74 results about "Enzyme specificity" patented technology

Enzyme substrates and associated technology of the present invention are provided. An enzyme substrate of the invention may comprise a biologically functional fluorescent dye and an enzyme-specific substrate moiety attached in such a way that the functionality of the functional dye is diminished. An enzymatic reaction may cleave at least a portion of the substrate moiety from the enzyme substrate to provide a more functional product dye. This product dye may be nonfluorescent or weakly fluorescent, in general, and relatively fluorescent, in a particular condition, such as when bound to a partner biological molecule or an assembly of partner biological molecules. An enzyme substrate of the present invention may thus be useful in fluorescence detection, and / or in any of a variety of useful applications, such as the detection of enzymatic activity in a cell-free system or in a living cell, the screening of drugs, or the diagnosis of disease.

The present invention provides a method for preparing bean dregs soluble dietary fiber by zymotechnics. Firstly, to dry the fresh bean dregs at the temperature of 60 to 80 DEG C for 12 to 14 hours, then to comminute until passing 40 mesh sieve, and to confect into ferment culture medium according to the ratio of 1 : 20 to 25 of bean dregs : water, and regulating the pH value to 4 to 4.5, then to disinfect at the temperature of 121 DEG C for 15 to 20 min to obtain liquid; to inoculate leeb wood mildew into the culture medium according to the 3 to 4 percent of the liquid volume ratio, then to shake bed culture at the speed of 140 to 160 r / min for 3 to 4d at the temperature of 28 to 30 DEG C. After sterilization, filtering and concentrating, to obtain deposit by non-water ethanol deposition, then to dry the deposit in vacuum to produce finished product. The present invention condition is geniality, equipment is simple, enzyme specificity is high, and can improve activity component amount maximum, has no pollution to circumstance, the SDF content in the bean dregs is increased greatly.

The present invention relates to prodrug molecules comprising conjugates of an antiproliferative drug, a protease specific cleavable peptide, and, optionally, a targeting peptide, with the prodrugs being substantially inactive prior to degradation of the cleavable sequence by proteolytic enzymes abundant within or in close proximity to the target cancer cell. Also, pharmaceutical compositions of the conjugates and the use of these compositions for treatment of cancer are disclosed.

The invention discloses a quantitative analysis method aiming at DNA methylation monitoring. The quantitative analysis method comprises the following steps: (1) designing a hairpin type DNA secondary structure according to a DNA methylation palindromic sequence, and modifying a methylene blue electroactive material on the structure; (2) reacting hairpin type DNA with transmethylase and methylation adenosine to synthesize a methylated hairpin type DNA; (3) performing specific enzymolysis on a DNA methylation site through DpnI enzyme to release DNA fragments connected with methylene blue; (4) performing sample monitoring through an ITO micro-electrode chip with negative electricity, and acquiring electrochemical signals through a multi-pass electric signal reading device; (5) recording a monitoring result; and (6) adding methylation suppression chemicals into a DNA methylation process to screen the DNA methylation suppression chemicals. The quantitative analysis method has the advantages of simplicity in operation, portability, low cost and the like, and an electrode does not need complicated modification.

The invention discloses an amino acid dehydrogenase mutant and an application thereof. The amino acid sequence of the amino acid dehydrogenase mutant is an amino acid sequence obtained by mutating anamino acid sequence represented by SEQ ID NO:1, and the mutation includes at least one of the following mutation sites: the 64thmutation site, the 94thmutation site, the 133rd mutation site, the 137thmutation site, the 148th mutation site, the 168th mutation site, the 173rd mutation site, the 183rd mutation site, the 191st mutation site, the 207th mutation site, the 229th mutation site, the 248thmutation site, the 255th mutation site and the 282nd mutation site; or the amino acid sequence of the amino acid dehydrogenase mutant is an amino acid sequence having the mutation sites in the mutated amino acid sequence and having a 80% or more homology with the mutated amino acid sequence. The amino acid dehydrogenase mutant has greatly improved enzyme activity, the enzyme activity is above 50times higher than that of a wild amino acid dehydrogenase, and the enzyme specificity is also correspondingly improved.

The invention discloses a preparation method of one kind of magnetic resonance imaging developers and two-photon imaging developers. The preparation method is characterized by comprising the steps of: firstly, synthesizing a lysine-cysteine-arginine-arginine-valine-arginine peptide fragment and a valine-arginine-cysteine-arginine-lysine-arginine peptide fragment, connecting the fragments with 2-amino-6-cyanobenzothiazole and then connecting a macrocyclic compound, and reacting with metal ions in a buffer solution to obtain five compounds, wherein the final product A1 of a first compound, the final product A2 of a second compound and the final product A5 of a fifth compound are magnetic resonance imaging developers, and the final product A3 of a third compound and the final product A4 of afourth compound are two-photon imaging developers. Because of being micromolecules, the magnetic resonance imaging developers and two-photon imaging developers have the advantages of good hydrophilia, easiness of preparation and high absorption; and the final product A1 of the first compound and the final product of the third compound have peptide fragments with specific enzyme specificity recognition, and have the advantage of high targeting.

There is provided a method for specifically determining a glycated β-chain N-terminal of glycated hemoglobin using enzymes without a separation operation, and a determination reagent kit therefor. A protease that cleaves a glycated amino acid and / or a glycated peptide from a glycated β-chain N-terminal without substantially cleaving a glycated amino acid or a glycated peptide from a glycated α-chain N-terminal of glycated hemoglobin or a fragment thereof is screened. The method of specifically determining a glycated β-chain N-terminal of glycated hemoglobin and the determination reagent kit are provided by using the protease obtained by the screening method. According to the present invention, a glycated β-chain N-terminal of glycated hemoglobin can specifically be determined without a separation operation.

The invention discloses a urease inhibitor determination method based on a fluorescence gold nano cluster. The urease inhibitor determination method is characterized in that urea is catalyzed to generate ammonium and carbon dioxide by utilizing the specificity of urease, the pH value of the system can be increased through the newly generated ammonium, the fluorescence of the gold nano cluster protected by N-acetyl-L-cysteine is extinguished, the urease inhibitor can prevent the process that the urea is decomposed by the catalyzing of the urease, the extinguishment of the fluorescence is inhibited, and the urease inhibitor determination method can be used for detecting the urease inhibitor. An F650 value is determined, the inhabitation rate is calculated, and the contents of IC50 of cysteamine and p-benzoquinone, respectively 2.8mu mol / L and 11.9mu mol / L, can be obtained through the fitting by software. The urease inhibitor determination method can be used for the high-throughput screening of the urease inhibitor.

The present invention relates to prodrug molecules comprising conjugates of an antiproliferative drug, a protease specific cleavable peptide, and, optionally, a targeting peptide, with the prodrugs being substantially inactive prior to degradation of the cleavable sequence by proteolytic enzymes abundant within or in close proximity to the target cancer cell. Also, pharmaceutical compositions of the conjugates and the use of these compositions for treatment of cancer are disclosed.

The present disclosure provides a method of producing enzyme-specific inhibitors or substrate binding partners comprising: identifying active site residues of the substrate in the enzyme substrate complex or in substrate binding partner-substrate complex; randomizing the active site residues to produce a combinatorial library of substrate variants; and selecting substrate variants that inhibit enzyme activity or bind substrate as substrate-specific binding partners. The present disclosure also provides ubiquitin enzyme specific inhibitors and ubiquitin variants that bind ubiquitin interaction motifs.

The invention discloses a micro-RNA-21 ultra-sensitive detection method based on double-enzyme signal cascade amplification. The method includes the steps: horseradish peroxidase enzyme-labeled probepreparation: coupling carboxylation polystyrene micro-spheres and horseradish peroxidase enzyme through DNA (deoxyribonucleic acid) single chains by an EDC (ethylene dichloride) carboxyl and amino coupling method; double-chain specific nuclease assisted target recycling: specifically cutting the DNA single chains complementarily paired with RNA (ribonucleic acid) to be detected in a probe by double-chain specific nuclease, triggering target circular reaction, releasing more horseradish peroxidase enzyme by cutting and amplifying signals; horseradish peroxidase enzyme signal amplification: collecting the horseradish peroxidase enzyme released in supernatant by cutting, adding a TMB (tetramethylbenzidine) chromogenic substrate, realizing naked-eye qualitative diagnosis by color change, and realizing enzyme-labeled quantification according to change of absorbance values. The difference of micro-RNA family members is effectively distinguished, and the method is suitable for community tumorscreening of high risk groups and has great application values in biomedical research and clinical diagnosis.

The present invention discloses a low fat dragon fruit pectin preparation method, and belongs to the field of pectin materials. The method is as follows: dragon fruit peel meal can be obtained by softening by water absorption, enzyme inactivation and crushing, a phosphate solution is added for microwave extraction to obtain a pectin extracting solution, esterase is added into the pectin extracting solution to obtain an enzymatic hydrolysate, the enzymatic hydrolysate is filtered by suction to obtain a filtration residue, the filtration residue is placed in an oven for drying to obtain low fat dragon fruit pectin; according to the method, an enzymic method is used for defatting, due to the enzyme specificity and high efficiency, production cycle is short, extraction efficiency is high, product quality is good, the manufacturing process is free of environmental contamination caused by use of a lot of acid and alkali, and substances harmful to human body may not be brought.

The invention discloses a urease activity fluorescence determination method based on a gold nano cluster. The method is characterized by comprising the steps of catalyzing urea to generate ammonium and carbon dioxide by utilizing the specificity of urease, wherein the newly generated ammonium can increase the pH value of a system, so that the fluorescence of the gold nano cluster protected by N-acetyl-L-cysteine is extinguished, the variation of fluorescence emission spectrum characteristics can be reflected, and the method can be used for determining the activity of the urease. F650 is in a linear relation with the concentration value of the urease in the range of 2.2 to 44U / L, and the detection limit is 0.55U / L. The urease activity fluorescence determination method is high in sensitivity, good in reproducibility and applicable to the determination of helicobacter pylori.

The disclosed technology provides a computational prediction modeling comprising a novel algorithm for prediction of glycosylation or to optimize biopharmaceutical production of proteins of therapeutic relevance. The model of the disclosed technology can be used to predict glycosylation changes based solely on the stating glycoprofiles in any host cells and known or suggested rules on enzyme specificity. Applications of the invention model are also provided.

The invention relates to a substrate and an internal standard used in mass spectrography detection. The inventive substrate is provided which includes a substrate compound of formula A - B<1> - B<2> - B<3>: wherein A is a sugar moiety; B<1> is a linker moiety allowing the conjugation of moiety A and the remaining structure of the substrate; B<2> contains a permanently charged element such as a quaternary ammonium group so as to increase proton affinities and ionization efficiencies for mass spectrometry analysis; and B<3> of various carbon length conferring specificities to targeted enzymes. Also provided is a process to detect lysosomal diseases by contacting a sample with the inventive substrate along with an internal standard which is isotope-labeled analog of the product cleaved by a targeted enzyme upon the substrate.

The invention discloses a novel preparation method of (S)-(+)-2,2-dimethylcyclopropanecarboxylic acid by biocatalysis. The method uses ethyl 2,2-dimethylcyclopropanecarboxylate as substrate and Novozym 435 lipase as biological catalyst, then biological catalyst asymmetric hydrolysis reaction is carried out on the mixture at a temperature of 25-45 DEG C and at pH ranging from 6.0-8.0 in a water phase reaction system, after the reaction is completed, the reaction solution is isolated to obtain (S)-(+)-2,2-dimethylcyclopropanecarboxylic acid. The asymmetric hydrolysis reaction utilizes Novozym 435 lipase to catalyze ethyl 2,2-dimethylcyclopropanecarboxylate, therefore, the enzyme specificity is strong, the catalytic efficiency is high, no side product is generated, and the optical purity and yield of the product are both higher than those of the product obtained by chemical methods.

The invention discloses a single photon emission computed tomography developer and a preparation method thereof. The method is characterized by respectively synthetising two peptides by the solid-phase synthesis to obtain corresponding initial products and final products, synthetising a single compound with a first kind feature structure (I) or a second kind feature structure (II) and forming the developer according to a needed dosage ratio. The imaging developer is micro-molecule, good in hydrophilicity and easy to prepare, and precursor micro-molecule of the developer has peptides specially recognized by specific enzyme, so that imaging areas can be targeted actively, and controlled self-assembly of radioactive nano-structures in living cells is realized to study activity of specific enzymes in tumor cells. Compared with conventional micro-nano systems used in early disease diagnosis, by the preparation method, micro-nano materials synthetised in vitro are not needed to be injected into living bodies for tumor diagnosis, problems of low intake and difficulty in targeting of conventional nano-materials are solved.

The invention discloses a prokaryotic recombinant expression and preparation method of lysyl endopeptidase. The preparation method comprises the following steps: importing encoding genes of recombinant lysyl endopeptidase with the amino acid sequence 2 into recipient escherichia coli cells, thus obtaining recombinant escherichia coli cells; carrying out inducible expression on the recombinant escherichia coli cells, collecting and crushing the recombinant escherichia coli cells, thus obtaining an inclusion body; and sequentially carrying out denaturation, renaturation, activation and purification to obtain the recombinant lysyl endopeptidase. The recombinant lys-C protease prepared by adopting the preparation method provided by the invention has high enzyme activity and relatively high urea tolerance. The preparation method has the advantages that the enzyme specificity is good, no animal-origin viruses exist, etc.; the production and preparation processes are simple and are low in cost; and the method can be applied to biopharmacy and proteomic analysis.

The invention relates to primers and kit for rapidly detecting African swine fever virus. Four primers are provided, the kit comprises the primers for detecting nucleic acid of the African swine fevervirus and 18 [mu]L of a reaction solution, and the reaction solution comprises the following reagents: 1.8 [mu]L of 40 mM KCl, 1.8 [mu]L of 100 mM(NH4)2SO4, 1.8 [mu]L of 80 mM MgSO4, 1.8 [mu]L of 1%Tween-20, 0.9 [mu]L of28 mM dNTPs, 1.8 [mu]L of 8000 U / mL Bst enzyme, 0.18 [mu]L of 60 U specific endonuclease FEN1, 0.9 [mu]L of 1 mM SYBRGREEN fluorochrome, 1.8 [mu]L of 4.0*10<-5> mol / L gold nanoparticles and 0.22 [mu]L of ultrapure water. Compared with the prior art, the primers and kit for rapidly detecting the African swine fever virus have the following advantages: four different specific primers are provided, so that the accuracy of detection results for the African swine fever virus is higher, accurate detection results can be rapidly given in combination with an improved LAMP technology and a microfluidic chip technology, and the purpose of jointing detecting multiple different indexes of the same sample can be achieved.

The present invention provides a method of detecting platelet thrombosis or organ injury in a patient suffering from disseminated intravascular coagulation (DIC) syndrome or systemic inflammatory response syndrome (SIRS) by analyzing a von Willebrand factor-degrading enzyme and / or a degrading factor thereof. A kit for detecting platelet thrombosis or organ injury in a patient suffering from DIC or SIRS which contains an antibody capable of binding specifically to a von Willebrand factor-degrading enzyme or its fragment and an antibody capable of binding specifically to a degrading factor of a von Willebrand factor-degrading enzyme or its fragment.

There is provided a method for specifically determining a glycated β-chain N-terminal of glycated hemoglobin using enzymes without a separation operation, and a determination reagent kit therefor. A protease that cleaves a glycated amino acid and / or a glycated peptide from a glycated β-chain N-terminal without substantially cleaving a glycated amino acid or a glycated peptide from a glycated α-chain N-terminal of glycated hemoglobin or a fragment thereof is screened. The method of specifically determining a glycated β-chain N-terminal of glycated hemoglobin and the determination reagent kit are provided by using the protease obtained by the screening method. According to the present invention, a glycated β-chain N-terminal of glycated hemoglobin can specifically be determined without a separation operation.

The present invention relates to a method for detecting gene polymorphism using labeled probes and specific primer sequences, and a kit comprising the specific probes and the specific primer sequences, and is suitable for use in the fields of biotechnology and medicines. The kit provides the specific primer sequences and the labeled probes for simultaneous detection of human MTHFR gene C677T site and A1298C site polymorphisms, and the kit contains a Taq enzyme, the specific primers, the specific probes and an internal standard system. The provided primers and kit are used for simultaneously detecting the MTHFR gene C677T site and A1298C site polymorphisms, and have advantages of strong specificity, high sensitivity, simple and rapid operation, high throughput, safety, objective result interpretation, etc.

The invention discloses a (R)-styrene glycol preparation method that utilizes site-directed mutagenesis to alter coenzyme specificity and stereoselectivity, belongs to the technical field of biocatalytic asymmetric transformation, and provides a recombinant Escherichia coli strain BL21 / pETSCR6768 with the preservation CCTCC NO of M208079, and an asymmetric transforming preparation method of (R)-styrene glycol. The preparation method leads Ser in position 67 and His in position 68 of a (S)-specific carbonyl reductase to mutate to Asp, constructs recombinant plasmid pETSCR6768, and feeds the Escherichia coli, thus obtaining the recombinant strain E.coli BL21 / pETSCR6768 which leads the coenzyme specificity of (S)-specific carbonyl reductase to change from NADPH to NADH; furthermore, the stereoselectivity of the product is altered from (S)-styrene glycol to (R)-styrene glycol, and the preparation method provides an effective way for preparing (R)-styrene glycol in high efficiency and low cost and has significant meaning for recognizing the stereoselectivity of functional zymoprotein in the molecule and protein level.

The invention discloses an esterase mutant and application thereof. The esterase mutant has a sequence with amino acid mutation of a sequence shown in SEQ ID NO: 1, and sites with amino acid mutationcomprise an N51G site. According to the esterase mutant, on the basis of esterase shown in SEQ ID NO: 1, mutation is conducted through a site-specific mutagenesis method, so the amino acid sequence ofthe esterase mutant is changed, the structure and function of protein are changed, and esterase with the mutation site is obtained through a directional screening method; therefore, the esterase mutants have the advantage that the enzyme specificity is greatly improved, and the enzyme activity is correspondingly improved, so the use amount of enzyme is greatly reduced, and the cost in industrialproduction is reduced.

The invention relates to a hybridoma for secreting an anti-recombinant schistosoma japonica enolase specific monoclonal antibody as well as a preparation method and application of the hybridoma, and belongs to the technical fields of gene engineering, preparation of monoclonal antibodies and immunologic diagnosis. The hybridoma JYG-1 capable of secreting the anti-recombinant schistosoma japonica enolase (Sj Enolase protein) specific monoclonal antibody is preserved in the Common Microbiology Center of CCCCM (China Committee for Culture Collection of Microorganisms) and has the preservation number of CGMCC NO. 9910. The hybridoma JYG-1 can secrete the anti-recombinant schistosoma japonica enolase (Sj Enolase) specific monoclonal antibody JYGENmb-1. The monoclonal antibody JYGENmb-1 can be combined with Sj Enolase protein specificity of schistosoma japonica, a sandwich ELISA method for detecting Sj Enolase protein is provided, and the monoclonal antibody can be applied to establishing various immunological methods for detecting Sj Enolase protein in schistosoma japonica infected human and animal blood and excreting secretion.

The invention relates to a method for detecting gene polymorphism with labeled probes and specific primer sequences and a kit including specific probes and the specific primer sequences, and is suitable for biotechnology and medical fields. The kit provides the specific primer sequences and the labeled probes for simultaneous detection of polymorphism of C677T and A1298C loci of a human MTHFR gene. The kit contains a Taq enzyme, the specific primers, the specific probes and an internal standard system. The primers and kit provided by the invention can be used for simultaneous detection of thepolymorphism of the C677T and A1298C loci of the MTHFR gene, and has the advantages of high specificity, high sensitivity, simple and fast operation, high throughput, safety and objective result interpretation.

The present invention is directed to a composition and method for detecting intestinal deficiency. In particular, a 13C-α-limit dextrin that is specifically hydrolyzed by brush border intestinal enzymes is disclosed herein. The 13C-α-limit dextrin as disclosed herein may be used as a substrate in a bi-phasic breath test to detect intestinal glucoamylase deficiency and / or abnormal brush border intestinal enzyme activity.

It is an object of the present invention to provide a fluorescence imaging probe capable of selectively visualizing target cells such as cells expressing β-galactosidase (lacZ expressing cells) at a single-cell level in a red fluorescence region, and of performing co-staining together with GFP.An intracellularly-retainable red fluorescent probe comprising a compound represented by the following formula (I) or a salt thereof:wherein: A represents a monovalent group cleaved by an enzyme; R1 representsa hydrogen atom, or one to four of the same or different substituents bonded to a benzene ring; R3, R4, R5, and R6 each independently represent —CFR10R11, —CF2R12, a hydrogen atom, a hydroxyl group, an alkyl group, or a halogen atom, wherein at least one of R3, R4, R5, and R6 is —CFR10R11 or —CF2R12;R2 and R7 each independently represent a hydrogen atom, a hydroxyl group, an alkyl group, or a halogen atom; R8 and R9 each independently represent a hydrogen atom or an alkyl group; R10, R11, and R12 each independently represent a hydrogen atom, an alkyl group, or an alkenyl group; X represents Si(Ra) (Rb), wherein Ra and Rb each independently represent a hydrogen atom or an alkyl group; and Y is —C(═O)— or —RcC(═O)—, wherein Rc is an alkylene group having 1-3 carbon atoms.

The invention provides a method for extracting sardine polypeptide. The method comprises the following steps: carrying out enzymatic hydrolysis step by step through trypsin and papain, then heating to kill enzymes, performing centrifugation, subjecting the supernate to rotary evaporation, and carrying out freeze-drying to obtain the sardine enzyme. The method is not limited by the enzyme specificity of enzymatic method, has the advantages of mild reaction conditions, wide raw material source, low cost, and simple operation, can be applied to industrial production, and has a high medical and economic value.

The invention relates to a calorimetric bionic competitive detection method for detecting pesticide Atrazine residue, and belongs to the field of biosensors. The method is used for rapid detection of pesticide Atrazine residue in foodstuff or water source samples, and S-MIP is used as an identification element and filled in a reaction column. A competitive binding is carried out between a sample to be measured which is treated by simple pretreatment and a certain amount of beta-lactamase labeled objects to be detected in a mobile phase on blotting sites on S-MIP. Thermal signals released by adding a substrate with enzyme specificity and content of the objects to be detected are in inverse ratio. A standard curve is established based on an optimal experiment condition. The calorimetric bionic sensing method for quantitative detection of the typical pesticide Atrazine with a specific 'amplification method' has the advantages of simple pre-treatment, high sensitivity, good specificity, and quantitative detection.