23 results about "Multifunctional Enzymes" patented technology

The invention discloses a CRISPR (clustered regularly interspaced short palindromic repeats)-based homogeneous chemiluminescence kit as well as a preparation method and a use method thereof, and belongs to the technical field of nucleic acid detection. The kit comprises a Cas enzyme, crRNA, a chemiluminescence reporter molecule, a free radical scavenger and hydrogen peroxide. The chemiluminescent reporter molecule comprises a reporter molecule modified with a chemical group for coupling, one end of the reporter molecule is connected with a peroxidase-like catalytic activity center for reacting with peroxide to generate active free radicals, and the other end of the reporter molecule is connected with a luminescent substrate for reacting with the active free radicals to generate luminescence. According to the kit, a multifunctional microplate reader can be used for collecting chemiluminescence signals for quantifying the concentration of target nucleic acid. The kit is simple in operation steps and short in detection time, does not need light source excitation, and avoids self-luminous interference of a biological sample matrix, so that the kit has more excellent sensitivity and detection dynamic range.

The invention discloses an enzyme-inspired hydrogen bond organic framework material as well as a preparation method and application thereof. The enzyme-inspired hydrogen bond organic framework material is formed by self-assembling Py-4DAT monomer molecules through hydrogen bonds. The multifunctional enzyme inspired hydrogen bond organic framework material is synthesized through bionic design, efficient capture, rapid detection and self-driven degradation of a nerve poison simulant DCP are achieved, and the bottleneck of a traditional porous material in the aspects of selective recognition and multifunctional synergy is broken through; the FDU-HOF-5 material has high stability, solution processability and real-time response capability, a functional textile prepared by loading the FDU-HOF-5 material can realize instant visual detection of DCP, a new material and a new strategy are provided for intelligence of chemical protection equipment, and the FDU-HOF-5 material has important application value in the field of chemical threat prevention and control.

The invention discloses a thioredoxin reductase detection method, which comprises the following steps: preparing a hairpin DNA probe HP solution, an H1 solution and an H2 solution, and carrying out annealing treatment; a pretreatment hairpin type DNA probe HP solution, a pretreatment hairpin type DNA probe H1 solution and a pretreatment hairpin type DNA probe H2 solution are obtained; mixing the three pre-treated hairpin DNA probe solutions, adding an NADPH solution and a sample to be detected, and incubating at 37 DEG C to obtain a solution to be detected; and collecting a fluorescence signal of the to-be-detected solution through a multifunctional microplate reader, and calculating the concentration of thioredoxin reductase through a linear equation: Y = 730.7 X + 2000.2, Y representing the fluorescence signal and X representing the logarithm LogCTrxR of TrxR concentration. According to the method disclosed by the invention, the detection sensitivity of the TrxR is remarkably improved, and the method is applied to imaging of the TrxR; the method has the advantages of good selectivity, high sensitivity, real-time monitoring and the like.

1. The name of the design product: multifunctional enzyme label instrument. 2. The use of the design product: for nucleic acid, protein concentration, enzyme activity analysis and other conventional molecular detection; signal transduction research, cell signal event active oxygen, modification detection; cell viability, apoptosis, killing and other overall level analysis. 3. The design points of the design product: in shape. 4. The picture or photo that best indicates the design points: perspective drawing.

The invention discloses a high-flux toxicity pre-evaluation substitution method based on an acetylcholin esterase target. By integrating an SH-SY5Y nerve cell endogenous AChE and recombinant exogenous AChE dual detection system and relying on an automatic screening system constructed by integrating core equipment such as an automatic pipetting workstation, a constant-temperature incubator and a multifunctional microplate reader, a high-flux toxicity pre-evaluation substitution method based on an AChE target is successfully established and is used for evaluating the neurotoxicity of a compound, and the method has the advantages of high sensitivity, high sensitivity, high sensitivity and the like. The method solves the limitation that a single screening method in the prior art cannot evaluate factors such as target inhibition, cell permeability and metabolic transformation at the same time, and the method is simple, convenient, rapid, high in accuracy and suitable for large-scale primary screening of the AChE inhibitor and early warning of neurotoxicity risks.

This invention relates to the field of enzyme network analysis technology, specifically a robust system for analyzing the functional balance of a multifunctional enzyme regulatory network. The system includes a network topology analysis module, a graph generation module, a flux balance analysis module, a functional simulation module, a robustness curve fitting module, and a report generation module. It identifies key nodes and vulnerable paths in the target regulatory network; constructs a structural feature association graph; performs flux balance analysis on the target regulatory network to obtain functional flux distribution data; simulates the functional operation of the target regulatory network and quantifies the output efficiency of the simulation results to obtain the core function maintenance degree; fits the perturbation trend of the core function maintenance degree to obtain a functional balance robustness curve; and analyzes the decay mode of the functional balance robustness curve to generate a robustness analysis report of the target regulatory network. This invention can improve the efficiency of functional balance robustness analysis of a multifunctional enzyme regulatory network.

This application relates to the technical fields of synthetic biology and enzyme engineering, and in particular to a computational-experimental closed-loop rational design method for polyketide synthases. The method includes: constructing a full-length FCHS model using AlphaFold3; identifying binding hotspots and verifying dynamic stability through molecular docking and kinetic simulations; and constructing a low-redundancy mutant library containing A, L, and F substitutions. This application solves the problems of difficult protein structure analysis and limited experimental throughput through a closed-loop process of computational pre-screening, dynamic verification, and evolutionary filtering, providing an efficient paradigm for the improvement of complex multifunctional enzymes.

The invention discloses a fusion protein and a fluorescent microplate reader method for high-flux cell apoptosis detection, apoptotic cells are marked by fusion protein of annexin A5 (AnxA5) and fluorescent protein (FPs), and the fluorescence intensity of the fusion protein of AnxA5 and FPs is detected by a multifunctional microplate reader. And judging the exposure condition of the phosphatidylserine PS on the surface of the apoptotic cell according to the fluorescence signal. The method disclosed by the invention has the characteristics of simplicity and easiness in operation and rapidness in detection, and the method for detecting the fluorescence intensity by the microplate reader is applied to the detection of the cell apoptosis, so that the high-throughput detection of the cell apoptosis is realized. Compared with a flow cytometer and a fluorescence microscope, the multifunctional microplate reader has the advantages of low price, easiness in operation, simplicity in maintenance and easiness in popularization. The AnxA5 apoptosis detection probe is wide in selection range, is not limited by excitation wavelength and emission wavelength of a fluorescent marker, can simultaneously detect a plurality of samples at one time, and is convenient and fast.

The application discloses a pseudomonas aeruginosa bacteriophage PAZ1 and application thereof, the bacteriophage PAZ1 has a preservation number of CCTCC NO:M 2024545; the application utilizes the bacteriophage to prepare a detection kit, the kit can effectively detect the pseudomonas aeruginosa in food samples, is coupled with nano magnetic beads to form a conjugate PhagePAZ1-MBs.The pseudomonas aeruginosa after enrichment and separation is lysed by CTAB, is reacted with a luminescent reagent and is read by a multifunctional enzyme label instrument, the number of the pseudomonas aeruginosa in the sample is determined, and the detection specificity of the method is good.The application utilizes the pseudomonas aeruginosa bacteriophage fluorescent detection kit to qualitatively and quantitatively detect the sample to be measured, the detection time is about 30 min, and the detection limit reaches 607 CFU / mL.The application expands the application range of the bacteriophage in water source food safety detection, and has a good application prospect.

The multifunctional enzymolysis equipment comprises an enzymolysis tank, a heat exchange jacket, a stirring device, a speed reducer and a control system, the top of the enzymolysis tank is provided with a feed port and a PH meter port, a flow meter can be mounted at the feed port, and a PH meter can be mounted at the PH meter port; a first discharge hole and a pressure manhole are formed in the bottom of the enzymolysis tank, and a pressure gauge can be mounted in the pressure manhole; a thermometer port and a sampling port are formed in the side of the enzymolysis tank, and a thermometer can be mounted at the thermometer port; the heat exchange jacket comprises a heat exchange pipeline surrounding the outer wall of the enzymolysis tank body, and a steam inlet and a condensed water outlet which are respectively communicated with the heat exchange pipeline; and the speed reducer is connected with a suspended stirring shaft of the stirring device. And the control system is connected with the flow meter, the PH meter, the pressure gauge, the thermometer and the speed reducer. According to the utility model, differences caused by manual operation can be eliminated, and the enzymolysis process can be monitored and controlled, so that the production efficiency and the product quality are improved, the cost is reduced, and the utilization rate of raw materials and energy is improved.

The utility model relates to the technical field of anti-aging raw material extraction, in particular to multifunctional enzymolysis extraction equipment which comprises a box body and a connecting unit, the connecting unit comprises an inner shell, a connecting bin, a discharging pipe, an electromagnetic valve, a temperature sensor, an electric heating wire, a mounting seat, a side door, a controller, a stirring assembly and a separating assembly, and the temperature sensor is fixedly connected with the box body; during use, a backing material and enzyme liquid are placed in the inner shell, then the side door is closed, the controller is clicked to start the electric heating wire, the temperature is increased by utilizing heat conductivity, the temperature sensor transmits the temperature in the box body to the controller in real time, the stirring assembly is connected with the stirring assembly, and the separation assembly is arranged below the discharging pipe. After the processing is completed, the separation assembly is used for filtering, and the stirring assembly is used for stirring and mixing the base material and the enzyme liquid, so that the efficiency of extracting the anti-aging raw material is improved, the complex technological process is shortened, and the workload of workers is effectively reduced.

The invention relates to the technical field of medical examination, in particular to a preparation method and application of a stable multifunctional enzyme compound quality control product, and the preparation method comprises the following steps: washing pig lungs with purified water, removing vascular connective tissues, cutting the pig lungs into small blocks with a knife, weighing and carrying out freezing pretreatment to obtain a first mixed solution; adding homogenate into the first mixed solution according to a weight ratio, and homogenizing to obtain a second mixed solution; refrigerating the second mixed solution or staying overnight, and fully extracting to obtain a third mixed solution; centrifugally filtering the third mixed solution, adding a salting-out drug and a protein protective agent, removing impure protein, centrifuging, and taking supernatant to obtain a fourth mixed solution; continuously adding a salting-out medicine into the fourth mixed solution, centrifuging, taking a precipitate, adding a dialysate into the precipitate, and fully dissolving to obtain a fifth mixed solution; dialyzing the fifth mixed solution, and collecting a solution in a dialysis bag to obtain an enzyme extracting solution; preparing a composite quality control product by using the enzyme extracting solution, and carrying out freeze-drying treatment on the composite quality control product to obtain composite quality control product freeze-dried powder.

The invention discloses a retinoic acid X receptor alpha activation effect molecule detection method based on a fluorescence polarization technology, and belongs to the technical field of biological analysis. According to the method, the change of a probe fluorescence polarization signal caused by the change of the binding state of the RXR alpha and the fluorescent nuclear receptor co-regulatory peptide is directly monitored, so that the rapid evaluation of the to-be-detected substance on the receptor activation effect is realized. According to the method, three core processes of background signal determination, to-be-detected substance signal detection and statistical analysis are completed on the molecular level by virtue of a conventional multifunctional microplate reader, so that the dependence on high-cost equipment or a complex cell model in the prior art is effectively avoided, and high efficiency, low cost and high flux of RXR alpha activator screening are realized. The method is suitable for early warning of toxicity of environmental compounds and efficient screening of RXRalpha targeted drugs, and a practical tool is provided for receptor function research and drug development.

The invention provides a method for biologically synthesizing 1 ', 4'-trans-abscisic acid diol by utilizing yarrowia lipolytica. According to the invention, a strain of recombinant yarrowia lipolytica capable of producing the abscisic acid at high yield is used as a chassis cell, and the synthesis of the 1 ', 4'-trans-abscisic acid glycol to the abscisic acid is blocked by knocking out sesquiterpene dehydrogenase BcAba4 in the chassis cell, so that the yarrowia lipolytica capable of producing the 1 ', 4'-trans-abscisic acid glycol is obtained; a citric acid transporter 1 coding gene CEX1 is further knocked out, ATP citric acid lyase coding genes ACL1 and ACL2, a peroxisome carnitine acetyltransferase coding gene CAT2, lipase coding genes TGL3 and TGL4 and a peroxisome multifunctional enzyme coding gene MFE1 are overexpressed, and an ERG9 gene promoter is replaced by a growth dependent promoter PD19. The supply of the precursor acetyl CoA is improved, the path of the precursor acetyl CoA to grease synthesis is reduced, the metabolic flux from FPP to squalene is reduced, and the accumulation capacity of 1 ', 4'-trans-abscisic acid diol in the recombinant bacteria is improved.

The invention discloses a flavin-dependent oxidase SavA and an application of the flavin-dependent oxidase SavA in asymmetric synthesis of 4-alkyl butenolactone. The SavA enzyme provided by the invention is a novel multifunctional enzyme with desaturation, hydroxylation and lactonization effects at the same time, and can catalyze conversion of fatty acyl thioester into single-configuration (4S)-4-alkyl butene lactone. The SavA enzyme provided by the invention is a flavin-dependent enzyme and naturally exists in a form of being combined with FAD, no exogenous FAD needs to be added in an in-vitro enzymatic reaction, no other coenzyme or cofactor is involved, only O2 is used as an oxidizing agent, the reaction process is green and efficient, and the SavA enzyme has the potential of being developed into an efficient tool enzyme for asymmetric biological catalytic synthesis of 4-substituted butenolactone.

This invention discloses a method for the quantitative detection of GDP-L-fucose based on high-performance liquid chromatography (HPLC) with a diode array detector. The method employs HPLC analysis under the following chromatographic conditions: an Agilent C18 column; mobile phase A consisting of 2.5–3.0 g / L 2.72 g / L potassium dihydrogen phosphate aqueous solution (containing 0.2%–0.3% tetrabutylammonium bromide, adjusted to pH 3.5–4.5 with phosphoric acid); acetonitrile as mobile phase B; gradient elution; and a diode array detector at a detection wavelength of 254 nm. This method exhibits low detection limits, good precision and accuracy, and not only provides spectral information for qualitative and quantitative analysis of GDP-L-fucose but also enables real-time monitoring of the reaction process during GDP-L-fucose synthesis. This provides an analytical method for improving the purity and yield of GDP-L-fucose and for screening the activity of multifunctional FKP enzymes.

The invention relates to the technical field of biology, in particular to a cellulase technology in the field of enzymology. The invention relates to the technical field of cellulose degradation, in particular to application of a GH5GH6 double-catalytic structural domain compound enzyme (containing a glycoside hydrolase family 5 (GH5) and a glycoside hydrolase family 6 (GH6)) in cellulose degradation. The invention relates to an application of a GH5GH6 double-catalytic structural domain compound enzyme in cellulose degradation. The GH5GH6 double-catalytic structural domain compound enzyme is derived from ship maggot symbiotic bacteria. The ship maggot symbiotic bacterium is a known strain Teredinibacter species, in particular to a Teredinibacter Turnerae strain, and the ship maggot symbiotic bacterium can be used for preparing the ship maggot symbiotic bacterium. According to the invention, a multifunctional enzyme compound catalyzed by double structural domains of GH5 and GH6 is obtained from a ship maggot symbiotic bacterium Teredinibacter, the compound is formed by arranging and connecting the GH5, the GH6 and 2-4 CBMs, three different functional structural domains of the GH5, the CBMs and the GH6 are integrated, and the efficiency and the stability of cellulose degradation are remarkably improved.

The invention discloses a molecular marker of a wheat grain storage protein regulatory gene TaMYB3R1-3D and application of the molecular marker, and belongs to the technical field of biology. The molecular marker is a group of KASP primers and comprises two reverse primers as shown in SEQ ID NO: 1 and SEQ ID NO: 2 (5'ends are respectively marked with recognition sequences of FAM and HEX fluorescent probes) and a forward primer as shown in SEQ ID NO: 3, a PHERAstar Plus multifunctional microplate reader is used for detecting a PCR (Polymerase Chain Reaction) product, and software KlusterCaller is used for carrying out genetic typing. The molecular marker has the advantages that the content of the storage protein of the wheat can be predicted by applying the KASP molecular marker developed by the invention, the cost is saved, the selection efficiency is greatly improved, the breeding process can be accelerated, and a useful molecular tool is provided for efficiently screening high-quality wheat strains.

The invention discloses a multifunctional enzyme microorganism plant boosting agent and a preparation method thereof, and belongs to the field of agricultural water-soluble fertilizers. The boosting agent is prepared from amino acid powder, brown sugar, potassium humate / potassium fulvate, monopotassium phosphate, an enzyme microbial agent, table vinegar and medical stone mineralized water in proportion. The preparation method comprises the following steps: dissolving the brown sugar with boiled medical stone mineralized water, cooling, adding the enzyme microbial inoculum for activation, and standing; adding table vinegar and the residual medical stone mineralized water, and controlling the temperature to ferment for 90-96 hours until the main fermentation is finished; and finally, standing the zymophyte liquid, adding amino acid powder, potassium humate / potassium fulvate and monopotassium phosphate, and uniformly stirring and chelating at 40-45 DEG C to obtain the plant boosting agent. The plant boosting agent is high in activity, green and pollution-free, has the effects of supplementing nutrients, improving soil, resisting diseases and the like, is suitable for various crops, is good in greenhouse planting adaptability, is proved to be remarkable in yield increasing and quality improving effects by field experiments, and is simple and convenient in preparation process, low in cost and suitable for industrial production.

This invention discloses a method for screening drugs against Japanese encephalitis virus, which uses the main protease NS3 of Japanese encephalitis virus. pro The activity reporter plasmid and the main protease NS3 of Japanese encephalitis virus pro Expression plasmids were used to screen for drugs against Japanese encephalitis virus; NS3 pro Active reporter plasmids and NS3 pro The expression plasmid was co-transfected into HEK-293T cells. The fluorescence intensity of green fluorescent protein (EGFP) was detected using an inverted fluorescence microscope and a multi-functional microplate reader to predict NS3. pro The activity of NS3, the main protease of JEV, can be used to screen for its activity. pro The purpose of this invention is to screen anti-JEV compounds using active reporter plasmids. The inhibitory effect of the compounds can be observed more intuitively and easily by utilizing fluorescence intensity, providing a powerful tool for screening broad-spectrum anti-JEV compounds.

The application discloses a wheat TaBRD2-7A gene molecular marker and application thereof, and belongs to the technical field of crop selection and cultivation. The molecular marker is a KASP marker, which is amplified by a primer group shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, the 5' ends of the two front primers are marked with FAM and HEX respectively, the PCR amplification product is detected by using a multifunctional enzyme marker, and genotyping is performed by using KlusterCaller software. The application has the advantages that: by applying the KASP molecular marker developed by the application, wheat excellent haplotypes can be identified in wheat line selection and breeding, not only the cost is saved, but also the selection efficiency is greatly improved, the breeding process is accelerated, and new possibilities are provided for efficient screening of excellent alleles of the TaBRD2-7A gene and cultivation of high-yield wheat varieties.

This invention belongs to the field of biomedical engineering technology, specifically relating to the construction and application of a screening model for influenza virus receptor binding inhibitors. Addressing the problem of influenza virus hemagglutinin (HA) being prone to mutation and having multiple subtypes, making it difficult to screen for broad-spectrum inhibitors, this invention replaces HA with elderberry lectin (SNA), which specifically recognizes α-2,6-sialic acid. Using MDCK cells overexpressing α-2,6-sialic acid, a fluorescently labeled high-throughput screening model is constructed by labeling SNA with fluorescein isothiocyanate (FITC). The relative fluorescence units (RFU) of the system are detected using a multifunctional enzyme-linked immunosorbent assay (ELISA) reader. Active compounds inhibit the binding of SNA to cell surface receptors, resulting in a lower RFU value, while inactive compounds show a higher RFU value. This invention provides key technical support for the development of novel inhibitors targeting influenza virus receptor binding.

A systematic pipeline is used to extract catalytically active pockets of the most diverse enzyme class—hydrolases, from the PDB database. A process extracts the 38029 hydrolase reactive centers (RC) and collates them into a publicly accessible active site collection (actiome; RC-Hydrolase). The process includes 128M pairwise shape comparisons across RC-Hydrolase using CADSEEK 3D Shape Search Engine to end up with 155,329 instances presented in a available, visually interactive dataset. Allowing comparisons of enzyme reactive centers across functional spaces (EC classification numbers) enables identification of enzyme backbones which can be minimally mutated to accommodate more than one type of catalytic activity to aid rational design of multifunctional enzymes. Such versatile enzyme backbones is leveraged by latest diffusion-based protein design models to design a library of structurally stable multifunctional enzyme pockets. Design of a bifunctional protease-nuclease shown as an example opens up a novel computational recipe for enzyme engineering.