594 results about "Isomerase" patented technology

The present application relates to the field of biotechnology, and in particular to an epimerase for synthesizing D-tagatose and its application, wherein the epimerase produces a mutation at position 100, or at position 125, or at position 127, or at position 131, or at position 268, or at position 270, or at position 271, or at position 308, or at position 339, or at position 340, or at position 342, or at position 366, or at position 402, or at position 437, or at position 440 in the amino acid sequence shown in SEQ ID NO. 142; through the above-mentioned method, the epimerase exhibits excellent epimerization activity at the 4th carbon position of D-fructose, can efficiently convert D-fructose into D-tagatose, and is beneficial to improving the synthesis efficiency of D-tagatose.

The invention belongs to the technical field of biology, and relates to an escherichia coli engineering strain for producing L-isoleucine through a methyl malic acid way as well as a construction method and application of the escherichia coli engineering strain. According to the invention, Escherichia coli BW25113 is used as a chassis strain, and a methyl malic acid synthase high-activity mutant gene cimA3.7, an isopropyl malic acid isomerase gene GsleuCD, a 3-isopropyl malic acid dehydrogenase gene AfleuB and a leucine dehydrogenase gene Lsleudh are over-expressed; a methyl malic acid absorption protein gene is over-expressed, and a branched chain amino acid transport system is modified to improve the exosome of the L-isoleucine; all methyl malic acid pathway related genes are integrated to a genome of a chassis strain, and a plasmid-free L-isoleucine production strain is constructed; the copy number of a key gene on a genome is optimized, and the yield of the engineering strain L-isoleucine is increased by introducing a non-oxidative glycolysis approach. The yield of the L-isoleucine after the engineering strain is fermented for 34 hours in a fermentation tank is 56.6 g / L.

According to the D-psicose 3-epimerase mutant and the application thereof, the D-psicose 3-epimerase mutant is obtained through protein C-terminal sequence modification, software analysis and sequence alignment of different sources of D-psicose 3-epimerase, the half-life period of the mutant enzyme at 60 DEG C, 70 DEG C and 80 DEG C is prolonged, and the activity of the mutant enzyme is improved. The substrate affinity and conversion efficiency of the enzyme to D-fructose as a substrate for synthesizing D-psicose are improved, and the Tm value of the mutant is 73.23 DEG C and is improved by 11.98 DEG C compared with that of a wild type; the half-life period at the temperature of 60 DEG C is 255.35 min and is 17.60 times that of a wild type, the problem that the half-life period of the wild type is too short at the high temperature is solved, the relative action time of the mutant enzyme is long, the yield of the product D-psicose is remarkably increased by applying the genetically engineered bacterium containing the modified enzyme for biotransformation, the product yield is increased by 4.3%, the equilibrium conversion rate reaches 33%, and the yield of the product is greatly increased. And a green, environment-friendly, sanitary and safe biological preparation method is provided for the preparation process of the D-psicose.

The invention discloses a mesophilic carotenoid-producing recombinant yarrowia lipolytica strain as well as a construction method and application thereof, the recombinant strain exogenously expresses mesophilic original fatty acid desaturase (FAD3) and isocitrate dehydrogenase (IDH2) mutants from phaffia rhodozyma, and efficient synthesis under the condition of 30 DEG C is realized; secondly, a metabolic pathway of the recombinant strain is optimized based on a metabolic flow push-pull strategy, and isopentenyl pyrophosphate isomerase (IDI), farnesyl pyrophosphate synthase (ERG20), plant alkene dehydrogenase (CarB) and bifunctional plant alkene synthase (CarRP) are overexpressed, so that the flux of a precursor is increased; finally, gene multi-copy of astaxanthin synthase (crtS) and cytochrome P450 reductase (crtR) from phaffia rhodozyma is carried out, and the carotenoid production capacity of yarrowia lipolytica is further improved. The construction method of the mesophilic yarrowia lipolytica is simple, efficient synthesis of carotenoid can be better achieved at the higher temperature, the fermentation cost is greatly reduced, and the industrial production potential is achieved.

The invention provides a keto-alcohol acid reductoisomerase mutant and application of the keto-alcohol acid reductoisomerase mutant in construction of genetically engineered bacteria for high yield of D-pantothenic acid. In order to solve the technical problem that in the prior art, the yield of D-pantothenic acid synthesized through microbial fermentation is not high, semi-rational design of enzyme is adopted, and a keto-alcohol acid reductoisomerase mutant with substrate specificity is obtained and applied to construction of genetically engineered bacteria for high yield of D-pantothenic acid. According to the invention, a genetically engineered bacterium with high yield of D-pantothenic acid is constructed by utilizing the keto-alcohol acid reductoisomerase mutant through a CRISPR-Cas9 gene editing technology, so that the yield of D-pantothenic acid is increased by about 12.04%, and compared with an original strain, the valine accumulation amount in obtained fermentation liquor is almost unchanged compared with that of a control, and the yield of D-pantothenic acid is greatly improved. However, the accumulation of branched chain amino acid isoleucine is less, so that the modification of the substrate specificity of the keto-alcohol acid reductoisomerase is achieved.

The invention discloses an inositol dehydrogenase mutant and a preparation method of D-chiral inositol, and belongs to the technical field of genetic engineering. The inositol dehydrogenase mutant is obtained by mutating an amino acid sequence as shown in SEQ ID NO.2 through the following mutations: the 200th amino acid is mutated into C from V, the 234th amino acid is mutated into Q from V, and the 256th amino acid is mutated into E from R. According to the invention, the amino acid sequence of wild type inositol dehydrogenase is mutated to obtain the inositol dehydrogenase mutant which is a high-activity mutant capable of catalyzing conversion of myo-inositol into 2-keto-myo-inositol; therefore, when the inositol dehydrogenase mutant and keto isomerase act together to prepare D-chiral inositol by taking myo-inositol as a substrate, the yield of the D-chiral inositol is effectively improved.

The specification relates to conjugates comprising one or more Dc and pharmaceutically acceptable salts thereof. The specification also relates the use of the conjugates for the treatment of diseases such as cancer, and intermediates useful for the synthesis of the conjugates.

The invention discloses a multi-target anti-tumor active rutaecarpin derivative or a pharmaceutical salt thereof. The structural general formula of the rutaecarpin derivative or the pharmaceutical salt thereof is shown as a formula I or a formula II: # imgabs0 #, wherein R1 is selected from C1-C5 alkoxy, hydroxyl and hydrogen; r2 is selected from hydrogen and halogen; according to the present invention, the enzyme inhibition activity and in vitro anti-tumor activity test results show that the compound of the present invention has strong inhibition activity on topoisomerase 1, topoisomerase 2 and c-MYC oncogenes, such that the new approach is developed for the deep research and the development of the new structure type anti-tumor drug, and the new strategy is provided.

The invention relates to the field of biological engineering and technology, in particular to an enzyme for synthesizing glabridin, a biological synthesis method of glabridin and a bacterial strain. The invention discloses a series of glabridin synthesizing enzymes (phenylalanine ammonialyase, cinnamic acid-4-hydrogenase, tyrosine ammonialyase, p-coumaric acid coenzyme A ligase, chalcone synthase, chalcone reductase, chalcone isomerase, isoflavone synthase, 2-hydroxyisoflavone dehydratase, 4 '-oxymethyltransferase, 4'-hydroxymethyltransferase and 4 '-hydroxymethyltransferase) derived from glycyrrhiza glabra for the first time. ) can be used in the preparation of a compound (e.g., isoflavone 2 '-hydrogenase, isoflavone reductase, Vissisoketoreductase, isoprenyltransferase, pterocarpin reductase, oxidative cyclase, demethylase, cytochrome P450 reductase, and the like). The de novo synthesis of the glabridin in a microbial cell factory is realized for the first time, and the application prospect of the glabridin biological manufacturing industry is greatly promoted.

The invention provides a keto-alcohol acid reductoisomerase mutant and application of the keto-alcohol acid reductoisomerase mutant in construction of genetically engineered bacteria for high yield of D-pantothenic acid. In order to solve the technical problem that in the prior art, the yield of D-pantothenic acid synthesized through microbial fermentation is not high, semi-rational design of enzyme is adopted, and a keto-alcohol acid reductoisomerase mutant with substrate specificity is obtained and applied to construction of genetically engineered bacteria for high yield of D-pantothenic acid. According to the invention, a genetically engineered bacterium with high yield of D-pantothenic acid is constructed by utilizing the keto-alcohol acid reductoisomerase mutant through a CRISPR-Cas9 gene editing technology, so that the yield of D-pantothenic acid is increased by about 12.04%, and compared with an original strain, the valine accumulation amount in obtained fermentation liquor is almost unchanged compared with that of a control, and the yield of D-pantothenic acid is greatly improved. However, the accumulation of branched chain amino acid isoleucine is less, so that the modification of the substrate specificity of the keto-alcohol acid reductoisomerase is achieved.

The present application relates to ketol reductoisomerase variants, polynucleotides encoding the variants of the present disclosure, L-valine-producing microorganisms comprising the ketol reductoisomerase variants of the present disclosure or the polynucleotides encoding the variants, and methods of producing L-valine using the microorganisms of the present disclosure.

The invention discloses L-arabinose isomerase, engineering bacteria and application, the amino acid sequence of the L-arabinose isomerase is shown as SEQ ID NO: 3, and the gene sequence for coding the L-arabinose isomerase is shown as SEQ ID NO: 2. On the basis of a genome of a Peribacillussp.S4 strain, a gene for coding the L-arabinose isomerase is excavated, and a stable prokaryotic expression system is constructed by performing codon optimization on the gene, so that the large-scale controllable production of the L-arabinose isomerase is realized, the yield of the enzyme is remarkably increased, the production cost of the enzyme is reduced, and the method is suitable for industrial production. A stable and economical enzyme source is provided for the production of D-tagatose; the L-arabinose isomerase expressed by the constructed engineering bacteria can significantly improve the yield of D-tagatose, and industrialization of D-tagatose is facilitated.

The invention discloses an inositol dehydrogenase mutant as well as a preparation method and application thereof, and belongs to the technical field of gene engineering. The inositol dehydrogenase mutant is obtained by mutating an amino acid sequence as shown in SEQ ID NO.1, and the mutation is that the 247th amino acid is mutated from K to R, or the 266th amino acid is mutated from E to D. The inositol dehydrogenase mutant disclosed by the invention has higher enzyme activity, and when the inositol dehydrogenase mutant and ketoisomerase are catalyzed to synthesize D-chiral inositol by taking myo-inositol and NADP < + > as substrates, the reaction is more inclined to a forward reaction, so that the yield of the D-chiral inositol and the conversion rate of the substrate myo-inositol are further improved.

The invention relates to the technical field of synthetic biology, metabolic engineering and cell culture. The present invention provides a cell for the production of a negatively charged, preferably sialylated, oligosaccharide wherein the cell is genetically engineered to have or express, preferably overexpress, a hydrolytic UDP-N-acetyl-D-glucosamine-2-epimerase. The invention further provides the use of said cells in culture or incubation. Also described are methods of producing negatively charged, preferably sialylated oligosaccharides using the cells, and purification of the negatively charged, preferably sialylated oligosaccharides.

The invention provides recombinant escherichia coli for producing chondroitin sulfate A and application of the recombinant escherichia coli, and relates to the technical field of genetic engineering. According to the recombinant escherichia coli disclosed by the invention, chondroitin synthase, chondroitin 4-O-sulfotransferase, N-acetyl glucosamine epimerase, UDP-glucose dehydrogenase and chondroitin sulfate degrading enzyme are overexpressed; wherein the amino acid sequence of the chondroitin sulfate degrading enzyme is as shown in SEQ ID NO. 5. The recombinant escherichia coli is inoculated into a culture medium for fermentation culture, the average molecular weight of the obtained chondroitin sulfate A is 800Da, and the yield is about 12.5 g / L. Therefore, low-molecular-weight chondroitin sulfate A can be obtained and the yield of chondroitin sulfate A can be increased by fermenting the recombinant escherichia coli disclosed by the invention.

The invention discloses a genetically engineered bacterium for producing glucuronic acid as well as a construction method and application of the genetically engineered bacterium, and belongs to the technical field of genetic engineering. According to the genetically engineered bacterium for producing glucuronic acid, an inositol oxidase gene MMIOX is integrated on a site of a uronic acid isomerase gene uxaC. According to the invention, an inositol oxidase gene MMIOX is integrated on a genome of a genetically engineered bacterium for producing glucuronic acid, and a uronic acid isomerase gene uxaC is knocked out at the same time, so that the genetically engineered bacterium can efficiently catalyze inositol reaction to generate glucuronic acid through the integrated inositol oxidase gene MMIOX; the glucuronic acid isomerase gene uxaC is knocked out, so that isomerization reaction of glucuronic acid can be avoided, and the yield of glucuronic acid is further increased.

The invention belongs to the field of gene engineering, and discloses a sucrose isomerase mutant, a high-yield isomaltulose engineering bacterium and a construction method and application thereof. According to the sucrose isomerase mutant obtained through rational design of an enzyme molecular structure, the enzyme activity and the isomaltulose conversion rate of the sucrose isomerase mutant are remarkably improved, compared with a wild enzyme, the specific enzyme activity of the sucrose isomerase mutant is improved by 2.29 times, the isomaltulose conversion rate is improved to 98.9% from 91.1%, the impurity content is lower, and therefore the sucrose isomerase mutant is more suitable for synthesis of isomaltulose. A recombinant engineering bacterium of the high-yield isomaltulose engineering bacterium is further constructed; the recombinant engineering bacterium capable of efficiently expressing the sucrose isomerase is constructed by utilizing ubiquitin-like protein modified molecular genes to be fused with sucrose isomerase mutant coding genes, the enzyme activity of the prepared sucrose isomerase can reach 40.5 U / mg wet cells, and the recombinant engineering bacterium has a great application prospect in industrial production.

The invention belongs to the field of biological pharmacy and biotransformation, and particularly relates to a tagatose-4-epimerase mutant, a coding gene and application. The tagatose-4-epimerase mutant disclosed by the invention is obtained by carrying out single mutation or combined mutation on an amino acid residue at a specific position of an amino acid sequence as shown in SEQ ID NO.1. Compared with wild type tagatose-4-epimerase, the mutant has the advantage that the catalytic activity is improved when the D-tagatose is prepared through conversion. The obtained tagatose-4-epimerase mutant has the advantages of being mild in reaction condition, green, environmentally friendly, single in product and easy to separate and purify, and the production cost is greatly reduced.

The invention discloses L-rhamnose isomerase and application thereof, and belongs to the technical field of genetic engineering. The amino acid sequence of the L-rhamnose isomerase is obtained by performing the following mutation on an amino acid sequence as shown in SEQ ID NO.1: the 258th amino acid is mutated from D to Q. Compared with the prior art, the L-rhamnose isomerase mutant D258Q is obtained by mutating wild type L-rhamnose isomerase, and compared with the wild type L-rhamnose isomerase, the L-rhamnose isomerase mutant D258Q is higher in enzyme activity and longer in half-life period, so that when the L-rhamnose isomerase mutant D258Q is used for catalyzing the conversion of D-psicose into D-allose, the L-rhamnose isomerase mutant D258Q has the advantages that the enzyme activity is higher, and the half-life period is longer; the catalytic capability is stronger and more durable, and the conversion efficiency of the reaction and the yield of D-allose are improved.

The invention discloses a mutant with improved enzyme activity of sucrose isomerase and application of the mutant, and belongs to the technical field of genetic engineering and enzyme engineering in molecular biology. A known sucrose isomerase parent is subjected to molecular modification by utilizing a site-directed mutagenesis technology to obtain mutant enzymes T522S / K340D, D222H / K340D, G467N / K340D, T522S, K340D, D222H and G467N, and the catalytic activity of the series of mutants provided by the invention is increased by 10%-25% compared with that of original bacteria; the isomaltulose conversion rates of the combined mutants T522S / K340D, D222H / K340D and G467N / K340D are respectively increased by 2.3%, 1.5% and 1.3% compared with those of original bacteria, and the strain has good stability, can be used for biological preparation of isomaltulose and has potential application prospects.

The invention relates to the technical field of gene engineering, in particular to an L-rhamnose isomerase mutant, a recombinant plasmid, a mutant strain and application. The amino acid sequence of the mutant is as shown in SEQ ID NO.4, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO.3. A specific mutant gene sequence is designed, a recombinant plasmid pET28a (+)-L-Rhi is successfully constructed, the gene sequence is connected between enzyme cutting sites BamH I and Hind III of an escherichia coli expression vector pET-28a (+) to construct a mutant expression vector (the recombinant plasmid pET-28a (+)-L-Rhi), the mutant expression vector is transformed into an escherichia coli BL21 competent cell, a mutant strain is obtained, and the recombinant plasmid pET-28a (+)-L-Rhi is obtained. The obtained mutant strain can overexpress L-rhamnose isomerase, the conversion rate of D-allose is remarkably improved, the thermal stability of the enzyme is remarkably improved through the design of mutation sites, and the half-life period is effectively prolonged.

The present disclosure relates to recombinant microorganisms, such as bacteria, e.g., E. coli, engineered to have improved flux and / or yield of the DXP pathway. Cells can be engineered to comprise nucleotide sequences comprising dxs polypeptides (EC 2.2.1.7); dxr polypeptides (EC 1.1.1.267); ispD polypeptides (EC 2.7.7.60); ispE polypeptides (EC 2.7.1.148); ispF polypeptides (EC 4.6.1.12); ispG polypeptides (EC 1.17.7.1 or EC 1.17.7.3); ispH polypeptides (EC 1.17.7.4); and idi polypeptides (EC 5.3.3.2). Cells can be further engineered to express redox polypeptides, e.g., ferredoxins, flavodoxins, and / or flavodoxin / ferredoxin--NADP reductases (EC 1.19.1.1 or EC 1.18.1.2), e.g., to increase the activity of the ispG and / or ispH polypeptides. Some or all of the nucleotide sequences can be heterologous to the cells. The recombinant microorganisms can be used in methods of isoprenoid production.

A method for recombinantly expressing feline serum albumin. A method for constructing an engineered strain for recombinantly expressing a feline serum albumin, wherein the method comprises: co-expressing a feline serum albumin, a protein disulfide isomerase, and regulatory factor HAC1 and / or vitreoscilla hemoglobin in a receptor yeast. The obtained strain is an engineered strain capable of expressing a recombinant feline serum albumin. The engineered strain can efficiently express rFSA. Compared with the direct and individual expression of rFSA, the expression level of rFSA in the co-expression strain is significantly increased, and the highest expression level can reach about 16 g / L. This lays a foundation for large-scale industrial production of rFSA.

The invention belongs to the field of biological pharmacy and biotransformation, and particularly relates to a tagatose-4-epimerase mutant as well as a coding gene and application thereof. The tagatose-4-epimerase mutant disclosed by the invention is obtained by carrying out single mutation or combined mutation on an amino acid residue at a specific position of an amino acid sequence as shown in SEQ ID NO.1. Compared with wild type tagatose-4-epimerase, the mutant has the advantage that the catalytic activity is improved when the D-tagatose is prepared through conversion. The obtained tagatose-4-epimerase mutant has the advantages of being mild in reaction condition, green, environmentally friendly, single in product and easy to separate and purify, and the production cost is greatly reduced.

The invention provides a method for converting D-fructose in jujubes into D-psicose by immobilized enzyme, jujube juice containing D-psicose and application, and particularly belongs to the technical field of beverage preparation. The method for converting the D-fructose in the jujubes into the D-psicose by the immobilized enzyme comprises the following steps: mixing the immobilized D-psicose-3-epimerase with jujube juice, carrying out sugar conversion, centrifuging and discarding precipitate to obtain jujube juice containing the D-psicose; the nucleotide sequence of the gene for coding the D-psicose-3-epimerase is as shown in SEQ ID NO. 1 (sequence identifier number 1). According to the method disclosed by the invention, the D-fructose in the jujubes can be effectively converted into the D-psicose.

The disclosure relates to the field of specialty chemicals and methods for their synthesis. In embodiments, the disclosure provides viable bacterial cells which comprise heterologous dual 3-hydroxy-acyl-ACP dehydratase / isomerases, etc. The disclosure further provides monounsaturated fatty acid derivative molecules produced by the viable bacterial cells which are non-native to the bacterial cells. The disclosure further provides methods for the preparation and production of non-native monounsaturated fatty acid derivative molecules such as e.g., an ω3-monounsaturated fatty acid derivative, an ω5-monounsaturated fatty acid derivative, an ω9-monounsaturated fatty acid derivative, an ω11-monounsaturated fatty acid fatty acid derivative, etc.

The invention relates to the technical field of gene engineering, in particular to an L-rhamnose isomerase mutant, a recombinant plasmid, a mutant strain and application. The amino acid sequence of the mutant is as shown in SEQ ID NO. 2, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO. 1. A specific mutant gene sequence is designed, a recombinant plasmid pET28a (+)-L-Rhi is successfully constructed, the gene sequence is connected between enzyme cutting sites BamH I and Hind III of an escherichia coli expression vector pET-28a (+) to construct a mutant expression vector (the recombinant plasmid pET-28a (+)-L-Rhi), the mutant expression vector is transformed into an escherichia coli BL21 competent cell, a mutant strain is obtained, and the recombinant plasmid pET-28a (+)-L-Rhi is obtained. The obtained mutant strain can overexpress L-rhamnose isomerase, the conversion rate of D-allose is remarkably improved, the thermal stability of the enzyme is remarkably improved through the design of mutation sites, and the half-life period is effectively prolonged.

The invention provides a recombinant Aspergillus niger strain with high yield of trans-aconitic acid, and belongs to the technical field of gene engineering. The recombinant aspergillus niger strain can simultaneously express aconitase and aconitic acid isomerase. According to the genetic engineering aspergillus niger strain provided by the invention, the trans-aconitic acid fermentation yield is generally higher than that of an original strain, and the strain with the highest yield can reach 51g / L on a shake flask level, so that the genetic engineering aspergillus niger strain has very high application value.

The invention belongs to the field of biosynthesis, and particularly relates to a saccharomyces cerevisiae engineering bacterium for producing glycosyl donor UDP-arabinose as well as a construction method and application of the saccharomyces cerevisiae engineering bacterium. According to the invention, UDP-Glc dehydrogenase, UDP-GlcA decarboxylase, UDP-Xyl-4-epimerase or UDP-Glc-epimerase are sequentially expressed in WAT11, so that a synthetic route of UDP-Ara is introduced into the saccharomyces cerevisiae for the first time; de novo synthesis of Ara-oleanolic acid is realized by introducing a synthetic route of arabinosyl transferase and oleanolic acid; the yeast containing the UDP-Ara synthetic pathway and the arabinosyl transferase gene can also utilize betulinic acid to generate Ara-betulinic acid. The invention solves the problem of yeast total synthesis of the arabinosyl donor, provides a new biotechnology platform for production of natural glycoside compounds, and has important theoretical value and wide application prospect.

The invention discloses a keto isomerase mutant as well as a preparation method and application thereof, and relates to the technical field of gene engineering. The keto isomerase mutant is obtained by performing the following mutations on an amino acid sequence shown as SEQ ID NO.2: the 34th amino acid is mutated from N to R; meanwhile, the 188th amino acid is mutated from I to K. The half-life period of the keto isomerase mutant obtained by mutating the amino acid sequence shown in SEQ ID NO.2 is prolonged, and the enzyme activity is improved, so that the yield of D-chiro-inositol is further improved.