375 results about "Isomerase" patented technology

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 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 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 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 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.

The disclosure provides method of treating cancer in a patient in need thereof, comprising safely and efficaciously administering to the patient PLX038, a long lasting-PEGylated prodrug of the topoisomerase I inhibitor. The disclosure further provides combination therapies of PLX038 with inhibitors of the DNA damage response (DDR).

The invention discloses a production method of fucosylated oligosaccharide. The core of the method is as follows: the GDP-D-rhamnose is converted into the GDP-L-fucose by using the GDP-D-rhamnose-3, 5-epimerase; the GDP-D-rhamnose is obtained by converting the GDP-D-mannose-4, 6-dehydratase and the GDP-4-keto-6-deoxy D-mannose reductase by taking the GDP-D-mannose as a substrate, and the GDP-D-rhamnose is obtained by taking the GDP-D-mannose as a substrate. The invention relates to a method for producing a fucosylated oligosaccharide, which is characterized in that a GDP-D-mannose-4, 6-dehydratase, a GDP-4-keto-6-deoxyD-mannose reductase, a GDP-D-rhamnose-3, 5-epimerase and a fucosyltransferase are expressed in a genetically modified host cell, and the fucosylated oligosaccharide can be produced by the way mentioned above.

The application belongs to the technical field of genetic engineering, and discloses a kind of saccharomyces cerevisiae for producing rare ginsenoside by seaweed biomass and a construction method and application thereof.The saccharomyces cerevisiae has the following characteristics: overexpression of agarase, neojuncanohydrolase, hydroxymethylglutaryl coenzyme A reductase, isopentenyl diphosphate delta isomerase, dammaradienol synthase, protopanaxadiol synthase, cytochrome P450 reductase, protopanaxatriol synthase and glycosyltransferase.The application combines enzymolysis of seaweed biomass with fermentation of rare ginsenoside, which not only endows wild-type yeast with the ability to degrade seaweed biomass that it originally does not have, but also effectively improves the yield of squalene and downstream terpenes (rare ginsenoside Rh1) by overexpression of tHMG1 and IDI1.The saccharomyces cerevisiae can effectively utilize seaweed biomass to obtain squalene and rare ginsenoside Rh1, and has the characteristics of simplicity, economy and ecological friendliness, can convert cheap biomass into high-value products, and provides a way for the development of marine bioeconomy.

The application provides a recombinant Aspergillus niger with high trans-aconitic acid yield and a construction method and application thereof, and relates to the technical field of genetic engineering. Specifically, the recombinant Aspergillus niger heterogeneously expresses aconitate isomerase TbrA and / or Adi1. The application provides a new engineering strain for efficient production of trans-aconitic acid, solves the source predicament of needing plant extraction or needing chemical synthesis in traditional production of trans-aconitic acid, and greatly improves the yield of trans-aconitic acid.

Described is an antibody-drug conjugate (ADC) having the formula A-L, wherein A is an antibody or an antibody fragment and wherein L is a linker, said linker comprising: as a first payload a topoisomerase I inhibitor which is cell-permeable, e.g., a camptothecin cytotoxic molecule which is cell-permeable; as a second payload a topoisomerase I inhibitor which is not cell-permeable, e..g., a camptothecin cytotoxic molecule which is not cell-permeable; and as a as a third payload a toxin or a cytotoxin, e.g., an auristatin such asMMAE (Monomethyl auristatin E). Moreover, described is a pharmaceutical composition comprising said ADC and at least one pharmaceutically acceptable ingredient. Further, described method of treating a patient suffering from, being at risk of developing, and / or being diagnosed for a neoplastic disease.

The invention belongs to the technical field of gene engineering and protein engineering, and particularly relates to thermal-stable D-psicose 3-epimerase as well as a preparation method and application thereof. According to the invention, a series of D-psicose 3-epimerase DAE mutants are constructed, and six mutants which not only can retain good catalytic activity, but also have high thermal stability are obtained through screening. The catalytic temperature of the three mutants A19D / A223P / V241I is increased by 25 DEG C compared with that of a wild type, while the three mutants A19D / A223P / V241I still show the optimal thermal stability, the catalytic activity of the three mutants is improved by 17.0%, and the three mutants show good industrial suitability. The D-psicose 3-epimerase DAE mutant with improved thermal stability provided by the invention is beneficial to improving the production efficiency of the DAE in industrial application of D-psicose, and provides powerful industrial enzyme guarantee for large-scale and low-cost preparation of D-psicose.

The invention belongs to the field of metabolic engineering, and discloses a genetically engineered bacterium for producing myo-inositol as well as a construction method and application of the genetically engineered bacterium. The genetically engineered bacterium takes escherichia coli as an original strain, and is obtained by performing the following gene editing on a genome of the escherichia coli: non-expressed lactose operon repressor protein, glucose phosphate dehydrogenase, acetokinase, glucose phosphate isomerase and glycerol repressor protein; and overexpressing inositol-1-phosphate synthase, inositol monophosphate, glucokinase, glucose permease and glycerol kinase. Compared with the prior art, the genetically engineered bacterium disclosed by the invention has remarkable advantages in the aspects of key enzyme expression rate, metabolism specificity, fermentation period and the like, and an efficient, stable, economical and feasible solution is provided for industrial production of myo-inositol.

The invention relates to the technical field of biology, in particular to a recombinant strain for producing glucuronic acid and application of the recombinant strain. The invention provides a recombinant strain for producing glucuronic acid, and the recombinant strain does not express uronic acid isomerase and overexpresses inositol oxidase. The technical scheme of the invention has the following advantages: (1) uronic acid isomerase uxaC from escherichia coli K12 is knocked out, so that glucuronic acid accumulation is increased; and (2) successfully integrating the target gene of the inositol oxidase from the mouse into the escherichia coli K12MG1655. And (3) when the engineering strain is used for catalyzing inositol, the content of glucuronic acid in a system after the reaction is finished is 52.50 g / L, and the conversion rate is as high as 97.23%.

The invention discloses a sclareol synthase mutant, a recombinant genetic engineering strain and application of the sclareol synthase mutant, and belongs to the technical field of biology. The sclareol synthase is modified through semi-rational design, the obtained sclareol synthase mutant has good catalytic activity, and the yield of sclareol can be remarkably increased; furthermore, the method is simple. The method comprises the following steps: integrating an HMG1 gene for coding HMG-CoA reductase, an IDI1 gene for coding isopentenyl pyrophosphate isomerase, an SsGGPPS gene for coding geranyl-geranyl pyrophosphate synthase, an SsLPPS gene for coding lybaidan diglycol diphosphate synthase, and an SsGGPPS gene for coding lybaidan diglycol diphosphate synthase; a recombinant genetic engineering strain obtained by the nucleic acid molecule for coding the sclareol synthase mutant can produce sclareol at high yield by taking glucose as a substrate, and has a remarkable industrial application value.

The invention provides recombinant escherichia coli for synthesizing glucuronic acid by using glucose and a method for producing glucuronic acid by using the recombinant escherichia coli, and belongs to the field of microbial metabolic engineering. The engineering bacterium is obtained by taking escherichia coli as an original strain and knocking out a glucuronic acid isomerase coding gene uxaC, a 6-phosphofructokinase I coding gene pfkA, a 6-phosphofructokinase II coding gene pfkB and a glucose-6-phosphate dehydrogenase coding gene zwf. The engineering bacterium disclosed by the invention has a good application prospect in industrial production of glucuronic acid.

The invention discloses a cellobiose 2-epimerase mutant as well as a gene, an expression vector, recombinant bacteria, a preparation method and application thereof. The mutant is obtained by performing the following point mutation on an amino acid sequence as shown in SEQ ID No.1: S12A, S24A, G131A, G176A, G224A, K46R, K69R, K72R, K91R, K103R, K201R, K219R, K223R, K241R, K263R, K266R, K273R, K284R, K325R, K335R, K349R and K387R. The invention further discloses a preparation method of the mutant. The invention further discloses a gene segment, a plasmid and a recombinant bacterium for recombinant expression of the mutant, and the gene segment, the plasmid and the recombinant bacterium are used for recombinant expression preparation of the mutant. The mutant can be used for continuously and stably catalyzing and preparing lactulose at high temperature, is relatively good in thermal stability and has a relatively good industrial application prospect.

The invention discloses saccharomyces cerevisiae with high yield of salidroside as well as a construction method and application of the saccharomyces cerevisiae. According to the recombinant saccharomyces cerevisiae, ribulose-5-phosphate isomerase RKI1 and transketolase TKL1 are expressed in host bacteria, so that precursor supply is increased; reverse methyltransferase ARO2 and phenylalanine decarboxylase ARO10 are expressed at the same time, and a tyrosine branch pathway is adjusted; then, UDP-glycosyl transferase of different sources is expressed, and iterative site-specific mutagenesis is carried out on the UDP-glycosyl transferase to enhance conversion of hydroxytyrosol to salidroside; the salidroside production performance of the recombinant strain is verified on the basis of a precursor supply pathway, a tyrosine regulation pathway and key enzyme screening, and the salidroside production capacity of the saccharomyces cerevisiae is further improved.

The invention discloses a recombinant bacterium capable of increasing the yield of alpha-bisabolol as well as a preparation method and application of the recombinant bacterium, and belongs to the technical field of microorganisms. The invention aims to improve the yield of alpha-bisabolol and enhance the tolerance of a host to an organic solvent. The invention provides a recombinant bacterium for improving the yield of alpha-bisabolol. Escherichia coli is used as a starting strain; the method comprises the following steps of: overexpressing an acetyl CoA acyltransferase / HMG-CoA reductase mvaE gene, an HMG-CoA synthetase mvaS gene, a 2-methyl citrate dehydratase prpD gene, a mevalonate kinase ERG12 gene, a mevalonate 5-phosphate kinase ERG8 gene, a mevalonate 5-diphosphate decarboxylase ERG19 gene and an isopentenyl diphosphate isomerase idi gene, so as to obtain a recombinant vector; the gene is obtained from an alpha-bisabolol synthase gene of artichoke, a farnesyl diphosphate synthase ispA gene and an alpha-bisabolol synthase CcBOS gene of artichoke. The industrial process of synthesizing alpha-bisabolol by a biological method is promoted.

The application discloses a D-allulose 3-epimerase mutant, a preparation method and application thereof, and relates to the fields of genetic engineering and enzyme engineering. The amino acid sequence of the D-allulose 3-epimerase mutant is shown as SEQ ID NO. 1. The specific enzyme activity of the D-allulose 3-epimerase mutant provided by the application is increased from 10.21 U / mg of a control (before mutation) to 15.29 U / mg at 80 DEG C and pH 6.0; and the equilibrium conversion rate is increased from 29.98% of the control (before mutation) to 36.26% when the substrate is 700 g / L of fructose. The D-allulose 3-epimerase mutant provided by the application has higher catalytic activity, and has a very broad application prospect in the efficient production and preparation of D-allulose.

The invention discloses recombinant saccharomyces cerevisiae, a 11-seed oil fermentation product as well as a preparation method and application of the 11-seed oil fermentation product, and belongs to the technical field of synthetic biology and fermentation product preparation. According to the recombinant saccharomyces cerevisiae disclosed by the invention, a caffeic acid coenzyme A ligase 4CL coding gene, a chalcone isomerase CHI coding gene, a flavanone-3-hydroxylase F3H coding gene and a chalcone synthase CHS mutant coding gene are over-expressed, and the chalcone synthase CHS mutant is preferably Y69H / H71Y / Q161K. The preparation method of the 11-seed oil fermentation product comprises the following steps: fermenting a recombinant saccharomyces cerevisiae fermentation solution in the presence of a sophora flower bud extract and caffeic acid to obtain a first fermentation solution; fermenting the lactic acid bacteria fermentation liquor in the presence of 11 seed oil to obtain second fermentation liquor; and mixing the first fermentation liquid and the second fermentation liquid, continuing fermentation, and respectively collecting an oil phase and a water phase after fermentation is finished, so as to obtain a fermentation product. The product provided by the invention has multivitamin antioxidant activity.

This invention provides a ketolic acid reductase mutant and its application in constructing genetically engineered bacteria that produce high levels of D-pantothenic acid. To address the technical problem of low D-pantothenic acid yield in existing microbial fermentation, this invention utilizes semi-rational enzyme design to obtain a substrate-specific ketolic acid reductase mutant, which is then applied to construct genetically engineered bacteria that produce high levels of D-pantothenic acid. Furthermore, this invention employs CRISPR-Cas9 gene editing technology to construct genetically engineered bacteria that produce high levels of D-pantothenic acid using the aforementioned ketolic acid reductase mutant, increasing D-pantothenic acid production by approximately 12.04%. Compared to the starting strain, the accumulation of valine in the fermentation broth remained almost unchanged compared to the control, but the accumulation of the branched-chain amino acid isoleucine was lower, achieving substrate-specific modification of the ketolic acid reductase.

A specific topoisomerase inhibitor, a use as an antibody drug conjugate, and a preparation method therefor, which relate to the technical field of medicinal chemistry. The inhibitor is a compound A or a tautomer, a mesomer, a racemate, an optical antipode, a diastereoisomer, or a mixture form thereof, or a pharmaceutically acceptable salt thereof; the structure of the compound A is such that the compound may also be further prepared to obtain an antibody drug conjugate, the antibody drug conjugate has good solubility and pharmaceutical properties, and the conjugation process does not result in precipitation, the antibody drug conjugate exhibits obvious in-vivo anti-tumor activity, and shows markedly stronger anti-tumor activity when compared with a control sample.

The invention belongs to the technical field of gene engineering, and in particular relates to a D-psicose-3-epimerase mutant subjected to Spytag / SpyCatcher cyclization modification and an application of the D-psicose-3-epimerase mutant. The preparation method comprises the following steps: carrying out multi-site simultaneous mutation on 109th aspartic acid and 160th serine of wild type D-psicose-3-epimerase to obtain a mutation intermediate, respectively connecting a Spytag tag and a SpyCatcher tag to the N end and the C end of the mutation intermediate, and carrying out cyclization modification, so as to obtain the wild type D-psicose-3-epimerase, wherein the Spytag tag and the SpyCatcher tag are respectively connected to the N end and the C end of the mutation intermediate; when fructose is used as a raw material to produce D-psicose and host bacteria are escherichia coli, the reaction of catalyzing 600 g / L fructose to produce D-psicose is performed for 4 h, and the conversion rate can reach 35.7%; when the host bacteria are bacillus subtilis, the reaction for catalyzing 500 g / L fructose to produce D-psicose is performed for 4 h, and the conversion rate reaches 36.5%; compared with a wild type D-psicose-3-epimerase strain of bacillus subtilis, the conversion rate of the strain is improved by 13.5%.

Disclosed herein are genetically engineered Kluyveromyces sp. cells capable of producing D-allulose. The genetically engineered Kluyveromyces sp. cells comprise an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3-epimerase enzyme at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or100% identical to at least one of SEQ ID NOs:249-256, 258, and 259; and an exogenous polynucleotide sequence encoding an allulose-6-phosphate phosphatase enzyme at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NOs:87, 89, 190, 123, 105, 107, 115, 83, 95, 113, 117, 119, 121, 127, 131, 137, 145, 169, 173, 179, and 183.

This invention discloses a ketoisomerase mutant and a method for preparing D-chiral inositol, belonging to the field of genetic engineering technology. The ketoisomerase mutant is obtained by mutating the 73rd amino acid of the amino acid sequence shown in SEQ ID NO.3 from L to N. The ketoisomerase mutant L73N obtained by this invention further enhances enzyme activity. When catalyzing the reaction of muscle inositol to synthesize D-chiral inositol together with inositol dehydrogenase, it can further improve the conversion rate and the yield of D-chiral inositol.

Disclosed herein are genetically engineered Kluyveromyces marxianus cells capable of producing D-allulose with increased talitol formation. The engineered cell may comprise a genetic modification resulting in overexpression of a native talitol dehydrogenase enzyme; an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3-epimerase enzyme at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NOs:249-256, 258, and 259; and an exogenous polynucleotide sequence encoding an allulose-6-phosphate phosphatase enzyme at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NOs:87, 89, 190, 123, 105, 107, 115, 83, 95, 113, 117, 119, 121, 127, 131, 137, 145, 169, 173, 179, and 183.

This invention discloses a ketoisomerase mutant and its application in the preparation of D-chiral inositol, belonging to the field of genetic engineering technology. The ketoisomerase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.1, specifically by mutating amino acid position 62 and / or amino acid position 175 of the amino acid sequence shown in SEQ ID NO.1. After the above-mentioned mutation of the wild-type thermostable ketoisomerase, the resulting ketoisomerase mutant not only retains the advantages of the wild-type thermostable ketoisomerase—its heat resistance and susceptibility to temperature fluctuations—but also exhibits higher enzyme activity. When co-catalyzing the reaction of muscle inositol to D-chiral inositol with inositol dehydrogenase, it can further improve the conversion rate and yield of D-chiral inositol even at high substrate concentrations.