205 results about "Recombinant enzyme" patented technology

The present invention relates to the intrathecal (IT) administration of recombinant enzyme to treat lysosomal storage disorders. In an exemplary embodiment, intrathecal administration of human α-L-iduronidase (rhIDU) injections in MPS I affected animals resulted in significant enzyme uptake, significant rh-iduronidase activity in brain and meninges and a decrease of glycosaminoglycan (GAG) storage in cells of MPS I subjects to that of normal subjects. Intrathecal administration proved more effective than intravenous treatment at alleviating MPS I symptoms, indicating it is a useful method of treating lysosomal storage disorders.

The present invention relates to recombinantly modified enzymes, which exhibit increased NAD(H) affinity compared to a unmodified or wildtype enzyme, gene sequences or polynucleotides that code for the recombinantly modified enzymes, plasmids and microorganisms that contain these gene sequences; as well as, methods of making and methods of using the enzymes of the present invention.

The invention discloses a CRISPR / Cpf1 gene editing system and application of the system in mycobacteria. The CRISPR / Cpf1 gene editing system comprises a recombinant vector carrying optimized FnCpf1 genes and pCR plasmids, wherein the sequence of the optimized FnCpf1 genes is shown in SEQ ID No:1, and the recombinant vector is a colibacillus-mycobacterium shuttle vector containing recombinant enzyme gp60 and gp61 genes; the pCR plasmids contain promoter-driven direct repeat sequence-spacer sequence-direct repeat sequence units, wherein the spacer sequence contains target sequence connection sites. When the CRISPR / Cpf1system is used in the mycobacteria for CRISPR / Cpf1 assisted homologous recombination, high recombination efficiency can be obtained, and gene editing of the mycobacteria can be achieved.

The present invention relates to the use of recombinant homoserine transsuccinylase enzymes with altered feedback sensitivity (MetA*) and possibly recombinant S-adenosyl methionine synthetase enzymes with reduced activity (MetK*) for the production of methionine, its precursors or derivatives thereof.

The present invention is directed to a recombinant enzymes having alcohol and aldehyde dehydrogenase activity which comprises one or more recombinant polypeptides selected from the group consisting of polypeptides which are identified by SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 and chimeric recombinant polypeptides that are a chimeric combination of at least two of the following amino acid sequences identified by SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 and functional derivatives of the polypeptides identified above which contain addition, insertion, deletion and / or substitution of one or more amino acid residues, wherein said enzymatic polypeptides have said alcohol and aldehyde dehydrogenase activity. DNA molecules encoding the recombinant polypeptides, vectors comprising such DNA molecules, host cells transformed by such vectors and processes for the production of such recombinant enzymes are provided. Furthermore, the recombinant enzymes having alcohol and aldehyde dehydrogenase activity are used for obtaining aldehydes, ketones or carboxylic acids, and specifically, 2-keto-L-gulonic acid an intermediate for the production of L-ascorbic acid (vitamin C).

Isolated and / or recombinant enzymes that include surface binding domains, surfaces with active enzymes bound to them and methods of coupling enzymes to surfaces are provided. Enzymes can include large and / or multiple surface coupling domains for surface coupling.

The invention discloses a construction method and an application of a corynebacterium gene continuous knockout system, belonging to the technical field of genetic engineering. The corynebacterium gene continuous knockout system comprises a template plasmid and a temperature-sensitive expression plasmid, wherein the template plasmid provides a kan fragment (also known as a kan box) with loxp or variant loxL / R sites at two ends; and the temperature-sensitive expression plasmid carries a Cm resistance gene cat and a temperature-sensitive C. glutamicum replicon, expresses a Cre recombinant enzyme and is used for removing a Km resistance gene kan. The corynebacterium continuous knockout system disclosed by the invention can be generally used for continuous gene transformation of corynebacterium and has the advantages of simplicity and convenience in operation and high efficiency. By utilizing the knockout system, gene (continuous) knockout of three major typical subspecies genomes of the corynebacterium can be successfully completed, and the system is proved to be applicable to research and production of corynebacterium metabolites.

Methods of producing an unsaturated free fatty acid comprising at least 18 carbon atoms are provided. In some embodiments, the methods comprise culturing an engineered microorganism in a culture medium, wherein the engineered microorganism comprises at least one recombinant enzyme selected from acyl-lipid desaturase delta-9 (EC:1.14.19.1), acyl-lipid desaturase delta-12 (EC:1.14.19.6), acyl-lipid desaturase delta-15 (EC:1.14.19.-), and thioesterase (EC:3.1.2.14). Engineered microorganisms comprising at least one of those recombinant enzymes are also provided. The methods and organisms can be used to produce at least one free fatty acid selected from oleic acid, linoleic acid and α-linolenic acid.

The invention discloses clone and expression of an Alpha-glucosidase (abbreviated as AGLU enzyme) gene and belongs to the fields of enzyme gene engineering and enzyme engineering. The invention obtains the aglu gene with SEQ ID NO of 1 through an Aspergillus niger (A.niger) WX-07 total DNA, and aglu cDNA uses an expression vector of plasmid pPIC9K and an expression parasitifer of pichia stipitis (P.pastoris), thus realizing extracellular dissolubility expression of the aglu gene; and the aglu cDNA totally has 2880 nucleic acid and encoding of 960 amino acid, constructs eukaryotic expression plasmid and transforms Pichia pastoris KM71 for AGLU enzyme expression. The recombinant enzyme has transnucleosidation activity, can generate isomalto oligosaccharide through maltose transnucleosidation, is applicable to the requirements of industrial application including food, feeding stuffs and medicaments and the like, and can be applied into the industrial production of isomalto oligosaccharide.

The invention relates to incision-type sodium alginate lyase as well as an encoding gene and application thereof. The amino acid sequence of the incision-type sodium alginate lyase AlgL-5 is as shown in SEQ ID NO.2. A gene for encoding the incision-type sodium alginate lyase AlgL-5 is as shown in SEQ ID NO.1. Main oligosaccharide products generated in a process of degrading sodium alginate by using the incision-type sodium alginate lyase AlgL-5 include unsaturated disaccharide, unsaturated trisaccharide, unsaturated tetrasccharide and unsaturated pentasaccharide. Pentasaccharide is the smallest unsaturated oligosaccharide substrate of lyase, and disaccharide is the smallest unsaturated oligosaccharide product of lyase. The recombinant enzyme is stable in property and has certain industrial application potential.

A medical prosthetic device or medical implant containing a metal material (A) selected from the group consisting of titanium or an alloy thereof, zirconium or an alloy thereof, tantalum or an alloy thereof, hafnium or an alloy thereof, niobium or an alloy thereof and a chromium-vanadium alloy, wherein surface parts of the metal material (A) are coated with a layer of a corresponding hydride material (B) selected from titanium hydride, zirconium hydride, tantalum hydride, hafnium hydride, niobium hydride and chromium and / or vanadium hydride, respectively, said device or implant being characterised in that the layer of hydride material (B) comprises one or more biomolecule substances (C) associated therewith. The device or implant exhibits improved biocompatibility. The metal material (A) is preferably titanium. The biomolecule substance (C) may be selected from the following types of substances: Natural or recombinant bio-adhesives; natural or recombinant cell attachment factors; natural, recombinant or synthetic biopolymers; natural or recombinant blood proteins; natural or recombinant enzymes; natural or recombinant extracellular matrix proteins; natural or synthetic extracellular matrix biomolecules; natural or recombinant growth factors and hormones; natural, recombinant or synthetic peptide hormones; natural, recombinant or synthetic deoxyribonucleic acids; natural, recombinant or synthetic ribonucleic acids; natural or recombinant receptors; enzyme inhibitors; drugs; biologically active anions and cations; vitamins; adenosine monophosphate (AMP), adenosine diphosphate (ADP) or adenosine triphosphate (A TP); marker biomolecules; amino acids; fatty acids; nucleotides (RNA and DNA bases); and sugars.

The invention relates to a construction method and an application of high-yield gamma-aminobutyric acid recombinant escherichia coli / pET-28a-1pgad, in particular to a genetic engineering bacterium construction method, recombinant enzyme enzymology property study and an application to the conversion of L-glutamic acid for producing gamma amino butyric acid (GABA), which belongs to the technical field of biology in the fermentation engineering. Firstly, lactobacillus plantarum GB 01-21 glutamic acid decarboxylase (GAD) genes are obtained through polymerase chain reaction (PCR) amplification, recombinant plasmids pET-28a-1pgad are constructed, in addition, the successful expression is realized in E.coli BL21(ED3), secondly, Ni column affiliation chromatography purification is adopted on crude enzyme liquid for obtaining recombinant GAD, in addition, the enzymology property of the recombinant GAD is primarily studied for guiding the optimization of conversion conditions, finally, conversion experiments are carried out on a 5L fermentation tank, the GABA accumulation concentration can reach 204.5g / L, the mol conversion rate is 97.92 percent, and good foundation is made on the further industrial application.

The invention relates to a normal-temperature and constant-temperature nucleic acid amplification method using a fluorescence probe. The normal-temperature and constant-temperature nucleic acid amplification method includes: under constant temperature, single-strand binding protein partially opens the double-strand parental strand into a single strand; recombinant enzyme is combined with primer to form complex, the complex is combined to the parental strand under the action of auxiliary protein, and the fluorescence probe is combined with a complementation area; DNA polymerase is combined to the 3' tail end of the primer to perform sub-strand extension; exonuclease identifies the tetrahydrofuran locus on the fluorescence probe under a double-strand state, fluorescent groups and quenching groups are separated after enzyme digestion to release fluorescence; after the fluorescence probe is cut off, the 3'-OH end originally sealed by probe 3' tail end modification is exposed, and DNA polymerase can continuously extend to form a sub-strand; qualitative and semi-quantitative detection can be performed on a to-be-detected sample by detecting the shape of an amplification curve and the strength of a fluorescence signal. The nucleic acid amplification method has the advantages that normal-temperature and constant-temperature detection can be performed, and qualitative and semi-quantitative detection can be performed on nucleic acid to-be-detected objects.

A preparation with the activities of laccase, xylanase and peroxidase for paper-making, environment production, food and feed industries is prepared through cloning new genes of laccase, xylanase and peroxidase, configuring their expression carrier, introducing it to yeast or mycelial fungus for expression, checking the activity of recombinant enzymes, and mixing said recombinent enzymes together.

Heparin is synthesized from a polysaccharide comprised of a 1-4 glycosidically linked alternating polymer of uronic acid and glucosamine residues, wherein the uronic acid is selected from iduronic and glucuronic acid, wherein the glucosamine is partially N-sulfated; by a series of selective reactions catalyzed by recombinant enzymes.

The invention belongs to the technical field of food industry biology, and relates to an issatchenkia terricola acetaldehyde dehydrogenase gene and an expression vector thereof. The sequence of the gene is SEQ ID No. 16, the open reading frame sequence of the gene is SEQ ID No. 17, and the amino acid sequence coded by the gene is SEQ ID No. 18. The gene is inserted into the expression vector to transform escherichia coli for IPTG (isopropyl beta-D-1-thiogalactopyranoside) inducible expression. The expressed recombinant enzyme can catalyze acetaldehyde oxidation to generate acetic acid. The invention provides a novel high-activity acetaldehyde dehydrogenase gene; and the gene realizes recombinant expression in the escherichia coli, overcomes the defect of low yield of acetaldehyde dehydrogenase produced by fermenting the issatchenkia terricola XJ-2, can provide a large amount of acetaldehyde dehydrogenase for researching alcohol relieving products, and lays a foundation for constructing safe acetaldehyde dehydrogenase gene engineering bacteria.

The invention concerns aspergillus niger 9891 CGMCC NOú‘0991, which produces alantin endonuclease, and clones alantin endonuclease gene. The result of target gene fragment sequence analysis indicates that open reading frame of gene not containing signal peptide is 1485bp, and codes 509 amino acid, the said protein molecular weight is 55.9KD. The homolog of said gene with counterpart of aspergillus niger (Ohtak.et alú¼ 1998)and Aspergillus ficuum(Uhmú¼t.ú¼et alú¼1998) are respectively 92úÑ and 95úÑ. The alantin endonuclease gene is inserted into Pichia pastoris expression vector, thus recombinant of transformed Pichia pastoris is obtained. The said gene expresses in Pichia pastoris, and expressed product has its function. The expression amount of good recombinant I3-50 is 84 times higher than alantin endonuclease of initial strain. Recombinant yeast is induced and fermented. The analysis about recombinant enzyme shows that its optimum PH is 5.5ú¼ and optimum reaction temperature is 55íµ..

The present disclosure relates to filamentous fungal host strains and recombinant DNA constructs for creation and use thereof. The filamentous fungal host strains are particularly useful for achieving reliable expression of recombinant enzymes and variants.

The invention discloses a method for constructing a recombinant expression vector simultaneously expressing a plurality of genes. An MISSA system consists of a strain and a vector, wherein the strain comprises a donor strain and an acceptor strain; the vector comprises a donor vector and an acceptor vector; replication initiation protein carried by a chromosome of the donor strain is responsible for replicating and amplifying a suicidal donor vector, and the carried conjugal transfer protein Tra is responsible for conjugal transfer of the donor vector; the acceptor strain can induce and express two sets of locus specific recombinant proteins, namely Cre recombinant enzyme and lambda phage locus specific recombinant proteins; when the donor strain is mixed with the acceptor strain, the donor strain and the acceptor strain are conjugated, and the donor strain is transferred into the acceptor vector because the donor vector in the donor strain carries an oric element; and the donor vector and the acceptor vector undergo recombination reaction in the acceptor strain under the action of the two sets of locus specific recombinant proteins, so that a target gene carried by the donor vector is assembled onto the acceptor vector. The process is repeated, so a plurality of target genes can be assembled onto the acceptor vector according to the preset direction and order.

The invention relates to the genetic engineering field, especially to a strain Bispora sp.MEY-1 which produces an acidic xylanase and the acidic xylanase XYL10A got from the strain having an amino acid sequence shown as SEQ ID NO.1 or 2, a gene for coding said xylanase having a nucleotide sequence shown as SEQ ID NO.1 or 2, and application of a recombinant vector, a recombinant strain and a recombinant enzyme containing said gene. The xylanase of the invention has the following advantages: the optimum pH 2.4, the optimum temperature 80 DEG C, good pH stability and thermostability, specific activity 5437 U / mg, good protease resistance and easy for industrial fermentation production. The product of the invention can be widely used for the animal feeding-stuffs, the food, the medicament, the brewing and the energy industry as a novel enzyme preparation.

The invention provides a 2-deoxy-D-ribose-5-phosphate aldolase (DERA enzyme) which is capable of catalyzing an aldol condensation reaction at high performance. Compared with a wild type escherichia coli DERA enzyme, the 2-deoxy-D-ribose-5-phosphate aldolase has improved substrate resistant ability and more efficient catalytic ability. If the DERA enzyme provided by the invention is used as a catalyst, the problem of the inactivation of the DERA enzyme caused by a high-concentration chloroacetaldehyde substrate can be solved, and simultaneously, the volume of addition of the enzyme in the production process is reduced. The invention also provides a nucleic acid for coding the DERA enzyme, a recombinant expression vector containing the nucleic acid, a recombinant expression transformant containing the recombinant expression vector, a method for preparing a recombinant DERA enzyme and a catalyst for forming an optically active statin intermediate through catalysis.

The invention discloses a method to introducing multiple DNA fragment in DNA carrier, which comprises the following steps: augmenting two or more goal DNA fragment with oligonucleotide isogeneic limb through polyose chain-reaction; transforming to permissive state cell with recombinant enzyme vector and armed reforming carrier; getting the reforming carrier with one step through consangnuinity reconstruction of oligonucleotide; making the first fragment 5' end of goal DNA fragment possesses the same sequence with armed replacing position left side of target carrier; setting the last 3' end possesses the same sequence with armed replacing position left side of target carrier; making each fragment 5' end possesses the reversing complementary sequence with the last fragment 3' end from the second fragment. The method of this invention is simple, which can be a general method to reform DNA carrier.

The invention provides a neosporosis rELISA antibody assay kit. The neosporosis recombinant enzyme-linked immunosorbent assay (rELISA) antibody assay kit is characterized in that designing a primer set according to a neospora caninum NcSRS2 gene of the GenBank, introducing the primer set to an enzyme digestion site, removing a signal peptide zone of an NcSRS2 protein, extracting a DNA from Xinjiang cow positive serum, carrying out polymerase chain reaction (PCR) amplification of the DNA to obtain a tNcSRS2 genetic fragment having length of 975bp, cloning the tNcSRS2 genetic fragment into a pMD-18-T carrier, carrying out enzyme digestion and sequencing, further directionally connecting the desired fragment obtained by the previous step to a pGEX-4T-2 expression carrier, transferring the pGEX-4T-2 expression carrier with the desired fragment into escherichia coli, carrying out inducible expression and purification, wherein expressed fusion protein molecular weight is about 62kDa, and carrying out ELISA antigen coating by the expressed fusion protein to obtain the neosporosis rELISA antibody assay kit. A use method of the neosporosis rELISA antibody assay kit comprises the following steps that a coated enzyme label reaction plate is taken out, is subjected to first washing, is enclosed, is subjected to second washing, is added with a sample needing to be detected, is subjected to third washing, is added with a secondary antibody, is subjected to fourth washing, and undergoes a color reaction; and when the color reaction is finished, an OD450 / 630 value of the coated enzyme label reaction plate is read and a result is determined. Compared with a commercial ELISA assay kit, the neosporosis rELISA antibody assay kit has a higher coincidence rate above 95% The neosporosis rELISA antibody assay kit also is suitable for the ELISA of a neospora caninum antibody in ruminant serum and blood plasma.

Provided is a production method whereby it is possible to produce a powder containing crystalline trehalose dihydrate with starch as a starting material with high powder-to-starch yields. More specifically, provided is a production method for a powder containing crystalline alpha, alpha-trehalose dihydrate and having at least 98.0mass% of alpha, alpha-trehalose on an anhydrous basis, said method including: a step in which liquefied starch is subjected to the actions of a starch debranching enzyme, cyclomaltodextrin glucanotransferase, an alpha-glycosyl trehalose producing enzyme derived from a micro-organism belonging to the genus arthrobacter, and a trehalose releasing enzyme derived from a micro-organism belonging to the genus arthrobacter, and then to the action of glucoamylase, to obtain an alpha, alpha-trehalose-containing saccharide solution; a step in which alpha, alpha-trehalose dihydrate is crystallized from the saccharide solution; and a step in which the crystallized alpha, alpha-trehalose dihydrate is extracted by centrifugation, aged, and dried. The production method is characterized in that a natural or recombinant enzyme derived from a micro-organism belonging to the genus paenibacillus or a variant of such an enzyme is used as the cyclomaltodextrin glucanotransferase such that the content of alpha, alpha-trehalose in the saccharide solution is over 86.0mass% on an anhydrous basis, without having to undergo a column chromatography fractionation step.

The invention relates to a gene knockout targeting vector, which comprises a positive selective gene. Two ends of the positive selective gene are provided with a recombinant enzyme recognition sequence; the other end of the recombinant enzyme recognition sequence is respectively connected with a 5' homologous arm and a 3' homologous arm of a knockout gene; promoters for promoting the expression of the positive selective gene are not arranged in the two homologous arms; and the outer sides of the homologous arms are provided with negative selective gene. The targeting vector transfects cells, positive clones are screened through a culture medium containing a medicament corresponding to positive selective gene, and the obtained positive clones have high enrichment efficiency; and the cell screening method is simple without massive manpower and material resources, the subsequent cell freezing and identification are greatly facilitated, and the cost of the gene targeting is greatly reduced.

The invention discloses a method for inserting a target DNA fragment into a vector. The method comprises the following steps of: 1) designing and synthesizing a primer for amplifying the target DNA fragment and amplifying by using the primer to obtain the target DNA fragment, wherein 3 to 20 bases on the outmost side of the primer are completely complementary with the outmost side of a linear vector; and 2) mixing the target DNA fragment and the linear vector, and adding a recombinant enzyme with the excision enzyme activity and the homologous recombination enzyme activity from ends 3' to 5' for reaction to obtain a recombinant vector into which the target DNA fragment is inserted. The method has the advantages of dual effects of excision and homologous recombination, more flexible selection for the number of bases at the recombinant ends by selecting the number of homologous bases of a PCR product and the linear vector in a wider range (namely 3 to 20 bases), no need of DNA ligase, linearization for the target vector only, no need of enzymatic treatment on the PCR product, direct mixing with a target vector, one-stop solution for all the problems within 20min, realization of perfect seamless connection, simple operation and low cost.

The invention discloses a method for improving the thermal stability of enzyme. The method is used for obtaining the recombinant enzyme with the improved thermal stability through fusion expression of parents small peptide and the recombinant enzyme, wherein the sequence of the optimized parents small peptide is DWLKAFYDKVAEKLKEAFKVQPYLDDWLKAFYDKVAEKLKEAF. The method has the advantages of being remarkable in effect, simple in process and convenient to promote. The method serves as a new method and idea for quickly improving the thermal stability of the industrial enzyme.