Enzyme Immobilization And Enzyme Activity - Fluorescence Analysis

Enzymes, as biological catalysts, have high catalytic activity and selectivity. With the development of scientific technology, enzyme is employed with a growing application. Due to the low stability under the condition of high temperature, organic solvent and harsh pH, enzyme can hardly be widely used in the industry. Moreover, it is hard to separate dissolved enzyme from the system, and the cost to use is raised. Immobilized enzyme is designed to solve the problem. Immobilized enzymes have many advantages, including high catalytic efficiency, specifity, easy recycling, reusability, consecutive operation, easy preparation and enhanced stability. With the above mentioned advantages, immobilization will be a promising method for further application of enzymes.As a class of zero-dimensional graphitic nanomaterials less than 10 nm, graphene quantum dots (GQDs) exhibiting fluorescence have recently aroused increasing interest. Compare to two-dimensional graphene nano sheet and one-dimensional graphene nanoribbons, GQDs exhibit larger BET area, higher carrier mobility, better mechanical flexibility, stronger chemical durability, and more environmental protection. Due to quantum confinement and edge effects, GQDs is arousing the interest of subjects like chemistry, physics, materials and biology. Today, GQDs is increasingly used in biological medicine, photoelectric device of solar power, luminous diode and enzyme sensors.Amino PGMA/EDMA beads is synthesized and characterized with SEM and mercury porosimetry. Two different methods are used in immobilization of a-amylase and (3-glucosidase onto the beads, then optimized conditions of immobilization and stabilization of immobilized enzyme is studied. AChE and a-glucosidase are immobilized onto GQDs surface by crosslinking of EDC/NHS. Then fluorescence nanosensor is established for enzyme assay and inhibitor screening. The main research contents and conclusion are shown as follows:1. Mono-disperse method is used in the preparation of PGMA/EDMA beads. After amination with ethanediamine and activation with glutaraldehyde, PGMA/EDMA beads showed commendable a-amylase immobilization capacity of 35.1 mg/g carrier. The optimized condition and stability is studied. Compared with free form, immobilized a-amylase had increasement of 12.94 mg/mL for Km and 0.124 mmol mL-1 min-1 for vmax, improved acid resistance (the optimal pH from 7 to 5), presented better thermal stability by retaining 61% activity than 40% at 90℃, and displayed high operational reusability by retaining 58% of its initial activity after nine uses. Moreover, less than 10% of the free enzyme and more than 80% of the immobilized enzyme retained activity after 180 min pre-incubation at 50℃.2. Cross-linked enzyme aggregates β-glucosidase (CLEAs BGL) is immobilized through adsorption, precipitation and crosslinking process on nano-sized carrier for potential application in biofuel production with immobilization conditions optimized. The Km and vmax of CLEAs BGL are obtained as 7.67 μM and 0.639 μmol mL-1 min-1. Compared with soluble form, CLEAs BGL showed better affinity to the substrate, had the optimal pH shift from 5 to 5.5 and optimal temperature shift from 50 ℃ to 60 ℃, presented better thermal stability by retaining more than 80% activity at 70 ℃, and displayed high operational reusability by retaining more than 90% of its initial activity after nine uses. Maximum cellobiose hydrolysis by CLEAs BGL was achieved in 1 h, which was much quicker than 4 h of soluble form. The impressive performance of CLEAs BGL in the present study indicate a promising way for food industry and potential bioethanol production.3. Acetylcholinesterase (AChE) is immobilized onto GQDs through the cross-linking effect of EDC/NHS with the intensity of GQDs enhanced. Hg2+is employed to quench reagent, then the enhanced fluorescence of GQDs could be effectively quenched. Thiol compounds released by acetylthiocholine iodide (ATCh) under AChE catalytic hydrolysis could interact with Hg2+ through the formation of Hg-S bonds, GQDs-Hg2+ diassociated, resulting in the fluorescence recovery of GQDs. The fluorescence recovery ratio of GQDs was proportional to the dose of AChE. According to fluorescence "turn-on-off" mechanism, a fluorescence nanosensor for AChE detection is established, and an analytical method for detecting AChE was constructed with a linearity range of 10-5～10-2 U and a limit of detection of 2.3×10-4 U. In addition, two traditional AChE inhibitors were employed to verify the feasibility of the system. Because of the enhanced fluorescence and lower amount of GQDs, lower IC50 values of 63.76 and 14.07 nM were obtained for paraoxon and tacrine, respectively. The developed protocol provides a new and promising platform for assaying AChE activity and screening its inhibitors with low cost and high sensitivity.4. Following the optimized condition of fluorescence enhancement, a-glucosidase was immobilized onto GQDs by cross-linking effect of EDC/NHS. With the addition of substrate pNPG, the fluorescence of GQDs was quenched by released pNP. With a linearity between quenching ratio and dosage of a-glucosidase, an analytical method for detecting a-glucosidase was constructed. Finally, acarbose was employed as model inhibitor to inhibit the enzyme activity. GQDs fluorescence nanosensor provides a highly sensitive assay for AChE activity and potential screening for its inhibitors.