28 results about "Specific substrate" patented technology

The invention discloses a glycosyl transferase mutant and an application of the glycosyl transferase mutant in synthesis of rebaudioside. According to the invention, single-point mutation and multi-point mutation are carried out on the basis of a glycosyl transferase amino acid sequence as shown in SEQ ID NO: 1, and a mutant with improved catalytic activity is obtained. The catalytic activity, the substrate specificity and / or the substrate specificity of the glycosyl transferase mutant are / is changed, and the catalytic activity of enzyme to a specific substrate can be remarkably improved by mutation at a specific site. And carrying out induced expression and protein purification on the obtained mutation sequence to obtain the mutant enzyme. The mutant enzyme is used as a catalyst, and UDPG is used as a glycosyl donor, so that the catalytic reaction efficiency of substrates such as stevioside ST, rebaudioside A (RebA) and rebaudioside D (RebD) can be obviously improved. According to the glycosyl transferase UGT76G1 mutant constructed by the research, the catalytic activity of the glycosyl transferase UGT76G1 mutant is improved, and efficient production of rebaudioside M is realized by optimizing a reaction system.

The invention discloses an artificial sensor element combination, nucleic acid for coding the artificial sensor element combination, a recombinant expression vector containing the nucleic acid, a host cell and application of the artificial sensor element combination. The artificial sensor element combination comprises a polypeptide chain 1, a polypeptide chain 2 and a specific substrate, and the method is used for high-throughput detection of protein solubility, stability and enzyme activity. When the artificial sensor element combination is used for detecting protein properties, the problems of inaccuracy, false positive and the like of soluble detection of a conventional protein soluble sensor caused by dynamic characteristics of protein expression, folding and aggregation processes can be at least solved.

The invention discloses a method, medium and device for constructing a general prediction model for classifying mutant enzyme-substrates, and belongs to the technical field of bioinformatics, protein engineering and machine learning, the method comprises the following steps: step 1, expanding data to construct a data set, specifically comprising positive data screening and negative data generation, 2, building a multi-task coupling framework; and 3, optimizing the performance of the prediction model. The invention further provides a medium and a device carrying the model, and the accuracy of interaction prediction is improved through joint optimization of protein and small molecule characterization. Mutation sites capable of remarkably improving the catalytic efficiency are screened out, the activity of the enzyme is optimized, the catalytic ability of the mutant enzyme to different substrates is predicted, and the design of the enzyme with specific substrate selectivity is facilitated.

This invention discloses a quantitative detection kit for arginine peptidase, a qualitative detection kit for arginine peptidase, and their usage methods. The quantitative detection kit utilizes the specific substrate Nα-benzoyl-DL-arginine-4-ylbenzamide hydrochloride, which can be hydrolyzed by arginine peptidase to 4-ylbenzamide. 4-ylbenzamide has a maximum absorption peak at 405 nm, and the specific arginine peptidase activity can be rapidly detected at room temperature using spectrophotometry. The qualitative kit utilizes the specific substrate Nα-benzoyl-DL-arginine-β-naphthamide hydrochloride, which can be hydrolyzed by arginine peptidase to p-β-naphthamide. β-naphthamide combines with 4-(dimethylamino)cinnamaldehyde to form a compound that is visible to the naked eye as a pink color. The disulfide bond cleaving agent cuts the mucus disulfide bonds, exposing bacterial enzyme sites, and the synergistic effect of the enzyme activator enhances the reaction rate of arginine peptidase hydrolysis of the substrate, significantly improving the detection sensitivity of arginine peptidase in tongue coating samples.

The present disclosure provides recruitment of a ULK initiation complex (e.g., a ULK1 initiation complex) used to induce selective autophagy, a process by which the degradative mechanism is targeted to specific substrates. Chimeric compounds for binding to the ULK initiation complex, comprising a ULK complex binding moiety, a linker, and a target binding moiety, are provided, as well as a target of interest.

Provided are a biosensor for detecting Csk kinase activity in cells and a detection method thereof, relating to the technical field of biosensors. The biosensor fuses N-terminal ECFP, an SH2 domain of Src, a flexible linker having 15 amino acids, a substrate polypeptide, and C-terminal YPet; the substrate polypeptide is selected from one of a Src kinase C-terminal tail peptide chain FTSTEPQYQPGENL, a Fyn kinase C-terminal tail peptide chain FTATEPQYQPGENL, and a Csk specific substrate peptide EEEIYFFF. An FRET biosensor provided by the present invention and relating to a fluorescence resonance energy transfer (FRET) technology and gene encoding provides a powerful tool for visualizing signal molecules in living cells with high temporal-spatial resolution, can specifically visualize the dynamic state of the Csk kinase in the cells, and provides a visualization tool for further exploring the action mechanism of in-vivo Csk.

This invention discloses a glycosyltransferase mutant and its application in the synthesis of rebaudioside. Based on the amino acid sequence of the glycosyltransferase shown in SEQ ID NO:1, this invention performs single-point and multi-point mutations to obtain mutants with enhanced catalytic activity. The catalytic activity, substrate specificity, and / or substrate specificity of the glycosyltransferase mutant are altered; mutations at specific sites can significantly improve the enzyme's catalytic activity for specific substrates. The mutant enzyme is obtained by inducing expression and purifying the protein from the obtained mutant sequence. Using the mutant enzyme as a catalyst and UDPG as a glycosyl donor, the catalytic reaction efficiency for the substrates steviol glycoside ST, rebaudioside A (RebA), and rebaudioside D (RebD) can be significantly improved. The glycosyltransferase UGT76G1 mutant constructed in this study improves its catalytic activity, and the efficient production of rebaudioside M is achieved through optimization of the reaction system.

The invention relates to the technical field of enzyme activity detection, in particular to a standardized Glu-C incision enzyme activity spectrum detection method. The method provided by the invention realizes rapid, accurate and low-cost enzyme activity detection by using a specific substrate Z-Phe-Leu-Gl-4-pNA and optimized reaction conditions, and specifically comprises the following steps: diluting a Glu-C endonuclease sample, mixing the diluted Glu-C endonuclease sample with a substrate solution, putting the mixture into a preheated Tris-HCl buffer solution, monitoring absorbance change at the wavelength of 405nm, and calculating the enzyme activity through a specific formula. A standardized Glu-C incision enzyme activity detection system is established, the detection time is short, the cost is low, the detection method has a wide linear range, a low detection limit and high precision, and a technical basis is provided for establishing an industrial unified standard.

Embodiments described herein relate to compounds for detecting wounds (e.g., chronic wounds or infected wounds), including compositions, substrates, kits, dressing materials and articles, and systems containing these compounds. Other embodiments relate to methods for using these compositions, kits and systems for diagnostic assays, and for diagnosing and / or detecting chronic or infected wounds based on enzymatic conversion of specific substrates contained in the compositions. Additional embodiments relate to methods for characterizing wounds based on the expression of multiple markers, and using this information to treat, manage and follow up patients with chronic or infected wounds.

The invention belongs to the technical field of biotechnology and enzyme engineering, and relates to a molecular modification method for improving aldehyde ketoreductase substrate specificity, which comprises the following steps: establishing a substrate library of aldehyde ketoreductase AKR13B3 and analyzing kinetic parameters; the method comprises the following steps: performing loop structure analysis and amino acid sequence comparison on a constructed composite structure of aldoketoreductase AKR13B3, coenzyme NADPH and substrate 3-keto-DON to determine a mutation site, and screening mutants with increased substrate specificity; according to the invention, the geometrical shape of the inlet ring region of the AKR13B3 substrate binding bag is designed to change the substrate preference, so that the catalytic activity of the enzyme to a specific substrate is increased; in addition, a feasible scheme is provided for designing the aldoketoreductase with good substrate specificity, and huge hope is provided for developing efficient enzyme preparations.

The present application relates to the field of bioengineering, and particularly relates to genome, expression module, recombinant strain and application thereof in preparation of rhodiolin. The present application provides a genome comprising one or more of expression gene, knockout gene, overexpression gene and inhibition gene. The present application constructs a strain capable of utilizing sugarcane molasses as the only carbon source to generate rhodiolin, thereby reducing the cost of materials; then the metabolic network of the microorganism is restructured through metabolic engineering, the metabolic flow is optimized, the metabolic flux among different metabolic modules is reasonably distributed, and the efficient synthesis of the target product is promoted; finally, the mining and screening of enzymes are crucial, the enzymes with higher activity and higher specificity for specific substrates are mined and screened based on the combination of the trained large language model and the machine learning model and the structural information of the substrates, which significantly improves the titer and conversion rate of rhodiolin generation, and at the same time, reduces the generation of by-products and the accumulation of precursor substances, and will promote the large-scale production and application of rhodiolin.

The present application belongs to the technical field of biotechnology and enzyme engineering, and relates to a molecular modification method for improving substrate specificity of aldehyde ketone reductase, which is achieved by constructing a substrate library of aldehyde ketone reductase AKR13B3 and analyzing kinetic parameters, determining a mutation site by performing loop structure analysis and amino acid sequence alignment on a complex structure of aldehyde ketone reductase AKR13B3, coenzyme NADPH and substrate 3-keto-DON, and screening a mutant with increased substrate specificity, the present application designs the geometric shape of the entrance loop region of the substrate binding pocket of AKR13B3 to change substrate preference and realize the increase of catalytic activity of the enzyme on specific substrates, in addition, the present application provides a feasible scheme for designing aldehyde ketone reductase with good substrate specificity, and provides great hope for developing high-efficiency enzyme preparations.

The application provides a glucose dehydrogenase mutant, a preparation method, application and freeze-dried powder thereof, and relates to the technical field of biotechnology.The glucose dehydrogenase mutant provided by the application has an amino acid sequence as shown in SEQ ID NO.1.The glucose dehydrogenase mutant has better stability compared with a wild type, and has more specific substrate specificity, and can be used for preparing a blood glucose detection product.

The invention belongs to the technical field of protein research engineering, and discloses a transporter screening method based on an artificial intelligence technology, and the method is based on the artificial intelligence technology, according to protein sequence characteristics, whether protein belongs to transporter is predicted, and the transporter is predicted to be outward transporter, inward transporter or bidirectional transporter. And predicting whether the transporter can transport a specific substrate or not, and screening out the transporter with a specific function by integrating calculation results of the three steps. Compared with a conventional single-scale universal machine learning model, the method provided by the invention provides a more comprehensive prediction solution for screening of transport proteins, and the cost consumed by a wet experiment is greatly reduced.

Provided is a method of controlling a substrate transfer system transferring substrates by controlling a first transfer device and a second transfer device comprising: a process of acquiring process module-specific substrate transfer positions for each of the plurality of process modules, wherein the process module-specific substrate transfer positions are substrate transfer positions of the mounting parts of the first transfer device for allowing the second transfer device to place substrates at centers of the stages, the second transfer device transferring substrates from the mounting parts of the load lock module to the stages of the process modules.

A multi-step process for the preparation of a protein myceliated composition, in which a particular substrate comprising cereals and legumes is colonized with the mycelia of a filamentous fungus. In the process solid fermentation of the substrate is carried out. The process provides a composition that resembles actual meat and that can be used as meat analogue.

The invention discloses a cross-protease substrate cleavage site prediction method and system, and belongs to the technical field of bioinformatics. A mapping relation between protease and a substrate cleavage site is learned based on a space structure of protein, protease interaction information is integrated into modeling of a protease-substrate cleavage site map, and an advanced multi-granularity protein map representation technology is combined. And a universal and explainable substrate splitting site recognition model of the cross-protease is established. The invention also discloses a system for predicting the substrate cleavage site of the cross-protease. Compared with other protease specific models, the method has high universality and accuracy. The model can reflect potential space structures of a plurality of protease specific substrate cleavage sites, provides a function of predicting the substrate cleavage sites of more than 100 proteases, and has an important promotion effect in the field of recognition of the protease substrate cleavage sites.

The present application belongs to the field of biomedical detection technology, and particularly relates to a reaction solution for detecting trypsin-like transpeptidase activity and application thereof. The present application uses BAPNA as a specific substrate, which can produce a detectable signal change in the transpeptidation reaction, and a high-concentration amino group-glycine amide is added as a receptor molecule to enhance the transpeptidation reaction efficiency. Therefore, the present application solves the technical problem that the enzyme activity cannot be detected by the immunization method, has high reaction efficiency, stable and reliable detection process, shortened operation time (within 15 minutes), simple operation, strong practicability, can be used for evaluating the severity of acute allergic reaction, providing scientific guidance for the diagnosis of mast cell-related airway inflammation and mast cell activation syndrome, monitoring the effect of allergic treatment, and has clinical value in airway inflammation typing, differential diagnosis and efficacy monitoring through induced sputum or BAL fluid trypsin activity determination.

The invention discloses taxus chinensis-sourced dioxygenase as well as a preparation method and application thereof, and belongs to the technical field of natural product biosynthesis. The dioxygenase TcOGD1 and the dioxygenase TcOGD2 are identified and obtained from taxus chinensis var mairei, and both the dioxygenase TcOGD1 and the dioxygenase TcOGD2 can participate in oxidation modification and skeleton rearrangement reaction of taxane natural products. The TcOGD1 shows strong catalytic activity and wider substrate adaptability, in-vitro synthesis of the 5 / 7 / 6 type taxane taxupine J with the potential of reversing multidrug resistance of tumors is realized, and a new class of 5 / 7 / 6 type taxane compound is obtained. The TcOGD2 can produce selective oxidation products for specific substrates. The dioxygenase can efficiently catalyze conversion of various taxane structures in vitro, the framework type and structural diversity of taxane natural products are expanded, a key tool is provided for synthesis of taxane drug lead compounds and new drug development, and the dioxygenase has good application prospects.

The present invention discloses a method, medium and device for constructing a general prediction model for classifying mutant enzymes - substrates, belonging to the technical fields of bioinformatics, protein engineering and machine learning. The method includes step 1 of constructing a data set through data augmentation, specifically including positive data screening and negative data generation, step 2 of building a multi-task coupling framework, and step 3 of optimizing the performance of the prediction model. The present invention also provides a medium and a device carrying the model. By jointly optimizing the characterization of proteins and small molecules, the present invention improves the accuracy of interaction prediction, screens out mutant sites that can significantly improve the catalytic efficiency, optimizes the activity of enzymes, predicts the catalytic ability of mutant enzymes for different substrates, and helps to design enzymes with specific substrate selectivity.

The present disclosure provides recruitment of a ULK initiation complex (e.g., a ULK1 initiation complex) used to induce selective autophagy, a process by which the degradative mechanism is targeted to specific substrates. Chimeric compounds for binding to the ULK initiation complex, comprising a ULK complex binding moiety, a linker, and a target binding moiety, are provided, as well as one or more targets of interest. The present disclosure also provides methods of using such compounds or compositions thereof.

The invention relates to improved limonene synthases for biosynthetic production of limonene. engineered limonene synthases with improved characteristics compared to reference limonene synthases are provided. Some engineered limonene synthases have improved limonene production yield. Other engineered limonene synthases have switched enantioselectivity. Still other engineered limonene synthases have improved yield with specific substrates. Some engineered limonene synthases have improvements in a combination of one or more of limonene production yield, enantioselectivity and substrate specificity. Also provided herein is a method of screening limonene synthase, e.g., for characterization of limonene synthases. Further provided is a method of producing limonene using the engineered limonene synthases provided herein.

The present invention relates to the field of biological detection technology, and in particular to a specific substrate of cathepsin S, its application and a kit containing the same. The present invention provides a specific substrate of cathepsin S, ACA-HPVGAPWD-Lys(DNP)-DLys-NH2, which is highly specific to cathepsin S. The kit prepared therefrom can be used for the early diagnosis of dry mouth disease belonging to Sjögren's syndrome. The sample is collected by a special saliva collector, and then mixed with the specific substrate and 2× enzyme reaction solution. The enzyme activity of the sample is determined according to the fluorescence intensity of the reaction solution, thereby diagnosing whether the patient suffers from Sjögren's syndrome. The detection method of the kit is highly sensitive, and non-invasive and non-invasive detection and diagnosis can be performed at an early stage of the onset of the disease. The patient can be effectively controlled as soon as possible due to early diagnosis, thereby reducing the pain and suffering caused by the development of the disease; and the kit is easy to use, has a fast detection speed, does not require high-end instruments and high-end technicians, has a low detection cost, and is suitable for large-scale production.

. There is great interest in developing strategies to identify proteolytic substrates. Synthetic peptide libraries are among the most widely used to screen substrates-based activity, however, this technique is limited by the size of libraries and is time-consuming. Several combinatorial library-based display technologies such as phage, bacteria and yeast display, can identify better substrates for protease activity with higher-throughput sequencing and enrichment of specific substrates over multiple rounds of selection. Even though these profiling techniques provide greater coverage of protease cleavable substrates, such cells are less capable of accessing difficult to deliver sites, pose immunogenicity concerns, and have not been applied to discriminate on-from off-target activity in vivo. Disclosed herein are in vitro and in vivo methods for screening protease substrates.

The invention discloses a human trophoblast organ-like model and a construction method and application thereof. The method comprises the following steps: firstly, digesting and separating trophoblast cells from human placenta villus, inoculating the trophoblast cells into matrigel, and adding an ADMEM / F12 culture medium containing specific growth factors for culturing to form trophoblast-like organs; then morphological observation is carried out, and expression of P-gp, BCRP and MRP1 / 2 is detected from mRNA and protein level to carry out model verification; a co-incubation method is adopted, a plurality of ABC transporter specific substrates are adopted, and the influence of verapamil, KO143 and probenecid on the transportation of the ABC transporter specific substrates is researched; and finally, the method is applied to evaluation of substrate transport inhibition of a clinical drug on the ABC transporter protein. The human trophoblast organ model is constructed for the first time and is applied to ABC transporter mediated drug transport research, the operation method is efficient and rapid, and the organ model can be widely applied to in-vitro high-throughput screening of clinical drugs on ABC transporter inhibitors. The method has a wide application prospect.

The present invention discloses a method for preparing a highly active deoxycytidylic acid deaminase mutant, which relates to the field of bioengineering technology and includes the following steps: 1. Synthesis of deoxycytidylic acid deaminase parent and mutant genes; 2. Screening of superior mutants; 3. Purification of deoxycytidylic acid deaminase and mutants; 4. Determination of enzyme activity. The present invention introduces multiple mutation sites through site-directed mutagenesis technology to optimize the catalytic performance of deoxycytidylic acid deaminase, introduces the required mutations at specific positions of the enzyme to achieve precise regulation of the catalytic performance of the enzyme, significantly improves the catalytic activity and reaction rate of the enzyme, and thus improves the efficiency of the entire biocatalytic process. Site-directed mutagenesis technology enhances the thermal stability and pH stability of the enzyme so that it can maintain catalytic activity under a wider range of conditions. Optimizing the amino acid sequence of the enzyme can make it better adapt to specific substrates or reaction conditions, thereby improving the activity of deoxycytidylic acid deaminase in the dCMP deamination reaction.

The present invention belongs to the field of bioinformatics and discloses a method for predicting kinase-specific substrate proteins that regulate yeast autophagy. The method comprises the following steps: S1: performing temporal transcriptome, proteome, and phosphoproteomic analysis on normal samples, gene knockout samples, and plasmid complementation samples before and after starvation induction; S2: screening for interacting genes, recorded as set A; S3: extracting the transcriptional expression changes, protein expression changes, phosphorylated protein expression changes, and the protein sequence composition corresponding to each gene in set A as features; S4: establishing and training a prediction model; S5: optimizing the model and scoring the genes to predict kinase-specific substrate proteins that regulate yeast autophagy. The method of the present invention can predict kinase-specific substrate proteins that regulate yeast autophagy before traditional experimental verification, effectively narrowing the screening scope and reducing the workload of experimental verification.

The invention belongs to the technical field of biomedical detection, and particularly relates to reaction liquid for detecting trypsin transpeptidase activity and application of the reaction liquid. BAPNA is adopted as a specific substrate, detectable signal change can be generated in the transpeptidation reaction, then high-concentration amino group-glycinamide is added as an acceptor molecule, and the transpeptidation reaction efficiency is enhanced. Therefore, the technical problem that the enzyme activity cannot be detected by an immunoassay is solved, the reaction efficiency is high, the detection process is stable and reliable, the operation time is shortened to be within 15 minutes, the operation is simple and convenient, and the practicability is high; the kit can be used for evaluating the severity of acute anaphylaxis, provides scientific guidance for diagnosis of mast cell related airway inflammation and mast cell activation syndrome, monitors the anaphylaxis treatment effect, and has clinical value in the aspects of airway inflammation typing, differential diagnosis and curative effect monitoring by inducing phlegm or BAL liquid trypsin activity determination.