High Expression Of Glucose Oxidase In Pichia Pastoris And Its Preparation For Enzyme Electrode

Glucose Oxidase?GOx?is an important enzyme involved in the process of carbon metabolism.It catalyzes glucose oxidation to gluconic acid and hydrogen peroxide with high efficiency and specificity.Glucose and gluconic acid are widely used in food and medical industry.In diagnosis,glucose concentration is often used as a human heath indicator.Gluconic acid is used as a food flavoring agent to regulate the taste,and its salt products are used as health products to supply human with trace ions.The aim of this study is to achieve the high expression of glucose oxidase using a Pichia pastoris expression system.There are two ways to achieve the high expression of glucose oxidase in Pichia pastoris.First,We can use G418 to select Multi-copyies strains in Pichia pastoris.Then,We can use pPICZ?A to select Multi-copyies strains in Pichia pastoris.Furthermore,a glucose biosensor was fabricated using glucose oxidase and TiO₂ nanocrystals for rapid detection of glucose.The main research contents were as follows:?1?High glucose oxidase expression system in Pichia pastoris1)Multi-copy selection of glucose oxidase in Pichia pastoris by G418In this chapter,the glucose oxidase gene obtained from Aspergillus Niger was used to construct the pPPIC9k-GOx recombinant vector,and then transferred to GS115 yeast by using Sac I site linearization.Recombinants were screened by MD plates and then the recombinants will be plated on YPD plates containing geneticin G418 for multiple-copies selection.G418 was selected as geneticin resistance screening reagent,and eleven G418concentration gradients of 0 mg/mL,0.25 mg/mL,0.5 mg/mL,0.75 mg/mL,1.0 mg/mL,1.25mg/mL,1.5 mg/mL,1.75 mg/mL,2.0 mg/mL,3.0 mg/mL,4.0 mg/mL were set to performmultiple copies of Pichia pastoris screening.After 3-5 days of culture,the transformants were randomly selected to select 21 strains of single colony with better growth in G418 plates.The recombinant yeasts were selected to be used for the determination of crude enzyme activities after 120 h culture.By using fluorescence quantitative PCR?q-PCR?method to determine the gene copies of recombinant yeast,we successfully obtained four copies GOx recombinant.The results showed that enzyme activity of four-copy recombinant was the highest and the enzyme activity was 363 U/mL for 120 h culture.2)Multi-copy selection of glucose oxidase in Pichia pastoris by pPICZ?AIn this chapter,we chose pPICZ?A as the expression vector,constructed one to six copies in vitro using BamH I and BgI II restriction enzyme,and then screened the GOx multi-copy recombinants using YPDZ plates.The results showed that in a certain range of copy number increases easily in X33 endocrine expression.Beyond this range,the higher the number of copies increased,the higher the enzyme activity decreased.All copies of the strains were in methanol-induced culture after 120 h.We found that four copy-reconbinant was the highest and enzyme activity was 350 U/mL.?2?Preparation and characterization of TiO₂ nanocrystals glucose oxidase electrodeGlucose electrochemical biosensors are a fast and reliable method of detecting glucose concentration.Nanomaterials have been widely used in the modification of glucose electrode to enhance electron transfer efficiency and effective on enzyme active center.In this research,TiO₂ nanocrystals with inherent peroxidase activities were firstly found synthesized by an alcohol-thermal method.And the synthesized TiO₂ nanocrystals were further explored for glucose electrochemical sensing.This TiO₂ nanocrystals facilitates direct electron transfer from hydrogen peroxide to the electrode.The strong interaction between GOx and TiO₂nanocrystals greatly improves the stability during the substrate reactions sequentially.The TiO₂ nanocrystals and glucose oxidase were immobilized on the surface of Glassy Carbon Electrode?GCE?by physical adsorption method,and the nanocrystal TiO₂ glucose oxidase biosensor?Nafion/TiO₂/GOx/GCE?was made by covering the surface with perfluorosilsulfonic acid?Nafion?film.The linear range of glucose by this sensor are 0.5-30mmol/L respectively,and the detection limits is 6.3×10^-5 mol/L?S/N=3.0?.The results showed that nanocrystal TiO₂ glucose oxidase biosensor?Nafion/TiO₂/GOx/GCE?can maintain the biological activity of glucose oxidase and promote its redox reaction to glucose.It has good selectivity for glucose detection and can be used for rapid detection of glucose concentration.