22 results about "Urate oxidase" patented technology

The invention discloses a urate oxidase mutant and application thereof in improving the thermal stability of urate oxidase, and belongs to the technical field of biology. The urate oxidase mutant disclosed by the invention is T68L / T75S / E222D / K299E, T68L / T75S / K299E or T75S / K299E, and the urate oxidase mutant disclosed by the invention is T68L / T75S / K299E. According to the method, the key sites influencing the thermal stability are mined in the modes of directed evolution, high-throughput screening, multiple virtual screening and the like, and the stability is improved through mutation. And finally, a plurality of key sites and mutants influencing the stability are excavated, and the thermal stability is improved.

Provided is a method for improving the stability of urate oxidase. The inventors have discovered that combining an active ingredient urate oxidase or chemically modified urate oxidase with a stabilizer xanthine through a non-covalent bond can significantly improve the in vivo and in vitro stability of urate oxidase and chemically modified urate oxidase in the form of a tetramer, thereby effectively improving the stability of urate oxidase.

The invention discloses a uricase mutant. The uricase sequence from aspergillus flavus and the uricase sequence from candida are taken as templates, multiple rounds of random mutation are carried out, sequencing verifies that the key amino acid site of the catalytic center is mutated, and the catalytic activity is enhanced. In-vivo and in-vitro uric acid reducing activities of the two uricase mutants are further verified, and the two uricase mutants can significantly reduce the content of uric acid and are expected to become new uricase drugs for treating hyperuricemia.

The invention belongs to the technical field of uric acid detection, and relates to a preparation method and application of a uric acid and pH dual-electrode detection sensor. The preparation method comprises the following steps: mixing a soluble manganese salt with a complexing agent, adding a soluble hexacyanoferrate solution, aging and standing at room temperature, centrifuging, drying, and pyrolyzing at high temperature to obtain the Mn2O3 / Fe2O3 material. Coating on the surface of a first working electrode, and then continuously dropwise adding a mixed solution containing a cross-linking agent and urate oxidase; and uniformly mixing a chloroauric acid solution with a hydrochloric acid solution and an aniline solution with a hydrochloric acid solution, carrying out electro-deposition on a second working electrode, and drying at low temperature to obtain the sensor. The sensor designed based on the double working electrodes shows excellent stability, reproducibility and anti-interference performance in sweat uric acid monitoring. The sensor can realize rapid identification and dynamic response of uric acid in a real sweat sample, and is expected to be used for integration of wearable equipment to realize continuous gout early warning.

The application provides a uric acid oxidase secretion signal peptide and application thereof, and relates to the technical field of genetic engineering. In view of the problem that uric acid oxidases reported in the current literature are difficult to secrete in Pichia pastoris, the endogenous signal peptide of Pichia pastoris is screened, a signal peptide with strong secretion capacity is obtained, and the signal peptide is optimized in a targeted manner, a signal peptide mutant with strong secretion capacity is obtained, uric acid oxidase can be better secreted to the extracellular, and then the extracellular enzyme activity of uric acid oxidase is improved.

The application provides an electrochemical uric acid test paper and a uric acid detection method. The electrochemical uric acid test paper has simple structure, low cost, easy manufacturing process, rapid reaction, sensitive detection, wide storage temperature, wide use temperature, wide effective period, accurate result, simple operation, etc. The uric acid detection method distinguishes the test current of ascorbic acid by different time and different excitation signals, subtracts the current signal of ascorbic acid from the current signal of uric acid and ascorbic acid, eliminates the interference of ascorbic acid, is simple to operate and low in cost, does not need to add other material components, does not need the participation of uric acid oxidase and ascorbic acid oxidase, directly distinguishes the current signal by different voltages, is short in test time, reduces the complex data correction process, maintains good repeatability of data, is simple in logic operation, basically has no influence on the repeatability of test signals, and can effectively remove the interference of ascorbic acid.

The invention discloses a urate oxidase gene derived from aspergillus cristatus and application thereof, and the urate oxidase gene is characterized in that a natural urate oxidase gene coding region sequence is obtained from the aspergillus cristatus, and the gene is optimized according to the codon preference of escherichia coli to obtain an optimized sequence. Connecting the optimized gene to a carrier through NdeI and BamHI restriction enzyme cutting sites, and constructing a wild type uox recombinant plasmid; the method comprises the following steps: replacing a sequence of uox of aspergillus cristatus for coding 243-site tyrosine (Y) with a sequence for coding lysine (K) through site-specific mutagenesis, constructing mutant Y243K recombinant plasmids, respectively transforming the recombinant plasmids into competent cells, culturing, and adding IPTG to induce target protein expression. And collecting the induced thalli, carrying out ultrasonication and centrifugation to collect the supernatant so as to obtain a crude enzyme, and purifying the crude enzyme through a nickel affinity column so as to obtain the purified UOX protein. Uric acid is used as a substrate to measure the enzyme activity of the purified UOX, and the result shows that the specific enzyme activity of the mutant Y243K is obviously higher than that of a wild type, and the activity is improved by 2.75 times, so that the mutant Y243K has higher application value.

The invention relates to the technical field of protein drugs, in particular to a urate oxidase variant and a glycosylation modification form thereof. An engineered N-glycosylation site is introduced into urate oxidase, and a pair of engineered disulfide bonds is additionally introduced into a tetramer interface, so that the obtained glycosylation modified urate oxidase variant can improve folding stability, in-vivo performance and immunological properties on the basis of maintaining enzymatic activity.

Provided is a method for preparing a polyethylene glycol-modified urate oxidase, at least 11 of the following amino acid sites of the polyethylene glycol-modified urate oxidase have a PEG modification: T1, K3, K4, K30, K35, K76, K79, K97, K112, K116, K120, K152, K179, K222, K231, K266, K272, K285, K291, and K293, and the preparation method includes: performing a coupling reaction between urate oxidase and polyethylene glycol, wherein the polyethylene glycol is provided in the form of an acidic solution, and a molar ratio of the urate oxidase to the polyethylene glycol is 1: (56 to 94), to obtain the polyethylene glycol-modified urate oxidase.

This invention discloses a uric acid-lowering drug composition, which comprises one or more of the following: levocarnitine, levocarnitine derivatives, pharmaceutically acceptable salts of levocarnitine or its derivatives, glucocorticoids, colchicine, nonsteroidal anti-inflammatory drugs, IL-1β inhibitors, drugs that inhibit uric acid production, drugs that promote uric acid excretion, and uricase. The composition has the effects of lowering uric acid, protecting liver and kidney function, and inhibiting leukopenia.

The application belongs to the technical field of biology and relates to a high-activity and high-humanization rate uric acid oxidase mutant. The high-activity and high-humanization rate uric acid oxidase mutant is a human uric acid oxidase mutant obtained by mutation of an inactivated human uric acid oxidase with an amino acid sequence of SEQ ID NO. 1, which has amino acid substitutions at positions 83, 119, 121, 151, 222, 232, 233, 240 and 252, also has amino acid substitutions at positions 208 and 219, and further has an amino acid substitution at position 112. The high-activity and high-humanization rate uric acid oxidase mutant has high homology with a theoretical amino acid sequence of human uric acid oxidase, has high activity, is more suitable for long-term use in the treatment of hyperuricemia and gout in the clinic, and can be used for preparing a drug for treating hyperuricemia and gout.

Provided is an mRNA-liposome complex. The mRNA-liposome complex includes a liposome and a nucleic acid. The nucleic acid includes at least one mRNA encoding urate oxidase or recombinant urate oxidase. The liposome includes at least two lipids, and the at least two lipids include a core lipid. The liposome has a pKa ranging from 6.0 to 6.3. A molar percentage of the core lipid in a total molar amount of the liposome is not less than 15%.

The application provides a fiber-based organic photoelectrochemical transistor uric acid sensor and a preparation method thereof. The fiber-based organic photoelectrochemical transistor uric acid sensor is obtained by growing Fe-MOF@PANI on the surface of a conductive fiber, then modifying uric acid oxidase and a biocompatible film solution as a gate electrode of the transistor, using the fiber loaded with a semiconductor material as a source-drain electrode of the transistor, and dropping a gel electrolyte. The organic photoelectrochemical transistor uric acid sensor uses a fiber-based material as an electrode, a photosensitive material and a sensing substance are modified on the gate electrode, the photosensitive material makes electrons jump to the surface of the electrode under light conditions, the channel current is regulated, and the sensing substance is used to detect uric acid. The uric acid sensor can work under zero gate bias, reduces the background signal and interference, shows a good current response to different concentrations of uric acid, and has great market promotion value.

The application provides an indole saturated ring compound or a derivative thereof, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a prodrug molecule thereof, or a deuteride thereof, or a tritium compound thereof, and an application thereof. The indole saturated ring compound or the derivative thereof provided by the application is a novel compound, the compound has good inhibitory activity on uric acid oxidase and uric acid transporter, is XOR / URAT1 dual inhibition, has good uric acid reducing effect, has good safety, has good pharmacokinetics, has high drug property, and can be used for preparing a uric acid reducing drug and used for preventing and / or treating gout or hyperuricemia.

The application provides a fusion uric acid oxidase and application thereof in a biosensing element. The fusion uric acid oxidase comprises a carbohydrate binding module and a uric acid oxidase, and the fusion uric acid oxidase can be made into a biosensing element for uric acid detection, and has the advantages of high sensitivity and good stability.

The present specification discloses a urate oxidase-albumin conjugate, a preparation method thereof, a urate oxidase variant contained in the urate oxidase-albumin conjugate, and a preparation method thereof. The urate oxidase-albumin conjugate is characterized in that three or more albumins are conjugated to the urate oxidase variant through a linker, thereby improving half-life and reducing immunogenicity. In addition, the urate oxidase-albumin conjugate can be used to prevent or treat various diseases, disorders and / or indications caused by uric acid.

The application discloses a heat-resistant uric acid oxidase TCI-UOX2 and application, and relates to the technical field of biology.The amino acid sequence of the heat-resistant uric acid oxidase TCI-UOX2 is shown as SEQ ID NO:1.The recombinant uric acid oxidase TCI-UOX2 shows the highest activity at 35 DEG C and pH 8, and still maintains about 100 percent relative activity at 5 DEG C to 40 DEG C.The enzyme still maintains about 100 percent residual activity after being incubated at the human physiological temperature of 37 DEG C for 12 hours.Under the anticoagulation environment of a high-uric-acid volunteer, the uric acid concentration can be reduced to the normal level of 360 micromoles per liter within 6 hours of reaction with the low-concentration 1 microgram per milliliter TCI-UOX2 enzyme solution.

The present application relates to a sweat uric acid detection patch based on a double-enzyme cascade catalysis and a preparation method and application thereof, the sweat uric acid detection patch comprising a bottom flexible substrate, a paper-based microfluidic channel, a top flexible packaging layer and a paper-based colorimetric unit, the bottom flexible substrate having a sweat permeation inlet, and the top flexible packaging layer having a transparent reading window; the paper-based microfluidic channel is arranged between the bottom flexible substrate and the top flexible packaging layer, and is used for transporting sweat from the sweat permeation inlet to the paper-based colorimetric unit; the paper-based colorimetric unit is loaded with a TMB chromogenic substrate and a double-enzyme cascade catalysis system; the double-enzyme cascade catalysis system comprises Fe single-atom nanoscale enzyme Fe-SANs and uric acid oxidase loaded on the surface of the Fe single-atom nanoscale enzyme Fe-SANs; wherein the Fe single-atom nanoscale enzyme Fe-SANs has FeN x active sites; the transparent reading window is arranged above the paper-based colorimetric unit, and is used for observing or collecting the color change of the paper-based colorimetric unit. The cascade catalysis process of the present application is conducive to improving the color development readability in the process of recognizing sweat uric acid.

The present specification discloses a urate oxidase-albumin conjugate, a preparation method thereof, a urate oxidase variant contained in the urate oxidase-albumin conjugate, and a preparation method thereof. The urate oxidase-albumin conjugate is characterized in that three or more albumins are conjugated to the urate oxidase variant through a linker, thereby improving half-life and reducing immunogenicity. In addition, the urate oxidase-albumin conjugate can be used to prevent or treat various diseases, disorders and / or indications caused by uric acid.

The invention relates to the technical field of biosensors, and discloses a metal nucleic acid framework nano cascade enzyme as well as a preparation method and application thereof. The preparation method of the nano cascade enzyme comprises the following steps: mixing nucleotide sequences as shown in SEQ ID NO.1-10 according to an equal molar ratio, and carrying out annealing reaction to obtain a DNA nanosheet of a metal template; and reacting the DNA nanosheet with a metal precursor to obtain the metal nucleic acid framework nano cascade enzyme. The metal nucleic acid framework nano cascade enzyme DNS-Fe has catalase activity, and after a corresponding aptamer is added, a target object can be accurately recognized in a plurality of interferents, and the accuracy is high; dNS-Pt / Fe has the activity of lactate oxidase, glucose oxidase and sarcosine oxidase, and DNS-Ag / Fe has the activity of urate oxidase, so that the cascade action can be realized by combining the DNS-Pt / Fe with the DNS-Fe, and the DNS-Ag / Fe replaces natural catalase to catalyze hydrogen peroxide to generate free radicals to be subjected to chromogenic reaction with a chromogenic substance, and the result is easy to observe.

The invention discloses a 4-carboxyl-3-fluorophenylboronic acid modified hydroxypropyl chitosan microneedle patch and a preparation method of the 4-carboxyl-3-fluorophenylboronic acid modified hydroxypropyl chitosan microneedle patch. The preparation method comprises the following steps: dissolving 4-carboxyl-3-fluorophenylboronic acid modified hydroxypropyl chitosan in deionized water, adding polyvinyl alcohol, mixing to form gel, adding the anti-inflammatory drug colchicine and the uric acid reducing drug urate oxidase, and uniformly mixing; a two-step injection molding centrifugal method is adopted, firstly, non-drug-loaded gel is injected into a microneedle mold for centrifugal drying, then, drug-loaded gel is injected into the microneedle mold and placed in a centrifugal machine to be centrifuged to remove bubbles, and the microneedle patch with pH responsiveness is obtained after the microneedle patch is dried to constant weight in a dryer. The microneedle responds to the pH change of the gouty arthritis part, so that the medicine is intelligently released, a series of side effects caused by abnormal plasma medicine concentration are avoided, the effective utilization rate of the medicine can be increased through the microneedle design that only the needle point carries the medicine, medicine waste is avoided, and disease management of gout patients is simplified.