41 results about "TNase activity" patented technology

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.

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 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.

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 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 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 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 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.

The present application relates to the field of proteins, in particular to a protein combination for detecting ADAMTS13 activity and application thereof.The protein combination for detecting ADAMTS13 activity provided by the present application is composed of two protein subchains Chain A and Chain B, wherein Chain A comprises a circularly arranged GFP as shown in SEQ ID NO.1, a V Part1 of a vWF variant as shown in SEQ ID NO.2 and a negative chain polypeptide as shown in SEQ ID NO.5; Chain B comprises a nanobody as shown in SEQ ID NO.6, a V Part2 of a vWF (D1596-R1668) variant as shown in SEQ ID NO.3 and a positive chain polypeptide as shown in SEQ ID NO.4.The protein combination for detecting ADAMTS13 activity provided by the present application can directly determine the ADAMTS13 enzyme activity in a blood sample, a biological sample or a chemical sample, and is suitable for clinical point-of-care testing, biological sample screening and drug development scenarios.

The invention discloses an artemisia annua alpha-farnesene synthase gene AaAFS as well as a mutant and application thereof, the alpha-farnesene synthase gene AaAFS is cloned from artemisia annua for the first time, the gene can specifically catalyze farnesyl pyrophosphate to synthesize alpha-farnesene, in order to further improve the enzymatic activity, AaAFS protein is analyzed, and the alpha-farnesene synthase gene AaAFS can be used as an alpha-farnesene synthase gene AaAFS. And single-site and multi-point mutants of a series of key sites are obtained. Experiments prove that a plurality of mutants (such as S387A, G292A, L487V and combinations thereof) can obviously improve the yield of alpha-farnesene, and the yield of triple mutants (S387A / G292A / L487V) is improved by about 300% compared with that of a wild type. The AaAFS gene and the high-performance mutant thereof provided by the invention provide core elements for green and efficient production of alpha-farnesene by means of synthetic biology, and have important application value in industrial and medicinal aspects.

The invention belongs to the technical field of biological engineering, and particularly relates to a method for improving the activity of beta-galactosidase (LacZ) by fusing a biological aggregate element and a recombinant engineering strain. According to the method disclosed by the invention, a biological aggregate protein element with phase separation capacity is introduced and is subjected to fusion expression with beta-galactosidase, and a target enzyme is induced to form a biological condensed state structure with catalytic activity in cells, so that the biological aggregate is highly enriched in a cell polar region; the local concentration and the reaction efficiency of the beta-galactosidase are effectively improved. According to the strategy provided by the invention, the enzyme activity of the beta-galactosidase is obviously improved and can be about 8 times. The method has the advantages of simplicity and convenience in operation, high universality, capability of being expanded to other functional proteins and the like, is suitable for a biosynthesis system combining high-efficiency expression and activity optimization, and has a wide industrial application prospect.

The application relates to the field of biomedical technology, in particular to a substrate modified peptide for detecting ADAMTS13 enzyme activity and a detection method and application. The application provides a substrate modified peptide for detecting ADAMTS13 enzyme activity, the amino acid sequence of the substrate modified peptide is shown as SEQ ID NO. 1, and the modification method is that a 5-FAM group is marked at the 4th amino acid and a Dabcyl group is marked at the 15th amino acid. The substrate modified peptide and the detection method provided by the application are not only high in accuracy, but also significantly improve the detection sensitivity.

The invention discloses alpha-L-arabinofuranosidase AraA02173 as well as a truncated body and application thereof, and belongs to the technical field of microorganisms. The invention provides amino acid sequences of AraA02173 and a truncated body thereof and a nucleotide sequence of a coding gene of the AraA02173. Characterization information is provided, and the characterization information includes that CBMs in the enzyme have a key maintaining effect on the temperature, pH stability and enzyme activity of an enzyme catalysis module of the CBMs. The purified enzymes of the AraA02173 and the truncation provided by the invention have obvious alpha-L-arabinofuranosidase activity, 4-nitrophenyl-alpha-L-arabinofuranoside can be degraded, the AraA02173 and the truncation also have beta-xylosidase activity, the specific enzyme activity of the AraA02173 is obviously superior to that of the truncation, and the AraA02173 and the truncation can be used for degrading 4-nitrophenyl-alpha-L-arabinofuranoside. Therefore, the alpha-L-arabinofuranosidase AraA02173 has great development potential, provides powerful theoretical support for preparing commercial enzyme preparations, and is of great significance in broadening related application of the alpha-L-arabinofuranosidase.

The invention discloses a Candida antarctica lipase B mutant and application 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; wherein compared with a wild type, the enzyme activities of the mutants Q11L, F118Y, V149T, L219N and T244D are all improved by 76.39%, 20.97%, 20.29%, 18.45% and 34.06% respectively, and the enzyme activities of the mutants Q11L, F118Y, V149T, L219N and T244D are respectively improved by 76.39%, 20.97%, 20.29%, 18.45% and 34.06%; 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.

Provided is a compound comprising the structure:(SIG)-(SI-MOD)m.In this compound, SIG is a signaling molecule, SI is a self-immolative structure bound to SIG such that SIG has a reduced signal relative to the signal of SIG without SI, MOD is a moiety bound to SI that is subject to modification by an activator, and m is an integer from 1 to about 10. With this compound, when MOD is modified by an activator, SI is destabilized and self-cleaved from SIG such that SIG generates an increased signal. Also provided is a method of determining whether a sample comprises an activator, using the above-described compound. Additionally provided is a method of determining whether a cell comprises a nitroreductase using the above-described compound where nitroreductase is the activator. Further provided is a method of determining whether a mammalian cell is hypoxic using the above-described compound where nitroreductase is the activator. A method of detecting a microorganism that comprises a nitroreductase, using the above-described compound where nitroreductase is the activator, is also provided. Also provided is a method of identifying nitroreductase in a sample, using the above-described compound where nitroreductase is the activator.

The application belongs to the technical field of biomolecule detection and analysis, and discloses a kind of POMs type mimic enzyme and its application.The POMs type mimic enzyme is a kind of transition metal substituted POMs, has better catalytic oxidation TMB ability, and spermine can produce enzyme activity inhibition to it for direct detection of spermine molecules.The transition metal substituted POMs is composed of 2 Keggin type three-void phosphotungstic acid polyoxy anion, 10 protons and 20 or 24 crystal water molecules.The spermine detection sensor constructed by the POMs type mimic enzyme is a new colorimetric detection biomolecule spermine strategy based on heteropoly acid, does not need to rely on complex pretreatment derivatization process and expensive instrument operation system, and can realize rapid, qualitative or quantitative determination of spermine in the system under ultraviolet detector or visual state.

The application discloses chalcone synthase mutants and application thereof, and belongs to the technical field of biology.The application obtains multiple CHS mutants, which are 1.1-2.96 times of the yield of wild-type synthesized naringenin, wherein the best mutant is CHS-3-15, and the yield of naringenin is 1.85 mM.Through in-vitro characterization and pure-enzyme activity comparison, the specific activity of CHS-2-7, CHS-2-12, CHS-3-15 and CHS-3-27 is 1.11-2.30 times of that of the wild type.To improve the synthesis of naringenin and reduce the synthesis of by-product CTAL, a specific chassis is constructed, and the CHIL protein capable of reducing by-products is co-expressed, the CHIL expression amount is optimized by optimizing the ribosome binding site sequence, and finally the yield of naringenin reaches 3.98 mM in a shake flask.

The invention discloses an amylosucrase mutant with improved thermal stability and application of the amylosucrase mutant. The amylosucrase as shown in SEQ ID No.1 is subjected to single-site or multi-site mutation, and the enzymatic activity and thermal stability of the mutant are improved to different extents along with superposition or increase of mutation sites. Wherein the four-site mutant V3 (D100S / L278M / G397 / Y427F) is optimal in performance, the optimal reaction temperature is increased by 5 DEG C compared with that of an initial enzyme, the half-life period at the residual temperature of 45 DEG C is 42.1 times that of the initial enzyme, the residual activity is increased by about 742%, and the enzyme activity is increased by about 157%. Due to the improvement of the thermal stability, the activity can be maintained under a wider temperature condition, so that the reaction is more effectively catalyzed, and the efficient synthesis of the pinose is realized.

The application provides feruloyl-CoA 6'-hydroxylase surface mutants and applications thereof. The feruloyl-CoA 6'-hydroxylase surface mutant is obtained by amino acid mutation of an amino acid sequence shown in SEQ ID NO. 1, and includes at least one of the following amino acid mutations: D102E, E163K, E190K, L196F, K199N. The application also provides an engineered bacterium for synthesizing coumarin compounds, which is constructed by using the feruloyl-CoA 6'-hydroxylase mutant. The feruloyl-CoA 6'-hydroxylase mutant has good enzyme activity and stability, and the enzyme activity is increased by more than 1.7 times, and can even be increased by 8.4 times. Therefore, the engineered bacterium can realize high-efficiency biosynthesis of scopoletin, fraxidin, fraxin, scopolin, fraxidin, fraxin A or chicoricin, and provides a reference for microbial synthesis of coumarin compounds.

The application discloses a high-enzyme-activity xanthan backbone cleavage enzyme mutant and a preparation method thereof. Microbacterium sp. ​ The application discloses a high-enzyme-activity xanthan backbone cleavage enzyme mutant and a preparation method thereof, wherein the wild-type xanthan backbone cleavage enzyme of XT11 is subjected to site-directed mutation, and the mutation point of the site-directed mutation comprises at least one of W753V, H755G and L810S. The xanthan backbone cleavage enzyme mutant of the application can directly degrade the xanthan backbone without side chain pretreatment, thereby simplifying an enzymolysis system, and the enzyme activity of the mutant after being treated by using an alkaline buffer system at 55 DEG C is obviously improved compared with the original enzyme, wherein the three-mutant W753V / H755G / L810S has the largest improvement range, and the three-mutant W753V / H755G / L810S overcomes the limitation that a traditional xanthan cleavage system depends on side chain pre-deconstruction.

The invention discloses application of xylanase, and belongs to the technical field of bioengineering. According to preferred codons of bacillus subtilis, a wild type xylanase (GH11 family) coding gene which is secreted by Cellulostridium morganii and is not represented is designed, and a genetic engineering technology is utilized, so that the wild type xylanase is subjected to recombinant expression in the bacillus subtilis. Through verification, the separated xylanase still has xylan hydrolysis activity under the conditions of high temperature (close to 100 DEG C) and strong alkali (pH 9-10.5), and shows good heat resistance and alkali resistance; and meanwhile, more than 60% of enzyme activity can still be maintained in a high-temperature (100 DEG C) environment within 1 hour, and the strain has relatively high thermal stability and has a good application prospect in extreme environments such as industrial production and the like.

The present invention relates to a small molecule lead compound against Cryptosporidium that targets the helicase of Cryptosporidium; it includes the following steps: S1. Detection of the enzymatic activity parameters of the drug target helicase (CpeIF4A) and the inhibitory effect of Roc-A thereon: expressing and purifying the CpeIF4A recombinant protein containing an MBP tag; detecting its enzymatic activity; detecting the inhibitory effect of Roc-A on the enzymatic activity; determining the affinity between Roc-A and CpeIF4A through a thermal shift experiment; S2. In vitro efficacy determination: evaluating the direct killing ability of Roc-A against sporozoites; determining the inhibitory effect of Roc-A on Cryptosporidium through an in vitro cell infection model, obtaining the EC50 value, and determining the intracellular stage at which it mainly exerts an inhibitory effect; detecting the toxicity of Roc-A to host cells, obtaining the TC50 value, and obtaining the in vitro anti-parasite selectivity index (SI); S3. In vivo efficacy test: establishing a C57BL / 6 IFN-γ gene knockout mouse infection model to evaluate the effectiveness and safety of Roc-A. The present invention determines that Roc-A has the potential for further development as an anti-Cryptosporidium drug, and confirms that targeting eIF4A can resist Cryptosporidium infection.

The application discloses a dienolactone hydrolase mutant and application thereof. The application provides a protein, which is a dienolactone hydrolase mutant, wherein the amino acid residue at the 184th position in the amino acid sequence shown in SEQ ID NO:2 is mutated, and the amino acid residues at other positions are not changed, so that a protein with PBAT hydrolase activity is obtained; the wild-type PmDLH is mutated by using structure analysis and site-directed mutagenesis technology, and the PmDLH mutant T184H is obtained. Compared with the wild-type PmDLH, the problem of low enzyme activity of the wild-type PmDLH is changed, the activity of the PmDLH in degrading the polyester plastic PBAT is effectively improved, the degradation effect of the PmDLH is improved, and the industrial application prospect is good.