59 results about "TNase activity" patented technology

The invention discloses a high temperature resistant xanthan gum lyase mutant and a preparation method thereof, wild type xanthan gum lyase comes from Paenibacillus anensis, site-directed mutagenesis is carried out on the wild type xanthan gum lyase to obtain the high temperature resistant xanthan gum lyase mutant, and the mutant comprises at least one of N66V, R292A and S676V. The enzymatic activity of the xanthan gum lyase mutant treated in a buffer solution with the pH value of 5.0 at the temperature of 65-85 DEG C for 2 h is remarkably improved compared with that of an original enzyme, and the enzymatic activity of N66V / R292A / S676V is improved by 3.8 times compared with that of the original enzyme at the temperature of 85 DEG C.

The invention relates to an anti-cryptosporidium micromolecular lead compound of a target cryptosporidium helicase. Comprising the following steps: S1, detecting enzyme activity parameters of a drug target helicase (CpeIF4A) and an inhibition effect of Roc-A on the drug target helicase: expressing and purifying a CpeIF4A recombinant protein containing an MBP tag; detecting the enzyme activity; detecting the inhibition effect of the Roc-A on the enzyme activity; determining the affinity between the Roc-A and the CpeIF4A through a heat transfer experiment; s2, in-vitro drug effect determination: evaluating the direct killing capability of Roc-A on sporozoites; the inhibition effect of Roc-A on cryptosporidium is determined through an in-vitro cell infection model, an EC50 value is obtained, and the intracellular period of the Roc-A mainly playing the inhibition effect is determined; detecting the toxicity of Roc-A to host cells to obtain a TC5 value), and obtaining an in-vitro insect-resistant selectivity index (SI); and S3, in-vivo drug effect test: establishing a C57BL / 6IFN-gamma gene knockout mouse infection model to evaluate the effectiveness and safety of Roc-A. The invention determines that the Roc-A has the potential of being further developed into the cryptosporidium-resistant drug, and verifies that the targeted eIF4A can resist cryptosporidium infection.

The invention relates to a pH-responsive copper hydrogen phosphate-loaded hydrogel with multiple enzyme-like activities and a preparation method and application thereof. The material mainly shows catalase-like activity in a neutral microenvironment, and can remove H2O2 and relieve oxidative stress; in a weakly acidic microenvironment, the compound mainly shows peroxidase-like activity and catalyzes H2O2 to generate. OH bacteria with higher toxicity; meanwhile, the inherent osteogenesis promoting performance of copper synergistically enhances bone regeneration, the biocompatibility, the oxidation resistance, the osteogenesis promoting capacity under the oxidative stress condition and the antibacterial efficiency under the weak acid condition of the SA / CuHP hydrogel are evaluated through in-vitro experiments, and a type II diabetes rat periodontitis model is established; the in-vivo antibacterial and osteogenesis promoting effects and the biological safety performance of the compound are verified, and a new strategy is provided for treatment of periodontitis with diabetes mellitus.

The invention belongs to the technical field of gene engineering, and particularly relates to an IsMHETase mutant with high catalytic activity and application of the IsMHETase mutant. The invention aims to solve the technical problem that the activity of the existing ISMETase is relatively low. The technical scheme of the invention is as follows: the IsMHETase mutant with high catalytic activity is a protein obtained by mutating IsMHETase of which the amino acid sequence is as shown in SEQ ID No.1. The invention also discloses a preparation method of the IsMHETase mutant with high catalytic activity. Six mutants are obtained after screening, and the enzyme activity is improved by 1.13-3.20 times compared with that of a wild type IsMHETase; the IsMHETase mutant obtained by the invention has higher enzyme activity, is more efficient when being used for biodegrading MHET micromolecules, simplifies the degradation steps, reduces the degradation cost, and provides a wide prospect for the application of the IsMHETase mutant in the industrial field.

The invention discloses a high-efficiency wide-targeting-range C-to-G double-enzyme synergistic base editor and application thereof, and belongs to the technical field of gene editing. The editor is a fusion protein comprising a Cas protein having reduced or lost endonuclease activity, a cytosine deaminase (CDA), and a cytosine DNA glycosylase (CDG). In order to solve the problems that an existing C-to-G editor is low in efficiency and limited in targeting range, the C-to-G editor synergistically and efficiently generates a base removal (AP) site on a target DNA through the dual effects of CDA and CDG, so that C-to-G base transversion is promoted. According to the editor, the C-to-G editing efficiency is remarkably improved, an editing window can be effectively expanded or moved, the targeting flexibility is greatly enhanced, meanwhile, high genome specificity is kept, and the editor shows strong application potential in various organisms such as yeast and plants.

The invention relates to the technical field of genes, in particular to preparation of a Trmt61a enzyme activity deleted zebrafish model, and the Trmt61a enzyme activity deleted zebrafish model is used for preparing a zebrafish strain which stably inherits trmt61aD181A-P2A-EGFP knock-in zebrafish strain. According to the invention, trmt61a D181A mutation site specificity is introduced into a zebra fish genome by using a CRISPR / Cas9 technology, and a zebra fish model with Trmt61a catalytic activity deficiency and tRNA m1A modification level significantly reduced is obtained. The zebrafish model fills the existing research blank, provides an available in-vivo research model for biological development regulation mechanism research related to tRNA m1A epigenetic modification and pathological mechanism analysis of diseases (such as malignant tumors and hematopoietic dysfunction), and can be directly used for exploring the core effect of the epigenetic modification in development and diseases.

Provided are a class of compounds, a stereoisomer, an optical isomer, a pharmaceutically acceptable salt, a prodrug, a solvate (for example, a hydrate) or an isotope derivative thereof. The compounds have a tri-heterocyclic structure (for example, the structure represented by formula (A)), which is a novel structure, thereby providing a new direction for the development of SOS1 inhibitor drugs. In-vitro enzyme activity inhibition activity studies show that the compounds have a relatively strong inhibition effect on SOS1 and can be used as a prospective compound for preventing and / or treating SOS1-mediated diseases. Moreover, said compounds also exhibit significant inhibitory activity on NCI-H358 cell proliferation. Furthermore, a specific synthesis method is provided. The synthesis method is simple in process, convenient to operate and beneficial to large-scale industrial production and use.

The invention discloses a phytase mutant with improved enzymatic activity and thermal stability as well as a preparation method and application thereof, and relates to the technical field of gene engineering and enzyme engineering. The amino acid sequence of the phytase mutant (named as Y111C / Q113L) is as shown in SEQ ID NO. 1. According to the invention, site-directed mutagenesis is carried out on amino acid residues which may influence the enzymatic activity and thermal stability of phytase YrAPPA in phytase YrAPPA, and mutants Y111C and Q113L with significantly improved enzymatic activity and thermal stability are constructed and screened. On the basis, the two-site mutant Y111C / Q113L with the phytase activity and the thermal stability further improved is further constructed and obtained. The enzyme activity and thermal stability of the phytase mutant provided by the invention are obviously improved, and the phytase mutant has a good application prospect in the field of animal feed additives.

The invention relates to a method for improving the activity of a Meyerozyma guilliermonii ECH strain for producing ethyl carbamate hydrolase in the technical field of microorganisms, which comprises the following steps: inoculating a bacterial liquid containing the Meyerozyma guilliermonii ECH strain into a liquid fermentation culture medium according to the inoculum size of 10-18%, adjusting the initial pH value to 4-6, and culturing for 3-5 days at the temperature of 25-35 DEG C; the liquid fermentation culture medium contains ethyl carbamate. According to the method, the activity of the strain for producing the ethyl carbamate hydrolase can be remarkably improved.

The invention relates to the technical field of biological detection, in particular to a UXS1 enzyme activity determination method. The method comprises the following steps: a) hydrolyzing a substrate UDP-GlcA by using UXS1 to generate UDP-xyl; b) adding XYLT1 to catalyze the UDP-xyl to generate UDP (User Datagram Protocol); c) converting the UDP into ATP (adenosine triphosphate); and d) quantitatively detecting ATP, and deducing the enzyme activity of UXS1 through the obtained signal intensity. The method can effectively reduce the cost of UXS1 enzyme activity detection, can help a pharmaceutical enterprise to quickly screen drugs of related targets at high throughput, and is beneficial to promoting the research and development process of related drugs.

The application relates to the technical field of enzyme engineering, in particular to a (-)-alpha-bisabolol synthase mutant and application thereof. On the basis of a wild-type (-)-alpha-bisabolol synthase (SEQ ID No: 1), three types of (-)-alpha-bisabolol synthase mutants are constructed through substitution, deletion and addition mutation modes, and the (-)-alpha-bisabolol synthase activity of homologous mutants with different consistency obtained through the above different mutation modes is investigated, and the yield ratio relative to the wild type when used for fermentative production of (-)-alpha-bisabolol is investigated. It is proved that the above mutants all achieve the same or higher (-)-alpha-bisabolol yield as the wild-type (-)-alpha-bisabolol synthase. It is proved that the above mutation modes and the obtained mutants and the recombinant cells for biosynthesizing the mutants can be used for batch production and popularization of (-)-alpha-bisabolol, thereby helping the development of many downstream industries such as beauty and medicine.

The invention discloses a Candida antarctica lipase B mutant and a preparation method thereof, and belongs to the technical field of biology. According to the invention, 11 mutants with improved enzyme activity are obtained, including 6 single site mutants and 5 combined mutants; compared with a wild type, the enzyme activities of the mutants Q11L, F71N, F118Y, V149T, L219N, T244D, S31T / F71N, F71N / F118Y, F71N / L219N, S31T / F71N / F118Y and F71N / F118Y / L219N are all improved, and are respectively improved by 18.45%-240.88%; according to the invention, the CALB mutant with high enzymatic activity is obtained, and the CALB mutant has great industrial application potential and economic value.

This invention relates to the field of gene technology, and more particularly to the preparation of a zebrafish model lacking Trmt61a enzyme activity, for the purpose of preparing a stable genetic model. trmt61a D181A -P2A-EGFP Knock-in zebrafish strains. This invention utilizes CRISPR / Cas9 technology to... trmt61a d181a trmt61a d181a The mutation site was specifically introduced into the zebrafish genome, resulting in the loss of Trmt61a catalytic activity and tRNA m 1 A zebrafish model with significantly reduced tRNA modification levels. This zebrafish model fills a research gap and provides a basis for tRNA m... 1 A provides a usable in vivo research model for studying the biological developmental regulatory mechanisms related to epigenetic modifications and elucidating the pathological mechanisms of diseases (such as malignant tumors and hematopoietic dysfunction), which can be directly used to explore the core role of this epigenetic modification in development and disease.

The present invention is applicable to the field of biomedical technologies, and provides the use of GYY4137 in the preparation of drugs against Cryptosporidium parvum. The GYY4137 inhibits the activity of Cryptosporidium parvum by inhibiting CpLDH. The present invention reveals that GYY4137 is a highly potential candidate drug for targeting CpLDH and inhibiting the survival of Cryptosporidium. At the nanomolar level, GYY4137 can completely inhibit the enzymatic activity of recombinant CpLDH. In addition, in vitro cell culture models have verified that GYY4137 inhibits the growth of parasites at the low micromolar level. These findings not only demonstrate the potential of GYY4137 for further development as an anti-Cryptosporidium drug, but also emphasize that the strategy of targeting CpLDH to combat parasitic infections is feasible.

The application discloses a Candida antarctica lipase B mutant and a preparation method thereof, and belongs to the technical field of biology. Eleven mutants with improved enzyme activity are obtained, including six unit point mutants and five combined mutants; compared with a wild type, the enzyme activity of the mutants Q11L, F71N, F118Y, V149T, L219N, T244D, S31T / F71N, F71N / F118Y, F71N / L219N, S31T / F71N / F118Y and F71N / F118Y / L219N is improved, and is respectively increased by 18.45% to 240.88%; the CALB mutant with high enzyme activity is obtained, and has great industrial application potential and economic value.

The invention discloses an LbuCas13a modified protein and an editing system thereof, the modification of the LbuCas13a modified protein comprises the following mutations: V411T, N414K and V421T which are introduced into a hairpin loop near an LbuCas13a nuclease active center and are responsible for being combined with a substrate RNA (Ribonucleic Acid), and the mutation sites are amino acid sequence 407-424 regions so as to enhance the electropositivity of the hairpin loop and the affinity of the hairpin loop with the RNA. According to the LbuCas13a-V411T-N414K-V421T mutant disclosed by the invention, directional point mutation and structural repeated design are carried out on a substrate RNA (Ribonucleic Acid) binding hairpin loop, so that the binding capacity of the LbuCas13a and a target RNA and the para-line cleavage activity are remarkably enhanced, and an experiment shows that the enzyme activity of the mutant LbuCas13a-V411T-N414K-V421T is improved by 36.7% compared with that of a wild type, and the activity of the repeatedly designed LbuCas13a-V411T-N414K-V421T-loop is improved by 168.6%. The technology has important significance on application of the LbuCas13a in molecular diagnosis, rapid screening of pathogens and public health monitoring.

The present invention belongs to the field of enzyme engineering technology, and specifically relates to a mutant of germaene A synthase and its application. The present invention obtains a series of chicory-derived germaene A synthase (CiGASlo) mutants by computer-aided design, constructs CiGASlo mutants by point mutation PCR method, further expresses recombinant protein and purifies, verifies enzyme activity by in vitro enzymatic reaction, and thus screens out CiGASlo mutants with significantly improved relative enzyme activity. The present invention can provide a CiGASlo mutant with significantly improved catalytic activity, which is helpful to promote and apply in the industrial production process of sesquiterpene compounds such as β-elemene. The present invention has the advantages of mild reaction conditions, high stereoselectivity, simple catalyst treatment steps, and environmental friendliness, and reduces equipment requirements, significantly reduces production costs, and shows a wide range of application prospects in industrial applications.

The invention belongs to the technical field of bioengineering, and particularly relates to an enzyme activity improving mutant of ergothioneine precursor synthetase and application of the enzyme activity improving mutant. According to the present invention, starting from the ergothioneine sulfoxide synthetase CtEgtB from the thermophilic photoacid bacillus thermophilus, based on Apla-Fold 3.0 structure prediction and artificial evaluation, five single-site mutants and combined mutants capable of potentially improving the enzyme activity are selected, and the enzyme activity experiment test is performed; according to the invention, beneficial mutation sites are screened, and the enzyme activity of the optimal mutant is 4.5 times that of an original enzyme. The optimal mutant crude enzyme is applied to a biological enzyme method, 2.3 g / L of ergothioneine sulfoxide is synthesized within 6 hours, and the method can be applied to industrial production of ergothioneine.

The invention discloses a preparation method and application of a mannose hexaphosphate kinase mutant. The method comprises the following steps: constructing a protein prediction model by utilizing AAindex based on PyPEF software; a mutation site combination is predicted through a protein prediction model, a mutant gene is generated according to the predicted mutation site combination, and the mutation site combination comprises H92L, E119R and A138R; the mutant gene is cloned to a pET-28a (+) expression vector and then is introduced into escherichia coli for expression and purification to obtain the mannokinase hexaphosphate mutant. The mutant overcomes the problems of low enzymatic activity, poor stability and the like, and the enzymatic activity of the synthesized M6P is improved by more than 15 times compared with that of a wild type.

The invention belongs to the technical field of biology, and relates to a bifunctional glutathione synthetase mutant and application thereof. The amino acid sequence of the bifunctional glutathione synthetase mutant is shown as SEQ ID NO.3. The enzyme mutant can efficiently catalyze L-glutamic acid, L-cysteine and glycine to generate glutathione, the concentration reaches 38.3 g / L, and the enzyme activity is improved by 4.2 times compared with that before mutation. The glutathione synthesized by using the bifunctional glutathione synthetase mutant provided by the invention has the advantages of high enzyme activity, short conversion time, simple and convenient process, low production cost and the like.

The invention discloses a homoserine dehydrogenase mutant and application of the homoserine dehydrogenase mutant. Compared with wild homoserine dehydrogenase Hom from corynebacterium glutamicum, the amino acid sequence of the mutant has one mutation type of A381D, A381L, A381I, A381P, A381V, A384R, A384D, I397C, I397D, I397G, I397S, I397R, I397V and I397A. Through enzyme activity determination and analysis of induced expression and purification, the enzyme activity of the mutant is remarkably improved compared with that of a wild control. Meanwhile, the feedback inhibition of L-threonine and L-isoleucine can be efficiently relieved. The homoserine dehydrogenase mutant, the coding gene of the homoserine dehydrogenase mutant and the host cell containing the coding gene provided by the invention can be industrially applied to produce L-homoserine and derived amino acids thereof.

The present invention belongs to the field of biotechnology, and discloses a BRAF V600E gene mutation detection method, detection system and kit. The detection system for BRAF V600E mutation disclosed in the present invention comprises a nickase, a guide RNA targeting a mutation site, and a fluorescent reporter system; the nickase has endonuclease activity. The present invention discloses a new application of Crispr technology in BRAF V600E mutation detection. The detection system and detection kit of the present invention can quickly and visually detect BRAF V600E mutations present in inflammation, autoimmune diseases or tumors, and have the characteristics of high sensitivity and high specificity, and have broad application prospects.

The invention provides the development and application of an EGFP reporter system for identifying N4BP1 endoribonuclease activity, relates to the field of biomedical technology, and uses a modified EGFP fluorescent protein to identify the endoribonuclease activity of N4BP1. Through experiments, it is found that the core sequence of the target gene FosC degraded by N4BP1 is determined, and this sequence is connected to the C-terminus of the mRNA of EGFP to construct a new EGFP-mRNA sequence. The N4BP1 overexpression plasmid is co-expressed in HEK293T cells, and then the endoribonuclease activity of N4BP1 can be determined by comparing the intensity of the fluorescence signal. The modified EGFP, due to the connection of the target sequence recognized by N4BP1, the expression amount of EGFP will change according to the amount of N4BP1 enzyme activity. By detecting the intensity of EGFP, the amount and enzyme activity of N4BP1 protein can be determined. Therefore, the GFP reporter system of N4BP1 can be used to detect the enzyme activity of N4BP1. Since the N4BP1 gene plays a key role in immunity, inflammation and tumors. The cell can detect the endoribonuclease activity of N4BP1 in related disease cells.

The present invention discloses a kind of LbuCas13a modified protein and its editing system, and its transformation is included in the introduction of the following mutations in the hairpin loop responsible for binding to substrate RNA near the LbuCas13a nuclease active center: V411T, N414K and V421T, and its mutation site is amino acid sequence 407-424 region, to enhance the electropositivity of the hairpin loop and the affinity with RNA. The present invention significantly enhances the binding ability of LbuCas13a to target RNA and collateral cleavage activity by carrying out directed point mutation and structural repetition design in conjunction with its substrate RNA hairpin loop, and experiments show that the enzyme activity of mutant LbuCas13a-V411T-N414K-V421T is improved by 36.7% compared with wild type, and the LbuCas13a-V411T-N414K-V421T-loopplus activity of repeated design is improved by 168.6%. This technology is of great significance to the use of LbuCas13a for molecular diagnosis, rapid screening of pathogens and public health monitoring.

The invention relates to the field of biological detection, in particular to a biosensor for sensitively and rapidly detecting Whara syndrome helicase activity and application, the biosensor comprises a substrate chain, crRNA, Cas12a protein and a report chain FQ; according to the invention, a DNA-DNA / DNA-RNA substrate chain with complementary sequences at two ends and a non-complementary'bubble 'structure with a specific length in the middle is constructed, the substrate chain can be specifically unwound by WRN, and released single-stranded DNA further activates trans-cleavage activity and non-specific cleavage fluorescence reporter groups of Cas12a, so that unprecedented high-sensitivity and high-specificity detection on WRN helicase activity is realized; the fluorescent probe can be applied to in-situ and real-time fluorescence imaging and visual analysis of WRN activity in living cells, and has wide application prospects in the fields of cancer mechanism research, WRN-targeted inhibitor high-throughput screening, clinical adjoint diagnosis and the like.

The present invention discloses a bifunctional enzyme, a bifunctional enzyme mutant and its application. The amino acid sequence of the bifunctional enzyme is shown in SEQ ID NO:1. The bifunctional enzyme is composed of a CKI enzyme and a CCT enzyme connected by a connecting peptide. The protein sequence of the CKI enzyme is shown in SEQ ID NO:2, the protein sequence of the CCT enzyme is shown in SEQ ID NO:3, and the amino acid sequence of the connecting peptide is shown in SEQ ID NO:4. The present invention has the following beneficial effects: (1) The bifunctional enzyme or mutant can replace the CKI enzyme and the CCT enzyme, so that the two-step reaction is combined into a one-step reaction. Since the intermediate product and the final product have low feedback inhibition on it, the enzyme activity is higher, which is more than 160% of the activity of the two enzymes, and is more suitable for in vitro enzymatic reactions. (2) When using the bifunctional enzyme for enzymatic reactions, only a single enzyme needs to be added to the reaction system, which reduces the production cost of the enzyme. (3) There is no need to consider the ratio of the enzymes in the reaction system, which makes the catalytic process simpler and more repeatable.

The invention discloses a lytic polysaccharide monooxygenase mutant FpLPMO9A-V123T and application thereof, and belongs to the field of enzymology, the optimal temperature of the mutant FpLPMO9A-V123T is 50 DEG C, and the optimal pH is 5. The enzyme activity of the mutant FpLPMO9A-V123T is 1.9 times that of a wild type, and the IC50 value of the mutant FpLPMO9A-V123T is 136.90 min and is far higher than that of the wild type and other mutants, so that the mutant has higher thermal stability. Due to the characteristics, the FpLPMO9A-V123T has remarkable advantages in actual application. According to the invention, the effect can be more effectively exerted, the production efficiency is improved, the cost is reduced, the higher thermal stability means longer service life and lower maintenance cost, and the competitiveness of the device in practical application is further enhanced.

The application discloses a glycosyltransferase UGT91C1 mutant and a method for catalyzing synthesis of rebaudioside D, and belongs to biological catalytic synthesis. The glycosyltransferase UGT91C1 mutant is any one of the following (A)-(C): A) a protein obtained by making any one or more mutations in the amino acid sequence shown in SEQ ID NO. 1: the 89th amino acid is mutated from N to Y; the 155th amino acid is mutated from M to L; the 274th amino acid is mutated from S to T; and the 361st amino acid is mutated from N to S; (B) a protein having 95% or 98% or above identity with the amino acid sequence defined in (A) and having the same function; and (C) a fusion protein obtained by connecting a tag to the end of the protein defined in (A) or (B). The glycosyltransferase UGT91C1 mutant is obtained by mutation screening from a wild-type glycosyltransferase UGT91C1, has higher enzyme activity and catalytic rate, and can efficiently synthesize rebaudioside D by taking rebaudioside A as a substrate.

Provided are a stable Phi29 DNA polymerase having high enzyme activity, and an encoding gene and an application thereof. The Phi29 DNA polymerase is obtained by substituting amino acid residue at at least one among the following 29 positions: position 17, position 96, position 97, position 99, position 123, position 140, position 148, position 158, position 159, position 171, position 203, position 204, position 213, position 217, position 224, position 250, position 270, position 309, position 310, position 320, position 344, position 345, position 347, position 369, position 402, position 416, position 509, position 515 and position 524 of a DNA polymerase shown in SEQ ID NO: 2.

The invention discloses a preparation method and application of a tumor-targeted photo-thermal / radiotherapy sensitizer, and belongs to the technical field of biomedical materials. The preparation method comprises the following steps: (1) preparing Fe3O4 (at) C nanoparticles; (2) dispersing the Fe3O4 (at) C nano-particles in a solution, adding platinum acetylacetonate, and depositing Pt nano-particles through thermal degradation in a protective gas atmosphere to prepare Fe3O4 (at) C (at) Pt nano-particles; and (3) combining the nano-iron platinum particles with a tumor targeting ligand in an electrostatic adsorption manner to prepare the tumor targeting nano-iron platinum particles. Compared with a traditional iron-based nano material, the prepared nano iron-platinum particles have better POD enzyme activity and CAT enzyme activity, and the Fenton catalytic capacity and the photo-thermal conversion capacity of the nano iron-platinum particles are higher. In addition, the hydrogel has good biocompatibility. The compound has no significant toxicity to normal cells, has the potential of targeting tumor sites, and has selective killing ability to tumor cells.