43 results about "Enzyme structure" patented technology

A Levodopa-selective sensor is provided. A continuous levodopa monitor is also provided. The levodopa sensor includes a sensor area having a working electrode with a levodopa-selective chemistry configured for at least partial implantation in a host. The sensor also includes at least one membrane adjacent the levodopa-selective chemistry. The at least one membrane includes an enzyme domain that at least one enzyme selected from a tyrosinase; a mutated tyrosinase with specificity towards I-3,4-dihydroxyphenylalanine; a dihydroxyphenylalanine 4,5-dioxygenase; a mutated dihydroxyphenylalanine 4,5-dioxygenase with specificity towards I-3,4-dihydroxyphenylalanine; and combinations thereof; a synthase enzyme with specificity towards I-3,4-dihydroxyphenylalanine; a mutated synthase enzyme with specificity towards I-3,4-dihydroxyphenylalanine; a 3,4-dihydroxyphenyl-acetaldehyde synthase; a mutated 3,4- dihydroxyphenyl-acetaldehyde synthase with specificity towards I-3,4-dihydroxyphenylalanine; a dioxygenase enzyme with specificity towards I-3,4-dihydroxyphenylalanine; and a mutated dioxygenase enzyme with specificity towards I-3,4-dihydroxyphenylalanine.

The present disclosure relates generally to transposase domains, in particular transposase domains comprising an amino terminal deletion, as well as transposase domains that form obligate heterodimers and transposase domains comprising a DNA targeting domain.

(1) An artificial nucleic acid molecule comprising (a) at least one of a 3'-untranslated region (3'UTR) element and / or (b) a 5'-untranslated region (5'UTR) element, and (2) a poly(A) tail is described. The artificial nucleic acid molecule may further comprise a polypeptide comprising a reverse transcriptase (RT) domain and optionally an endonuclease domain for genome modification. A system comprising the artificial nucleic acid molecule and a method of using the system are also described.

The invention relates to the technical field of enzyme extraction, in particular to an extraction process of papain. The invention discloses an extraction process of papain. The extraction process comprises the following steps: (1) taking supernate from immature papaya; (2) stirring and mixing the supernatant and the aqueous two-phase liquid, and standing for phase splitting to obtain a lower phase; (3) collecting a lower phase, and removing impurities through ultrafiltration to obtain a concentrated solution; and freeze-drying the concentrated solution to obtain enzyme powder. According to the extraction process disclosed by the invention, an organic solvent is replaced by a two-aqueous-phase liquid, mild phase splitting is realized through the synergistic effect of a surfactant and salt, and the damage to an enzyme structure is reduced; and further purifying and maintaining the enzyme activity by combining ultrafiltration and freeze drying.

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a streptavidin compound preparation with mucus removing and defoaming functions and a preparation process thereof. The preparation consists of streptavidin, sodium bicarbonate, dimeticone, silicon dioxide, polysorbate 80 and a filler / adhesive. The preparation method comprises the following steps: stirring dimeticone and silicon dioxide at 145-155 DEG C to react for 7-8 hours to form a compound through a step-by-step granulation process, performing wet granulation, mixing active ingredients at low temperature, and finally performing dry granulation and sub-packaging. The method is characterized in that 1) streptavidin and dimeticone synergistically decompose mucin and break bubbles, and double-view interference is synchronously eliminated; the pH value in the stomach is maintained to be 7-10 through sodium bicarbonate, the enzyme activity is guaranteed, step-by-step granulation avoids high-temperature damage to the enzyme structure, and 5-minute rapid disintegration is achieved through the 80-mesh sieve granule control technology. The preoperative process is simplified, the drug synergism is improved, and the preparation is suitable for gastroscopy and other scenes.

The application discloses a glutamate decarboxylase multi-site combined mutant and application thereof. The application finds key sites by analyzing the enzyme structure and amino acid sequence of glutamate decarboxylase from Bifidobacterium breve and human GAD65, finally the key amino acid residue sites in the catalytic region and the vicinity of the active center are reformed (C168M, V167F, Y165S, F304Y, Y308F), so that the catalytic activity and specificity of the glutamate decarboxylase are regulated, the reaction type is changed from decarboxylation to incomplete oxidation, and the catalysis of glutamic acid forms the oxidation byproduct succinamic acid. In the process of GAD producing succinamic acid, the side reaction is enhanced, and the main reaction is weakened, so that the produced GABA is further oxidized into succinamic acid. The biological catalysis method can effectively improve the screening probability of the mutant amino acid sites, improve the experimental efficiency and feasibility, and screen the mutant enzyme with enzyme activity obviously superior to the parent enzyme.

The present disclosure relates to biocatalytic ligation methods for generating oligonucleotides and fusion polypeptides for use in the methods. In particular, the disclosure relates to biocatalytic ligation methods that incorporate ATP regeneration and fusion polypeptides that include a polyphosphate kinase domain and an ATP-dependent nucleic acid ligase domain.

The invention belongs to the technical field of antibodies, and discloses a nano antibody for hot start of Taq DNA polymerase and a construction method of the nano antibody. The nano-antibody is a monovalent nano-antibody for sealing a 3 '-5' excision enzyme structural domain of Taq DNA polymerase, a monovalent nano-antibody for sealing a 5 '-3' polymerization active structural domain of Taq DNA polymerase or a bivalent nano-antibody obtained by connecting the monovalent nano-antibody and the monovalent nano-antibody through flexible peptide; the amino acid sequence of the monovalent nano antibody for sealing the 3 '-5' excision enzyme structural domain of the Taq DNA polymerase is shown as SEQ ID NO: 1, the amino acid sequence of the monovalent nano antibody for sealing the 5 '-3' polymerization active structural domain of the Taq DNA polymerase is shown as SEQ ID NO: 2, and the amino acid sequence of the flexible peptide is shown as SEQ ID NO: 3.

Described herein are crystalline forms of small molecule inhibitor compounds (1) of Prolyl Hydroxylase Domain-containing Protein (PHD) and pharmaceutical compositions thereof, and methods of their use in the treatment of diseases or conditions that would benefit from treatment with inhibitors of Prolyl Hydroxylase Domain-containing Protein (PHD). (1)

The invention relates to the technical field of bioengineering, and discloses a glucose dehydrogenase mutant G321R and application thereof. The method comprises the following steps: firstly, carrying out molecular docking on glucose dehydrogenase and a glucose substrate to obtain a series of amino acid sites possibly influencing the enzyme structure and the combination of the enzyme and the substrate, then carrying out virtual saturated mutation on amino acids of the sites, and screening out mutation possibly enhancing the enzyme stability and activity through molecular dynamics simulation. A plurality of amino acid sites and mutation schemes capable of improving the stability and activity of the glucose dehydrogenase are obtained through screening after large-scale expression of the glucose dehydrogenase, the stability and activity of mutants obtained through single-point mutation and multi-point combined mutation of the sites are improved to different degrees, and the mutants can be used for preparing the glucose dehydrogenase mutant. The compound has high activity, can improve detection sensitivity and accuracy when used for detecting blood glucose, has high stability, is suitable for preparing kits and the like, and has wide application prospects.

Described are compositions comprising (1) at least one of: (a) a 3 '-untranslated region (3' UTR) element and / or (b) a 5 '-untranslated region (5' UTR) element; and (2) an artificial nucleic acid molecule with a poly (A) tail. The artificial nucleic acid molecule may further comprise a polypeptide comprising a reverse transcriptase (RT) domain and optionally an endonuclease domain for genome modification. Systems comprising the artificial nucleic acid molecules and methods of using the systems are also described.

Provided are a highly efficient base editing system, a preparation method therefor and the use thereof. Specifically, provided is a fusion protein, comprising a nuclease domain and an editing enzyme domain, wherein the editing enzyme domain is inserted into an insertion site in the nuclease domain. The nuclease domain in the fusion protein has the function of a nuclease, the editing enzyme domain in the fusion protein has the function of an editing enzyme, and the nuclease domain is derived from a Cas protein or a Cas protein mutant. On the basis of an activation-induced cytosine deaminase (AID), a new AID mutant having a shorter sequence and higher base editing efficiency is obtained by means of screening, and then the highly active AID mutant is randomly inserted into nSaCas9 (D10A) KKH, such that a new cytosine base editor EB-TAM having high base editing efficiency and an expanded and narrowed editing scope is successfully obtained by means of screening.

The invention relates to the technical field of bioengineering, and discloses a glucose dehydrogenase mutant S334M and application thereof. The method comprises the following steps: firstly, carrying out molecular docking on glucose dehydrogenase and a glucose substrate to obtain a series of amino acid sites possibly influencing the enzyme structure and the combination of the enzyme and the substrate, then carrying out virtual saturated mutation on amino acids of the sites, and screening out mutation possibly enhancing the enzyme stability and activity through molecular dynamics simulation. A plurality of amino acid sites and mutation schemes capable of improving the stability and activity of the glucose dehydrogenase are obtained through screening after large-scale expression of the glucose dehydrogenase, the stability and activity of mutants obtained through single-point mutation and multi-point combined mutation of the sites are improved to different degrees, and the mutants can be used for preparing the glucose dehydrogenase mutant. The compound has high activity, can improve detection sensitivity and accuracy when used for detecting blood glucose, has high stability, is suitable for preparing kits and the like, and has wide application prospects.

Compounds comprising sulfonyl-purine or sulfonyl-purine analog bonds are described for use as electrophiles in sulfonyl-purine (SuPUR) chemistry. Also described is the use of SuPUR and / or SuPUR chemistry for proteome labeling of reactive amino acid residues and for developing new covalent therapeutic agents that selectively modify select amino acid residues (e.g., tyrosine or lysine residues) in biologically active proteins and peptides, such as amyloid-beta (Aβ)-binding alcohol dehydrogenase (ABAD), guanine nucleotide binding protein alpha stimulating activity polypeptide (GNAS), alpha / beta-hydrolase domain 10 (ABHD10), glutathione S-transferase pi 1 (GSTP1), glucosamine-phosphate N-acetyltransferase 1 (GNPNAT1), poly(ADP-ribose) polymerase 2 (PARP2), and acetyl-CoA acetyltransferase 2 (ACAT2) proteins.

The invention relates to the technical field of bioengineering, and discloses a glucose dehydrogenase mutant S334K and application thereof. The method comprises the following steps: firstly, carrying out molecular docking on glucose dehydrogenase and a glucose substrate to obtain a series of amino acid sites possibly influencing the enzyme structure and the combination of the enzyme and the substrate, then carrying out virtual saturated mutation on amino acids of the sites, and screening out mutation possibly enhancing the enzyme stability and activity through molecular dynamics simulation. A plurality of amino acid sites and mutation schemes capable of improving the stability and activity of the glucose dehydrogenase are obtained through screening after large-scale expression of the glucose dehydrogenase, the stability and activity of mutants obtained through single-point mutation and multi-point combined mutation of the sites are improved to different degrees, and the mutants can be used for preparing the glucose dehydrogenase mutant. The compound has high activity, can improve detection sensitivity and accuracy when used for detecting blood glucose, has high stability, is suitable for preparing kits and the like, and has wide application prospects.

The application discloses an enzyme turnover number prediction method based on a deep learning model and protein structure information, and the method comprises the following steps: constructing a dataset with unique enzyme sequence-enzyme structure-substrate combination and enzyme turnover number mapping in an offline stage, and training an enzyme turnover number prediction model; and in an online stage, real-time prediction of enzyme turnover number of a to-be-processed protein structure is realized through the trained enzyme turnover number prediction model. By adding protein structure information to enzyme turnover number prediction training and using a deep learning method to mine the potential relationship between the two, the model generalization effect can be effectively enhanced, and the problems of low prediction model accuracy and limitation to sequence similarity in the prior art are solved.

The application discloses a kind of nano bubble regulated hydrogen bond organic framework immobilized enzyme, and hydrogen bond organic framework immobilized enzyme is prepared by in-situ self-assembly and embedding method in water environment containing nano bubble from four (4-amidinophenyl) methane and four (4-carboxyphenyl) methane.The hydrogen bond organic framework is used to realize the immobilization of enzyme by in-situ embedding method, and a multi-level pore immobilized enzyme system based on nano bubble is constructed.Nano bubble can expand the pore size and pore volume of hydrogen bond organic framework as pore-forming template, while maintaining the stability of enzyme structure, significantly improving the mass transfer rate of substrate molecules in the framework, thereby enhancing the catalytic activity and substrate affinity of immobilized enzyme.The method effectively improves the enzyme catalytic reaction efficiency while maintaining the system immobilization efficiency, pH stability, temperature stability and reusability.The process is simple and suitable for the construction of multi-level pore immobilized system for various enzymes.

Provided herein are fusion proteins comprising transposase domains and DNA targeting domains. In particular, the DNA targeting domains may be targeted to the lipoprotein A (LPA) gene. Also provided are methods of making the transposase domains and fusion proteins, cells that are modified using the fusion proteins provided herein and methods of treatment using such cells.

The invention discloses a nucleotide detection method based on FEN1 mediated linear amplification reaction and a universal microarray and application. The method comprises the following steps: 1) triggering template activation by an invasive reaction based on FEN1 enzyme structure specific recognition, and further triggering linear isothermal amplification; 2) realizing colorimetric, optical, Raman spectrum and electrochemical results by utilizing multi-type signals formed by combining probes with various signal labels and spatial positioning output; and (3) constructing a standardized array, and enabling the microarray platform to have extremely strong adaptive compatibility to different targets through the universal design of a primer region and a recognition region. According to the detection method, the whole process from amplification to reading can be completed within 48 minutes, the sensitivity can reach 101 copies / mu L, and the detection method is suitable for rapid screening of methylation, gene mutation, SNP and pathogens, is particularly suitable for a microfluidic or paper-based chip integrated platform, and has the advantages of low cost, high throughput, simplicity and convenience in operation and the like.

The invention discloses a composite protective agent for liquid laccase and application of the composite protective agent, and belongs to the technical field of bioengineering. The protective agent takes a citric acid-disodium hydrogen phosphate buffer solution with the pH value of 4.5-6.5 as a solvent, and comprises propylene glycol, glycerol, sorbitol, cane sugar, maltodextrin, glycine, trehalose, potassium sorbate, diacetic acid and a mediator. The components form a stable microenvironment around enzyme molecules through a synergistic effect, the polyhydric alcohols and the saccharides stabilize an enzyme structure through hydrogen bonds and glassy state protection, the glycine plays a buffering role, and the mediator has dual functions of oxidation resistance and mediated catalytic reaction. Experiments prove that the protective agent can remarkably improve the thermal stability and storage stability of the liquid laccase, the enzyme activity retention rate reaches 86.7% after the liquid laccase is treated at 50 DEG C for 24 h, and the enzyme activity retention rate is still kept at 85.3% after the liquid laccase is stored at 37 DEG C for 180 days. The laccase is especially suitable for degrading aflatoxin B1, has a degradation effect obviously better than that of unprotected laccase, and can be widely applied to the fields of food industry, feed industry and the like.

The present invention addresses the problem of providing a substance having a stronger Ras inhibitory effect and / or a stronger anticancer effect. The problem is solved by a fusion protein comprising a Ras-cleaving / modifying enzyme domain and a Ras-binding domain.

Conjugation reagents for making, and targeted degrader conjugates made therefrom, comprising a Janus family kinase binder and a CD127 binding agent, are described. In particular, the conjugation reagents are compounds of Formula (I) where TM is a JANUS family kinase domain-targeting binder; L1 is a first linker; L2 is a second linker; and E3L is a E3 ligase ligand; wherein, TM, L2 and E3L are covalently linked to L1, and L2 comprises a reactive moiety for reacting with a binding molecule. (I)

This disclosure generally relates to transposase domains, in particular, transposase domains comprising amino terminal deletions, as well as transposase domains forming obligate heterodimers and transposase domains comprising DNA targeting domains.

Provided herein are methods and compositions comprising pharmacological inhibitors of HIF hydroxylases (e.g., or more inhibitors of Prolyl Hydroxylase Domain enzymes (PHDs) or Factor Inhibiting HIF (FIH)) for enhancing cyanide resistance in living organisms.

This invention proposes a method for screening and optimizing enzyme catalytic efficiency and stability based on protein language models and domain knowledge. The method includes: defining a set of allowable mutation intervals through multidimensional analysis; constructing a dual-source mutant library using a dual-source complementarity strategy based on protein language models and co-evolutionary models; using the dual-source mutant library as the initial population, employing a multi-model fusion catalytic efficiency prediction strategy as the population's overall fitness calculation method, and a dual-index verification strategy based on the SPIRED-Stab model for structural stability as the population screening method; iteratively obtaining the optimal mutant enzyme sequence through a semantically guided genetic algorithm; and verifying the structure and dynamic stability of the optimal mutant enzyme sequence based on three-dimensional structure prediction and molecular dynamics simulation to obtain an experimentally feasible final mutant enzyme sequence. This invention achieves improved optimization efficiency while ensuring the structural feasibility of key enzymes.

The invention discloses a glutamate decarboxylase multi-site combined mutant and application thereof. According to the invention, key sites are found by analyzing enzyme structures and amino acid sequences of glutamic acid decarboxylase derived from lactobacillus brevis and human GAD65, and finally key amino acid residue sites (C168M, V167F, Y165S, F304Y and Y308F) near a catalytic region and an active center are modified; therefore, the catalytic activity and the specificity of the glutamate decarboxylase are regulated and controlled, so that the reaction type of the glutamate decarboxylase is changed from decarboxylation to incomplete oxidation, and glutamic acid is catalyzed to form an oxidation byproduct succinamic acid. In the process of producing the succinamic acid from the GAD, the side reaction is enhanced, and the main reaction is weakened at the same time, so that the produced GABA is further oxidized into the succinamic acid. A biological catalysis method can effectively improve the screening probability of mutant amino acid sites, the experiment efficiency and feasibility are improved, and the mutant enzyme of which the enzyme activity is obviously superior to that of a parent enzyme is obtained through screening.

The invention relates to a structural analysis-based rational transformation method for improving enzymatic properties of beta-mannase under an alkaline condition, and belongs to the technical field of enzyme engineering and industrial biology. The method comprises the following steps: on the basis of a three-dimensional structure model of beta-mannase, identifying aromatic amino acid residues which are positioned in a catalytic center or near a substrate binding channel and play an important role in enzyme conformation stability and catalytic function; on the basis, establishing a negative limitation principle of rational design of catalytic activity, namely avoiding amino acid replacement of the aromatic amino acid residues in an enzyme engineering design process; on the premise of following the negative limitation principle, a substrate binding structure domain of the beta-mannase is constructed in series, so that the enzyme structure stability and the substrate binding capacity are synergistically improved, and the beta-mannase with improved stability and catalytic performance under the alkaline condition is obtained. According to the method, blind mutation is avoided, a rational design method for improving the enzymatic performance of the beta-mannase under the alkaline condition is provided, the rational design method has a clear structural basis and an engineering modification path, and the method has a good industrial application prospect.

The invention provides an efficient base editing system and a preparation method and application thereof. Specifically, the invention provides a fusion protein which comprises a nuclease structural domain and an editing enzyme structural domain, wherein the editing enzyme structural domain is inserted into an insertion site in the nuclease structural domain, and the nuclease structural domain in the fusion protein has a nuclease function; the editing enzyme structural domain in the fusion protein has an editing enzyme function; the nuclease structural domain is derived from a Cas protein or a Cas protein mutant. According to the present invention, based on activation induction cytosine deaminase AID, a novel AID mutant with characteristics of short sequence and high base editing efficiency is screened, and then the high-activity AID mutant is randomly inserted into nSaCas9 (D10A) KKH so as to successfully screen the novel cytosine base editor EB-TAM with characteristics of high base editing efficiency, wide editing range and narrow editing range.

The present application relates to the technical field of bioengineering, and discloses a glucose dehydrogenase mutant with enhanced stability and activity; firstly, molecular docking is carried out on glucose dehydrogenase and glucose substrate to obtain a series of amino acid sites which may affect enzyme structure and enzyme-substrate binding; then, virtual saturation mutation is carried out on the amino acids of the sites; through molecular dynamics simulation, mutations which may enhance enzyme stability and activity are screened; after starting wet experiment and screening through large-scale expression, multiple amino acid sites and mutation schemes which improve the stability and activity of glucose dehydrogenase are obtained; the mutants obtained through single-point mutation and multi-point combined mutation of the sites have different degrees of improvement in stability and activity; the high activity is used for detecting blood glucose, and can improve detection sensitivity and accuracy; the high stability is suitable for preparing reagent kits, and has a wide application prospect.

The invention provides a construction method and application of a P450 fusion protein. The construction method comprises the following steps: step (1), fusing abamectin oxidation type P450 Ema1 with a Rhodococcus P450RhF-RPs reductase structural domain by adopting a connecting peptide to obtain Ema1-Rhf; and (2) carrying out point mutation on the Ema1-Rhf, and constructing the P450 fusion protein. According to the invention, redox partners derived from Rhodococcus are connected with a structural domain of P450 Ema1 through a designed connecting peptide; the cytochrome P450 monooxygenase mutant further improves the enzyme activity in the process of catalyzing abamectin to be converted into methylamino abamectin, the maximum conversion rate can be improved by 2.2 times through determination, and the mutant achieves 90% conversion rate within 39.8 hours, which is far better than the expression of an unfused system. And the method has certain significance on industrial production of the emamectin benzoate.