Study Of Enzymatic Kinetics Based On Capillary Electrophoresis-based Immobilized Enzyme Microreactor

As in the1960’s, immobilized enzyme technology has unique advantages in the study ofenzyme analysis and attracted widespread attention. Enzyme immobilization technology bythe solution of the free enzyme combined with reaction system incompatible solid materials,the enzyme is fixed in the enzymatic reaction in a certain area to make it happen. Comparedwith the free enzyme, immobilized enzyme has shown several unique advantages, such asimproving enzyme stability, improve the enzyme reaction activity and repeat utilization, isadvantageous to the separation of substrate and product, etc. Immobilized enzyme reactorusing enzyme immobilization technology has become a research hotspot in recent years.Immobilized enzyme reactor not only set all the advantages of the immobilized enzyme, alsoto avoid the manual handle the trouble of the sample to reduce the likelihood of enzymesamples contaminated, in fermentation industry, biological medicine and biochemicalresearch is becoming more and more widely used.Capillary enzyme microreactor is capillary enzyme fixed matrix of an enzyme reaction device.Quartz capillary of bioactive molecules with a biological compatibility, and easily by physicaland chemical bond and so on the many kinds of way and the enzyme. At the same time, thecapillary also can be used as a separate analysis of channel, the enzyme reaction substratesand enzymatic product test on-line analysis. In addition, because the inner diameter of thecapillary is small, greatly reduces the consumed in the process of preparation of the dosage ofenzyme and substrate, especially for rare and expensive biological samples, greatly reduce thecost of the preparation of the enzyme reactor.For this paper， Graphene Oxide, as a support, use the large specific surface area, enzymecontent, good stability characteristics, through the layers by layer method immobilized on theinner wall of the capillary. And prepared two new capillary microreactor. The results provedthat the enzyme preparation by capillary reactor analysis of enzyme preparation is simple, notonly easy to operate, but also has good specificity, high stability, good enzyme catalyticactivity.The first part of work, we GO as a support to negatively charged will positively chargedTrypsin through Layer by Layer self-assembly (Layer by Layer) method and immobilizedon the inner wall of the capillary, preparation of a new type of capillary Trypsin enzymereactor. GO to the negatively charged a two-dimensional structure of nano materials,combining the characteristics of its large specific surface area, increase the load of positivelycharged trypsin and the stability of the electrostatic adsorption. By optimizing the preparationof capillary electrophoresis analysis of capillary enzyme reactor and enzyme reaction experiment conditions, system to study the performance of the enzyme reactor, thedetermination of the activity of the enzyme reactor, stability and reproducibility, etc. Underthe best conditions, the enzyme reactor to achieve the standard sample of peptides andproteins hydrolysis separation and analysis, online hydrolyzate characterization analysis bymass spectrometry. Online of BSA protein hydrolysis reaction (incubation time is only30minutes) and offline hydrolysis reaction of hatch (incubation time12h) mass spectrometrycharacterization results, show that the method has good application prospect in proteinanalysis.Further work, we were prepared to GO as a support of the negatively charged LDH capillaryenzyme reactor. Also use the Layer by Layer method of LDH successful immobilized on theinner wall of the capillary, prepared new type of capillary LDH enzyme reactor. The resultsshowed that the GO large specific surface area, strong function and hydrogen bonding,hydrophobic can steadily immobilized negatively charged LDH enzyme molecules. Weoptimize the preparation of capillary electrophoresis analysis of capillary enzyme reactor andenzyme reaction experiment conditions, system to study the performance of the enzymereactor, the determination of the activity of the enzyme reactor, stability and reproducibility,etc. Using the LDH enzyme microreactor, analyse the concentration of pyruvic acid in beer.The success of the enzyme reactor preparation and application, proving that the multipleinteractions between GO and enzymes (electrostatic adsorption, hydrophobic effect andhydrogen bonding interaction, etc.), and expand the application range of the LBL method inimmobilized enzyme method.