264 results about "Immobilized enzyme" patented technology

The invention discloses a high-stability hydroxysteroid dehydrogenase mutant, a co-immobilized enzyme construction method and application. According to the invention, a site-directed mutant library is constructed based on computer-aided design, and mutants with significantly improved thermal stability and greatly improved catalytic efficiency compared with wild type mutants are obtained through screening; meanwhile, a co-immobilized enzyme construction method based on electrostatic adsorption and chemical crosslinking is established, the hydroxysteroid dehydrogenase mutant and lactic dehydrogenase are co-immobilized, the co-immobilized enzyme can still keep high activity under high substrate concentration, and the conversion rate is still maintained to be 90% or above after 30 batches of co-immobilized enzyme are repeatedly used. And an important foundation is laid for industrial application of the hydroxysteroid dehydrogenase.

The invention discloses an enzyme immobilization method based on charge directed crosslinking, and belongs to the technical field of enzyme immobilization. The method sequentially comprises the following steps: reacting an enzyme with a modifier containing an anionic group to generate an electronegative modified enzyme; dissolving a protonated amino carrier in an acidic buffer solution to form a first solution, and dispersing a dissociation anion group-containing carrier and a modification enzyme in an alkaline buffer solution to form a second solution; mixing the two solutions, and constructing a gel network immobilized enzyme through electrostatic crosslinking; and finally, strengthening the gel network by using a multivalent metal ion solution. Directional anchoring of the enzyme in the gel is achieved through charge matching, the network stability and the enzyme microenvironment are synchronously optimized in combination with metal ion coordination, use of a covalent cross-linking agent is avoided in the whole process, conformation damage of an enzyme active center is avoided, the activity retention rate, the operation half-life period and the substrate mass transfer efficiency of the immobilized enzyme are remarkably improved, and the immobilized enzyme has good application prospects. The method is suitable for efficient immobilization of industrial enzymes such as beta-glucosidase, and has high biocompatibility and industrial potential.

The invention discloses an SAM hydrolase mutant and application thereof, the specific activation effect of halogen anions (F <->, Cl <->, Br <-> and I <->) on SAM hydrolase (containing MJ1651 wild type and mutant) is found for the first time, and the active sequence of the SAM hydrolase is F <-> > I <-> > Br <-> = Cl <->. According to the method, 400 mM of halide salt is added into a reaction system, so that the catalytic efficiency is improved in a breakthrough manner; the yield of wild type enzyme adenosine is improved by 20-300% (optimally, an F-system); the yield of the mutant R89C is soaring up by nearly 6 times under the action of KF; the activity of the immobilized enzyme is still more than 90% after the immobilized enzyme is repeatedly used for 5 times. The method is easy and convenient to operate, green production is achieved with water as the solvent (three wastes are reduced by 90%), the comprehensive cost is reduced by 41%-80 / kg, and an efficient solution is provided for industrial biological manufacturing of antiviral drug intermediates and the like.

Recipe for a double microencapsulated powder, characterized in that the recipe comprises the following components in parts by weight: 5-60 parts by weight of core material of group A, 5-40 parts by weight of core material of group B, 5-16 parts by weight of protein shell material, 3-19 parts by weight of polysaccharide shell material, 0.1-2 parts by weight of immobilizing enzyme, 1-10 parts by weight of filler, 1-4 parts by weight of pH regulator and 0-2 parts by weight of antioxidant.

The invention discloses a nicotinic acid immobilized enzyme conversion method, which comprises the following steps: taking wet thalli containing nitrilase, preparing a flocculation clear liquid, passing the flocculation clear liquid through an ultrafiltration membrane, carrying out embedding immobilization to obtain embedded particles not greater than 2mM, and after the embedded particles are hardened, separating and purifying the obtained conversion liquid by adopting a membrane separation method to obtain the nicotinic acid immobilized enzyme. Sequentially carrying out microfiltration, activated carbon decoloration, ultrafiltration, nanofiltration decoloration, acidification crystallization and centrifugation to finally obtain a nicotinic acid finished product. The immobilized enzyme prepared by the method disclosed by the invention can stably exist in nitrile substances, can be repeatedly used for many times, and is high in stability and low in enzyme activity loss rate, and a separated and purified product is free of impurities and high in yield.

The invention relates to the technical field of enzyme engineering, and particularly discloses a preparation method of immobilized sucrose phosphorylase (SPase) with low cost and high stability. According to the method, epoxy resin is synergistically modified through gallic acid (GA) and a polyethyleneimine-chitosan copolymer (PEI-CS), a covalent bond-hydrogen bond-ionic bond ternary cross-linked network is formed, and efficient immobilization of SPase is achieved. The preparation method comprises the following steps: preparation of a PEI-CS copolymer, preparation of a GA-PEI-CS modification liquid, modification treatment of epoxy resin, immobilization of SPase and determination of enzyme activity. The prepared immobilized enzyme has high enzyme activity retention rate (96.5%), wide temperature and wide pH stability (high activity is kept at 60 DEG C and pH 4.0-10.0), long reuse life (82.1% of enzyme activity is kept after 50 times of reuse) and low cost (26.7 yuan / kg), and can be widely applied to the fields of food industry, cosmetic industry and biological pharmacy.

The invention belongs to the technical field of natural product extraction and enzyme engineering, and particularly relates to a method for extracting red pomelo polyphenol based on an enzyme method. The method aims at solving the problems of high cost and the like caused by easy inactivation and difficult recovery of enzymes in most red pomelo extraction processes. Waste such as pericarp and pomelo seeds generated by red pomelo processing are used as carbon sources, a biochar material with a hierarchical porous structure is prepared through programmed heating carbonization activation, pectinase, cellulase and naringinase are immobilized by adopting a spatial zoning sequential immobilization strategy and then react with the red pomelo, and the biochar material with the hierarchical porous structure is prepared. The immobilized enzyme is rapidly recovered through magnetic separation to realize repeated utilization of the enzyme, and finally red pomelo polyphenol powder is obtained through multi-step treatment, so that recycling is realized, and the yield of polyphenol extracted by an enzyme method is improved.

The invention relates to the technical field of bioengineering, in particular to a process for continuously producing nicotine based on immobilized enzyme. The immobilized microbial agent comprises a composite carrier, an engineering strain and an immobilized auxiliary agent, in the process for continuously producing nicotine based on immobilized enzyme, amine oxidase of an engineering strain is subjected to site-specific modification, so that spatial structure deformation when the amine oxidase is combined with a carrier is reduced, and a three-dimensional network structure of hydroxyethyl chitosan-guar gum composite microspheres in the composite carrier is matched to provide an adaptive microenvironment; the activity loss of enzyme molecules caused by steric hindrance or structural change is reduced; in the immobilization auxiliary agent, gallic acid and an enzyme molecule sulfydryl form a coordinate bond to maintain an active center conformation, glutaraldehyde and polyethyleneimine enhance the binding stability through a crosslinking effect and charge regulation, and the maintaining effect of enzyme activity during continuous production is improved, so that the enzyme activity loss in the immobilization process is reduced, and the immobilization efficiency is improved. The stability of the enzyme in long-term continuous reaction is enhanced.

A method for immobilizing beta-glucanase based on a rice husk-sodium alginate composite carrier belongs to the field of enzyme engineering and biological materials, and comprises the following steps: 1) crushing and sieving rice husks; 2) mixing rice husks and sodium alginate according to a mass ratio of 1: 3-1: 6, and performing ultrasonic dispersion and magnetic stirring to form a carrier solution; (3) adding beta-glucanase into the carrier solution, and reacting for 35-50 minutes at the temperature of 30-35 DEG C; and 4) dropwise adding the enzyme-carrier mixed solution into a calcium chloride solution for curing and forming, and washing and drying to obtain the immobilized enzyme particles. By optimizing the ratio of the composite carrier, when the mass ratio of the rice husks to the sodium alginate is 1: 4, the catalytic activity of the prepared immobilized enzyme under unit mass is obviously higher than that of other ratio groups. According to the method, agricultural waste rice hulls are used as main raw materials, the cost is low, the process is simple, the prepared immobilized enzyme is high in activity, and a new way is provided for industrial application of beta-glucanase.

The invention belongs to the technical field of enzyme engineering and gene engineering, and particularly relates to a recombinant immobilized enzyme, an expression gene thereof and application of the recombinant immobilized enzyme in production of macrocyclodextrin. According to the preparation method, SpyTag and SpyCatcher peptides are fused to the N end of isoamylase and the N end of 4-alpha-glucanotransferase respectively, the 4-alpha-glucanotransferase modified by the SpyTag and the isoamylase modified by the SpyCatcher are obtained, and after spontaneous isopeptide bonds are formed between the SpyTag and the SpyCatcher, the self-assembled macrocyclodextrin production enzyme cluster is obtained. The obtained macrocyclodextrin production enzyme cluster forms an enzyme aggregate with a three-dimensional structure under the traditional glutaraldehyde-mediated catalytic action, namely the recombinant immobilized enzyme. The recombinant immobilized enzyme disclosed by the invention can be repeatedly utilized, is simple and convenient to operate, is easy to separate a converted product, and can be applied to industrial production of the macrocyclodextrin.

The invention discloses an immobilized enzyme with a Pickering emulsion stabilizing effect and a preparation method and application thereof. The preparation method comprises the following steps: adding shellac into an alkaline solution, stirring until the shellac is dissolved, preparing an aqueous solution, adjusting the pH value of the solution to 6-8, fully stirring to uniformly disperse shellac particles, and drying to obtain shellac nano-particle powder; the preparation method comprises the following steps: dispersing shellac nano-particle powder in water or a buffer solution, adding EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide), then adding lipase, and carrying out a coupling reaction to obtain the shellac nano-particle immobilized enzyme. The natural nanoparticle immobilized enzyme prepared by the invention not only can stabilize Pickering emulsion, and the emulsion can realize reversible emulsification-demulsification circulation under the stimulation of acid-base or gas, but also can be used for constructing a phytosterol ester efficient synthesis reaction system.

The invention provides a traditional Chinese medicine composition for improving spleen and stomach health and a preparation method thereof. The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 25 to 30 parts of rhizoma dioscoreae, 15 to 20 parts of rhizoma atractylodis macrocephalae, 8 to 15 parts of fructus aurantii, 10 to 15 parts of red dates, 8 to 12 parts of liquorice root, 10 to 15 parts of inulin, 6 to 10 parts of fructus amomi and 5 to 8 parts of cassia twig. The lauric acid modified fenugreek gum (LA-FG) is used as an emulsifier and an immobilized enzyme carrier to construct a water-oil two-phase enzymolysis system, so that the content of effective active ingredients in the traditional Chinese medicine composition, the product stability and the bioavailability are remarkably improved. In addition, the traditional Chinese medicine composition can effectively improve the gastric emptying rate and the small intestine propulsion rate, the level of motilin and anti-inflammatory factors IL-10 is improved, and the effect of improving spleen and stomach health is achieved through multi-target-point synergism. The method successfully breaks through double limitations of a traditional process and an existing scheme, effectively considers volatile oil protection, efficient enzymatic conversion and system stability, and provides a new path for promoting technical upgrading of the traditional Chinese medicine industry.

The invention discloses a method for catalytically synthesizing adenosine triphosphate by using microcrystalline cellulose carrier purification immobilized enzyme, which comprises the following steps: preparing bacterial suspension by using escherichia coli wet thalli and Tris-HCL buffer solution, crushing, centrifuging, and taking supernate to obtain crude enzyme; adding a microcrystalline cellulose carrier and the wet thalli into the crude enzyme liquid, and washing the immobilized enzyme after immobilization to obtain the immobilized enzyme; the method comprises the following steps: adding immobilized enzyme adenosine kinase and immobilized polyphosphate kinase into a substrate system with the volume of 1L according to a weight ratio, and starting a catalytic reaction under the conditions that the pH is 5.5-7.5 and the temperature is 20-40 DEG C; after catalysis is finished, the immobilized enzyme adenosine kinase and the immobilized polyphosphate kinase are subjected to solid-liquid separation with reaction liquid through a Buchner funnel, the immobilized enzyme continues to be subjected to catalytic reaction, and the method for preparing the immobilized enzyme is simple in mode, low in cost, easy to industrially produce, high in stability and capable of being repeatedly used for many times, and products are easy to separate.

The invention belongs to the technical field of organic synthesis, and particularly discloses a preparation method of N-isobutyl-4-chloropyrrolidone, and the method comprises the following steps: adopting an immobilized CAL-B enzyme as a catalyst, carrying out Michael addition in a water-phase buffer system, and synthesizing N-isobutyl-beta-alanine ethyl ester; a water / supercritical CO2 two-phase reaction system is adopted, and N-isobutyl-N-(chloracetyl)-beta-alanine ethyl ester is synthesized through amidation. A hydrotalcite solid base catalyst is adopted, Dieckmann condensation is carried out in a supercritical CO2 / cosolvent system, and N-isobutyl-4-chloropyrrolidone is synthesized. According to the preparation method of the N-isobutyl-4-chloropyrrolidone, the synthesis of the N-isobutyl-4-chloropyrrolidone is realized through three-step reaction, the defects of a conventional method are avoided, and the preparation method has the advantages of easily available raw materials, few reaction steps and high selectivity, and is suitable for industrial production.

The application discloses a heat-resistant magnetic chitosan immobilized enzyme, and relates to the technical field of immobilized enzymes. The immobilized enzyme carrier takes chitosan and sodium alginate as raw materials, utilizes the method of solubility under the freezing condition of an alkaline solution and hydrogen bond rearrangement, and prepares chitosan composite hydrogel which is excellent in mechanical properties and resistant to high temperature, and further prepares the immobilized heat-resistant enzyme by using a chemical crosslinking method. Based on this, the immobilized enzyme preparation method established in the present application is helpful to solve the problems that industrial enzymes are prone to inactivation under high-temperature conditions and are difficult to be continuously used.

This invention discloses a lipase mutant, an immobilized enzyme, and their applications, belonging to the field of bioengineering. The SpL enzyme mutant K193R / I134V described in this invention exhibits a specific activity of 7-ACA that is 1.6 times higher than before the mutation, and its half-life at 45℃, 50℃, and 55℃ is increased by 1.9 times, 2.6 times, and 3.8 times, respectively. Immobilizing the his-tagged SpL enzyme on a hydrophobically modified nickel-doped SBA-15 mesoporous molecular sieve preserves the enzyme's activity and improves its thermal stability and reusability. Using this immobilized enzyme to catalyze the production of D-7-ACA allows for at least 10 reusable batches, maintaining a conversion rate above 99%.

The invention belongs to the technical field of bioengineering, and relates to heparan sulfate defect type matrigel as well as a preparation method and application thereof. The preparation method comprises the following steps: providing natural matrigel; providing immobilized heparinase, wherein the immobilized heparinase can specifically catalyze and degrade heparan sulfate in the natural matrigel; carrying out contact reaction on the natural matrigel and the immobilized heparinase; and after the reaction is finished, separating the immobilized heparinase. According to the preparation method, the characteristics that the immobilized enzyme is convenient to separate and recycle are utilized, the problems that in a traditional solution enzyme method, enzyme residues exist, the reaction is difficult to accurately control, and subsequent purification steps are complex are solved, and a standardized heparan sulfate defective matrigel product which is controllable in component, free of residual enzyme and high in stability is provided. The method has wide application value in the fields of tumor mechanism research, anti-tumor drug screening, tissue engineering and the like.

The invention provides a method for high-throughput screening of an alpha-glucosidase inhibitor from boxthorn leaves based on a multi-shell MOF immobilized enzyme, and aims to overcome the bottlenecks of low throughput, tedious operation, high cost and the like of a traditional screening method. The core process of the method comprises the following steps: preparing a boxthorn leaf sample solution and pre-evaluating the in-vitro alpha-glucosidase inhibitory activity; a multi-shell MOF immobilized enzyme technology is coupled with a UPLC-QTOF-MS / MS technology, so that targeted screening, efficient separation and accurate identification of active components are realized; verifying the alpha-glucosidase inhibitory activity of the target compound; and predicting the binding site of the small molecule compound and the enzyme through a molecular docking technology. The method has the core advantages of high specificity, high sensitivity, high flux, repeated utilization of enzyme receptors and the like by depending on the inherent characteristics of an immobilized enzyme technology, can synchronously screen and identify various inhibitors, effectively integrates the resource advantages of a boxthorn leaf natural product and the high efficiency of a modern high-flux screening technology, and has a wide application prospect. And a key technical support is provided for diversified development of diabetes treatment medicines.

本发明公开了一种基于静电驱动的双酶级联放大荧光传感器及其制备方法和应用，所述纳米酶荧光传感器由探针Azo‑Bodipy 685、胆碱氧化酶（CHO）和氯化乙酰胆碱（ACh）组成。本发明基于静电吸附原理，提出静电络合共固定化‑酶级联反应协同策略，构建一种新的静电驱动荧光传感器，利用ACh的季铵盐正电荷与CHO表面负电荷的静电作用，在特异性荧光探针Azo‑Bodipy 685表面构建纳米尺度空间限域的酶反应微环境。当有机磷农药抑制乙酰胆碱酯酶（AChE）时，氯化胆碱生成减少导致CHO催化产生的过氧化氢量下降，荧光信号强度减弱，一系列荧光强度的变化可以实现检测AChE活性和测定有机磷农药残留含量。

The invention discloses a double-amino sequence functionalized graphene oxide immobilized enzyme as well as a preparation method and application thereof, and relates to the technical field of graphene oxide immobilized enzymes. The method comprises the following steps: grafting polydopamine to the surface of graphene oxide to obtain a GO-coated PDA material; reacting the GO-coated PDA material with an amino functional reagent in a first dispersion system, centrifuging, cleaning and drying to obtain a double-amino sequentially-functionalized graphene oxide material; the double-amino sequentially functionalized graphene oxide material is used for immobilizing a target enzyme; the amino functional reagent is polyethyleneimine and / or polyethanolamine. The preparation process of the double-amino sequence functionalized graphene oxide material provided by the invention is simple, and the process conditions are mild. The immobilized enzyme prepared from the material not only has better reusability, but also has better catalytic performance and stability than free enzyme when being practically used for preparing catalytic reaction products.

The application discloses a multi-enzyme immobilized enzyme for glucose-6-phosphate synthesis, which is obtained by immobilizing three enzymes, starch debranching enzyme mutant AcDBE_E131R, maltodextrin phosphorylase mutant AtMalP_N188D and glucose phosphomutase mutant TkPGM_F360P, on a resin. The application also discloses a preparation method and application of the multi-enzyme immobilized enzyme. The application screens enzyme mutants with good thermal stability and high catalytic activity by site-directed mutagenesis of starch debranching enzymes, maltodextrin phosphorylases and glucose phosphomutases from different sources, and prepares the multi-enzyme immobilized enzyme AcDBE_E131R+AtMalP_N188D+TkPGM_F360P. The multi-enzyme immobilized enzyme is used as a catalyst, maltodextrin is used as a substrate, and glucose-6-phosphate is efficiently synthesized by one-pot method, and the conversion rate reaches 71.2%. After 50 times of reuse, the relative enzyme activity of the multi-enzyme immobilized enzyme still remains 85.1%.

The invention relates to the technical field of biosynthesis, in particular to a preparation method of gamma-aminobutyric acid. The invention provides a method for preparing gamma-aminobutyric acid, according to the method, gamma-aminobutyric acid is catalytically synthesized by using a mutant of glutamate decarboxylase screened by the inventor, the problems of low catalytic efficiency, long conversion time and limited reuse times of enzyme are solved, the immobilized enzyme can be continuously subjected to at least 20 rounds of enzyme catalytic reaction, and the yield of gamma-aminobutyric acid is increased. And moreover, the catalytic efficiency is not obviously reduced, the substrate conversion rate can still be stabilized at 99% or above, and the method is suitable for industrial production.

The invention discloses a high-temperature-resistant beta-glucosidase inclusion body (at) MOF (Metal Organic Framework) catalyst as well as a preparation method and application thereof. The preparation method comprises the following steps: firstly, preparing a catalytic activity inclusion body (TsBgl1 CatIBs) of high-temperature-resistant beta-glucosidase, and then introducing the TsBgl1 CatIBs at the initial stage of synthesis of an iron-based MOF carrier under the conditions of a water phase and good biocompatibility, so that MOF crystals grow around an enzyme aggregate and encapsulate the enzyme aggregate in situ, thereby obtaining the high-temperature-resistant beta-glucosidase. Therefore, the immobilized enzyme catalyst with high enzyme activity retention rate, excellent thermal stability and good recoverability is obtained. The enzyme loading capacity of the TsBgl1 CatIBs coated MIL-100 (Fe) prepared by the invention can reach 12mg / g, and the encapsulation efficiency at the moment can reach 55%. The enzyme activity at 100 DEG C is 92.5% of the optimum enzyme activity of free TsBgl1CatIBs, and the equivalent enzyme activity is still kept after the TsBgl1CatIBs is stored at 4 DEG C for 40 days.

The invention provides a slow-release organic-inorganic compound fertilizer based on synergistic interaction of a carrier immobilized enzyme and a cofactor and a preparation method of the slow-release organic-inorganic compound fertilizer, and belongs to the technical field of organic fertilizers. According to the application, heat-insulation enzyme-loaded particles are added during pile building, a semi-coke-perlite low-heat-conduction framework forms a cold island in a reactor core, and a starch-polypeptide-sodium tripolyphosphate cross-linked membrane is used for firmly immobilizing enzyme, so that heat inactivation is prevented, fibrin is rapidly cracked, free ammonia is adsorbed, and the pile temperature is promoted to rapidly rise; then, dynamic humidity regulating particles are put, reversible water release-water absorption of montmorillonite is combined with glycerin, and a sorbitol hydrophilic film automatically and stably controls water content and supplements easily-degradable carbon, so that over-wet anaerobic or over-dry enzyme loss is avoided; the enzyme cofactor powder added in the later period slowly releases manganese, calcium and copper to serve as enzyme complement, alanyl glutamine serves as a molecular chaperone to repair damaged conformation, and copper and zinc are chelated to passivate heavy metal; the three agents sequentially realize'keep-alive, stable-alive ', prolong the high activity period of enzyme, accelerate lignin degradation, and reduce nitrogen volatilization, odor and heavy metal risks at the same time.

The invention provides immobilized cyclodextrin glucosyltransferase as well as a preparation method and application thereof and a preparation method of ascorbic acid glucoside, and belongs to the technical field of enzymes, the technical field of biological catalysis and the technical field of preparation of chemical raw material medicines. The preparation method of the immobilized cyclodextrin glucosyltransferase comprises the following steps: activating a carrier; mixing the activated carrier with a solution containing cyclodextrin glucosyltransferase, stirring, carrying out solid-liquid separation, and washing to obtain an immobilized enzyme; and mixing the obtained immobilized enzyme with a polyethylene glycol-polyethyleneimine copolymer solution, stirring, carrying out solid-liquid separation, and washing to obtain the immobilized cyclodextrin glucosyltransferase. The immobilized cyclodextrin glucosyltransferase prepared by the preparation method is high in catalytic activity and good in use stability, and when the immobilized cyclodextrin glucosyltransferase is applied to catalytic synthesis of ascorbic acid glucoside, high-content and high-yield ascorbic acid glucoside can be obtained.

The present application relates to the technical field of organic synthesis, in particular to a kind of immobilization method of biological catalyst and application in the synthesis of chiral pesticide.The immobilization method includes the following steps: uniformly mixing sodium alginate solution and sodium carboxymethyl cellulose solution to obtain carrier solution; biological catalyst is dissolved in buffer to prepare enzyme solution, then enzyme solution and carrier solution are fully mixed; mixed solution is added dropwise into calcium chloride solution, and gel microspheres are formed; gel microspheres are added into epichlorohydrin solution for crosslinking, and immobilized biological catalyst is obtained.The present application improves the shortcomings of poor stability, low loading rate and unstable selectivity of traditional immobilized carrier formed by sodium alginate and sodium carboxymethyl cellulose by adding epichlorohydrin for crosslinking, improves the stability of immobilized enzyme, realizes the repeated use of immobilized enzyme, greatly reduces the use cost of enzyme preparation, and is suitable for application and popularization in production practice.

The application discloses a high-stability immobilized mannanase and a preparation method and application thereof, and belongs to the technical field of enzyme immobilization. The preparation method uses PAMAM modified SBA-15 / calcium alginate composite microspheres as a carrier to fix mannanase in an enzyme solution by adopting an embedding-covalent crosslinking method, so as to obtain the high-stability immobilized mannanase. The preparation method uses the PAMAM modified SBA-15 / calcium alginate composite microspheres as the carrier to fix the mannanase by adopting the embedding-covalent crosslinking synergistic method, so that the pH tolerance, the circulation stability and the hypoglycemic efficiency of the enzyme are improved, and the problems of the traditional immobilized enzyme, such as large enzyme activity loss, easy leakage and poor stability under high temperature, are solved.

The invention belongs to the technical field of enzyme engineering, and particularly relates to an immobilized enzyme preparation containing D-psicose-3-epimerase cells as well as a preparation method and application of the immobilized enzyme preparation. The preparation method comprises the following steps: carrying out fermentation culture on genetically engineered bacteria containing thermal-stability D-psicose-3-epimerase to obtain wet thalli, mixing the thalli with egg shells and a diatomite carrier, immobilizing the obtained mixture on the carrier through a cross-linking agent, and carrying out separation, drying and granulation to obtain the immobilized enzyme preparation. According to the invention, the affinity between the immobilized carrier and microorganisms is stronger; the prepared immobilized enzyme preparation is high in mechanical strength and stable in enzyme activity, is used for preparing D-psicose through D-fructose isomerization, and is high in conversion efficiency. Under the condition of 60 DEG C, after the batch reaction is repeated for 10 batches, the conversion rate is still greater than 28%; 700g / L fructose solution is used as a substrate, the reaction is continuously carried out for 31 days, the average conversion rate is 28% or above, and the method has relatively high industrial advantages.

The invention discloses a method for rapidly screening and identifying an alpha-glucosidase inhibitor from stevia rebaudiana by combining a magnetic MOF immobilized enzyme technology with an ultra-high performance liquid chromatography-time-of-flight mass spectrometry technology. The method is based on an immobilized enzyme technology, screening of the alpha-glucosidase inhibitor in stevia rebaudiana is carried out by means of the affinity binding effect of active small molecules and enzyme and the magnetic separation characteristic of magnetic MOF immobilized alpha-glucosidase, and structural identification of an active compound is carried out by applying an ultra-high performance liquid chromatography-time-of-flight mass spectrometry technology. And finally, verifying the in-vitro alpha-glucosidase inhibitory activity of the screened active compound, and simulating the binding information of the two compounds by means of a molecular docking technology. The method provided by the invention has the characteristics of rapidness, high efficiency, accuracy, easiness in separation from a reaction system and reutilization of the target receptor immobilized alpha-glucosidase, is suitable for screening the alpha-glucosidase inhibitor from complex systems such as traditional Chinese medicines and natural products, and has a wide application prospect.