspheres was evenly distributed.The porous microspheres showed excellent loading capacity for laccase immobilization as high as 311.2 mg/g,which is higher than that of the same literature.The thermal stability,storage stability and recyclability of the immobilized enzyme on composite microsphere have also been significantly improved.After 50 days of storage,the immobilized enzyme retained75 % of the initial enzyme activity.The degradation of organic pollutants by immobilized enzymes is rapid and effective.The degradation rate of 2,4-DCP by immobilized enzyme can reach up to 62 % after 1 h.Thus,dopamine bionic composite microspheres are suitable for enzyme immobilization.The good operational stability and reusability of the microspheres also indicated the potential for industrial applications.(4)Preparation of polyvinyl alcohol composite beads and study on their immobilized enzymes: Polyvinyl alcohol(PVA)has its uniform and ordered pore structure and good biocompatibility,often used as an enzyme immobilized carrier.Enzymes are immobilized on PVA by hydrogen bonding between hydroxyl groups on the surface of PVA and amino groups of free enzyme.However,the thermal stability of PVA is poor.Proper amount of HNTs can improve the mechanical strength and thermal stability of the composites.It is shown that HNTs are ordered along the network of PVA.Composite beads have an open pore structure of micron holes and nanopores.The composite beads showed good loading capacity for laccase immobilization as high as 237.02 mg/g,which is higher than that of HNTs(21.46mg/g).Immobilized laccase on PVA/HNTs composite particles were significantly improved in pH tolerance,temperature tolerance,thermal stability and storage stability.In the immobilized laccase mediator system,the removal rate of non-phenolic compound by immobilized laccase can reached 93.41 % for 4.5 h and had good reusability.It shows that PVA/HNTs composite beads are an excellent carrier for enzyme immobilization,and have broad industrial application prospects inthe field of reactive dye degradation.