67 results about "Enzyme structure" patented technology

Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a single site within the genome of a cell or subject, e.g., within the human genome. In some embodiments, fusion proteins of Cas9 and nucleic acid editing enzymes or enzyme domains, e.g., deaminase domains, are provided. In some embodiments, methods for targeted nucleic acid editing are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of Cas9 and nucleic acid editing enzymes or domains, are provided.

Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a single site within the genome of a cell or subject, e.g., within the human genome. In some embodiments, fusion proteins of Cas9 and nucleic acid editing enzymes or enzyme domains, e.g., deaminase domains, are provided. In some embodiments, methods for targeted nucleic acid editing are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of Cas9 and nucleic acid editing enzymes or domains, are provided.

Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a single site within the genome of a cell or subject, e.g., within the human genome. The disclosure provides fusion proteins of nucleic acid programmable DNA binding proteins (napDNAbp), e.g., Cas9 or variants thereof, and nucleic acid editing proteins such as cytidine deaminase domains (e.g., novel cytidine deaminases generated by ancestral sequence reconstruction), and adenosine deaminases that deaminate adenine in DNA. Aspects of the disclosure relate to fusion proteins (e.g., base editors) that have improved expression and / or localize efficiently to the nucleus. In some embodiments, base editors are codon optimized for expression in mammalian cells. In some embodiments, base editors include multiple nuclear localization sequences (e.g., bipartite NLSs), e.g., at least two NLSs. In some embodiments, methods for targeted nucleic acid editing are provided.

The invention discloses an advanced treatment agent for textile printing and dyeing wastewater and a treatment method. The advanced treatment agent comprises a component A and a component B, the component A comprises modified graphene oxide and an enzyme treatment agent; the component B comprises magnesium aluminum silicate, hydroxyapatite, hydroxypropyl methyl cellulose and chitosan, and the hydroxyapatite is coated with the magnesium aluminum silicate to form a core-shell structure. By adopting a modified graphene oxide immobilized enzyme structure, harmful components in water can be effectively adsorbed, so that the effect of greatly improving sewage purification is achieved, and meanwhile, the adsorption effect of the harmful components can be further improved by matching with aluminum-magnesium silicate coated hydroxyapatite, hydroxypropyl methyl cellulose and chitosan; meanwhile, bonding with the modified graphene oxide is beneficial to agglomeration and flocculation of substances, so that the flocculation capacity is improved, and floc separation is facilitated.

The present invention relates to a functionally active mutated primase domain from an archaeal DNA primase belonging to the primase-polymerase family, comprising at least one amino acid substitution, wherein said mutated primase domain retains at least 50% of the template-independent terminal nucleotidyl transferase activity of the corresponding wild-type primase domain.

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 invention provides a high-activity pectin methylesterase mutant designed based on a diffusion model as well as preparation and application of the high-activity pectin methylesterase mutant. The amino acid sequence of the pectin methylesterase mutant PME-DS12 is shown as SEQ ID NO: 2. According to the invention, the structure of the pectin methylesterase is redesigned by utilizing an RFdiffusion generative diffusion model, and the mutant PME-DS12 of which the enzyme activity is remarkably improved is obtained by screening. Compared with wild pectin methylesterase, the enzymatic activity of the PME-DS12 is improved by about 6.3 times, and the PME-DS12 has higher industrial application value. The mutant has wide application prospects in the fields of food processing, spinning and the like. In particular, in the aspect of flavor substance generation, macromolecular substances in food are degraded by the enzyme, so that the generation of aroma lipid substances is provided, and the aroma quality of a product is improved; the method can also be used in the papermaking industry, including biological bleaching of paper pulp, reduces the use of chemical reagents, and reduces environmental pollution.

The invention discloses PDA / PtCuO CDs-Gel nano-enzyme composite gel and a preparation method and application thereof in preparation of an antibacterial therapeutic agent.The nano-enzyme composite gel is of a gel-wrapped nano-enzyme structure, PtCuO serves as a core of the nano-enzyme, a polydopamine layer is arranged outside the core, the polydopamine layer is modified with carbon quantum dots, and the carbon quantum dots are modified with PtCuO. The PtCuO is CuO nano particles of which the surfaces are modified with Pt nano particles. According to the nano-enzyme composite gel, H2O2 does not need to be added as an enzyme reaction substrate, the catalytic efficiency is high, the antibacterial performance is high, the cell penetrability and the photothermal conversion efficiency are high, and multi-mode antibacterial treatment can be achieved.

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 current invention relates to methods of targeted genetic alteration in cells, preferably plant cells, as well as to plant cells and plants thus obtained using at least a fusion protein comprising a site-specific nuclease domain and a deaminase domain, or a construct encoding the same. The method also provides for a composition and a kit comprising a combination of a first fusion protein comprising a cytosine deaminase domain and a second fusion protein comprising an adenine deaminase domain, preferably for use in the method of the invention. The method provides for targeted alteration of a DNA duplex in plant cells with increased efficacy.

The present disclosure relates to biocatalytic ligation methods for producing oligonucleotides; and to fusion polypeptides for use in said methods. In particular, the disclosure relates to biocatalytic ligation methods incorporating ATP regeneration, and to fusion polypeptides comprising a polyphosphate kinase domain and an ATP-dependent nucleic acid ligase domain.

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 invention discloses a preparation method of an in-situ encapsulated enzyme material based on a metal polyphenol network, and belongs to the field of immobilized enzyme preparation. The preparation method comprises the following steps: firstly, fully mixing glucose oxidase (GOx) and tannic acid (TA), and combining the glucose oxidase (GOx) and the tannic acid (TA) through hydrogen bonds and hydrophobic interaction to form a TA-GOx compound; then adding a FeCl3. 6H2O solution, oscillating by using a vortex oscillator, and forming a metal polyphenol network through a rapid coordination reaction to package the enzyme inside in a lossless manner, so as to realize the construction of an enzyme in-situ packaging system. The preparation method of the metal polyphenol network in-situ encapsulated enzyme material (20s vortex, room temperature and aqueous solution) is simple, overcomes the limitations that the enzyme is easy to fall off, the enzyme structure is irreversibly damaged and the accessibility of enzyme active sites is poor, and breaks through the size limitation of the enzyme. And the polyphenol and the metal salt are low in industrial price, so that the simple and economical enzyme encapsulation technology has a wide application range and industrial application.

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.

Provided herein are Rep proteins comprising mutated helicase domains, and methods using the Rep proteins for producing recombinant DNA-packaged AAV particles.

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.