Preparation And Properties Of Immobilized Pectinase Support

Development and application of the immobilization of enzymes are important fields in biotechnology. Enzyme immobilization offers advantages over free enzymes in choice of batch or stability, continuous processes, reusability of catalysts, controlled product purity and process effect, ease of enzyme survival in the reaction mixture and adaptability to various environment designs. Preparation of perfect immobilization enzyme demand both effective immobilized technology and ideal supports, therefore, fabrication of choiceness support materials based on modern biotechnology is an important task in this field.In this paper, a great variety of composite materials designed for the purpose have been introduced and discussed which were elaborated by the layer-by-layer assembly technique alternating deposition of synthetic polymers and mineral. Preparation of new kind of support materials through combining nanotechnology and bio-immobilized technology offer the immobilized enzyme a biofriendly microenvironment and improved activity. The relative experiments and results are summarized as follow:1. Well-defined amphiphilic block copolymers poly (styrene-b-acrylic acid) (PS-b-PAA) with controlled block length were synthesized using atom transfer radical polymerization (ATRP). Pectinase enzyme was immobilized on the well-defined amphiphilic block copolymers PS-b-PAA. The carboxyl groups on the amphiphilic PS-b-PAA diblock copolymers present a very simple, mild, and time-saving process for enzyme immobilization. Various characteristics of immobilized pectinase such as the pH, thermal stability and storage stability were valuated. Among them the pH optimum and temperature optimum of free and immobilized pectinase were found to be pH 6.0 and 65℃.2. Up to now, using the silica-coated chitosan support from layer by layer approach for pectinase immobilized has not been reported to our best know. In this study, pectinase was immobilized onto a new type of silica-coated chitosan support from layer by layer approach. Various characteristics of immobilized pectinase such as the pH stability, thermal stability and storage stability were valuated. The immobilized pectinase revealed acceptable pH stability over a broad experimental range. The colloidal stability is not impeded by the adsorbed proteins despite the fact that up to 247.8 mg of enzyme is adsorbed per gram of the carrier particles. The activity of immobilized pectinase adsorbed onto these particles is analyzed in terms of the Michaelis-Menten parameters. The Michaelis constant K_m differs only slightly from the K_m value of the native enzyme when the amount of adsorbed enzyme is raised despite the high local concentration of immobilized enzymes.3. The idea of preparing the surface of chitosan layer pre-absorbed on the "spherical polyelectrolyte brush" via layer by layer approach receives special relevance in immobilized enzyme technology. The colloidal stability was not impeded by the adsorbed proteins despite the fact that up to 316.8 mg of enzyme was adsorbed per gram of the cartier particles. The immobilized pectinase revealed acceptable pH stability over a broad experimental range of 3.0～4.5. The activity half lives for native and bound states of enzyme were found as 13.5 d and 30 d, respectively. Enzyme activity was found to be approximately 49.7% for immobilized enzyme after storage for 1 month.4. The idea of preparing the surface of Fe₃O₄ layer pre-adsorbed on the "spherical polyelectrolyte brush" via layer by layer self-assembly approach receives special relevance in enzyme technology. The silica-coated (PEI/Fe₃O₄)_n support presents a very simple, mild, and time-saving process for enzyme immobilization. These particles, premodified with the layer of magnetic nanoparticles to impart a magnetic property were repeatedly used as catalysts following their rapid and easy separation with a magnet.