254results about "Racemaces/epimerases" patented technology

This invention discloses a D-allulose-3-epimerase mutant, its host cell, and its application in the synthesis of allulose, belonging to the field of genetic engineering technology. This invention utilizes a mutant derived from Ruminococcus CAG55 (… Ruminococcus sp. Simultaneous mutations at positions 47, 72, 114, and 221 of wild-type D-allulose-3-epimerase (CAG55) yielded a series of D-allulose-3-epimerase mutants, significantly improving their catalytic activity and stability. The mutants obtained by simultaneous mutations at positions 47, 72, 114, and 221 exhibited the highest catalytic activity. The D-allulose-3-epimerase mutants provided by this invention have significant application value in the production of D-allulose.

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 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 relates to a NeuC mutant, a host cell containing the NeuC mutant and a method for producing sialic acid by fermentation. The invention discloses a NeuC mutant, a coding nucleic acid thereof, a vector, a host cell containing the vector, and a method for producing sialic acid through fermentation by using the host cell. Compared with wild NeuC, the NeuC mutant can obviously improve the yield of sialic acid. The sialic acid yield can reach 61 g / L after high-density fermentation culture is performed for 60 h in a 3L fermentation tank.

Provided are an Aeromonas dhakensis mutant strain with deletion of a surface polysaccharide synthesis gene, a complemented strain, a construction method therefor, and the use thereof. The mutant strain is obtained by deleting one or more of ugd, ugd2, and cap1J from the Aeromonas dhakensis wild strain A. dhakensis C160501.

The invention discloses a recombinant escherichia coli strain, a preparation method and application thereof, and a synthesis method of oryzanol A, and relates to the technical field of bioengineering. The recombinant escherichia coli strain takes E.coli BL21 (DE3) as a chassis cell, and is modified as follows: (a) knockout of a gene pfkA; or, the gene pfkA is knocked out, and an untranslated region of the 5'end of the gltA gene is mutated from the sequence as shown in SEQ ID NO: 1 to the sequence as shown in SEQ ID NO: 2; and (b) inserting exogenous genes agmA, agmB, agmC, agmE and agmF, so as to obtain the gene. The recombinant Escherichia coli strain is high in growth speed and short in fermentation period (within 72 hours), a culture medium which is cheap and easy to obtain can be adopted, and compared with an existing Escherichia coli system, the fermentation yield of oryzanol A is obviously increased.

The invention discloses a genetically engineered bacterium for high yield of L-alanine as well as a construction method and application of the genetically engineered bacterium, a dominant strain is obtained by knocking out an alanine racemase gene dadX and integrating coding sequences of alanine dehydrogenase from different sources, and the shake flask yield is 10.8 g / L; the activity of a corresponding mutant M18L / E75A / A78S / I266V is 4.3 times that of a wild type; 6 by-product related genes are knocked out, meanwhile, a multi-copy strain integrated with alanine dehydrogenase is constructed, and the yield of E.coli A-LB6 in a 5L fermentation tank is increased to 133.3 g / L. Through a dynamic regulation and control strategy, an oxygen-sensitive promoter Pomp * is adopted to replace a phosphoenolpyruvate carboxylase promoter, so that succinic acid byproducts are reduced by 85.8%, and the yield reaches 143.5 g / L after 48 hours of aerobic and anaerobic two-stage fermentation. And finally, by regulating and controlling the ATP synthetase gene cluster atp promoter, the yield of L-alanine in a 50 L fermentation tank reaches 151.3 g / L, and the saccharic acid conversion rate reaches 95.2%. The combination of polygene knockout and dynamic metabolism regulation effectively enhances the biosynthesis ability of alanine, and provides an efficient engineering strain for industrial production.

The present invention relates to the technical field of biotechnology, particularly to an enzyme combination, a genetically modified bacterium, and its use in the production of D-allulose. The present invention relates to Bacillus subtilis co-expressing glucose isomerase and D-allulose 3-epimerase from a specific source. The resulting modified bacterium is fermented to obtain a crude enzyme solution, which is then isomerized using a high concentration of glucose as a substrate. After separation, purification, and concentration, fructoglucose syrup and D-allulose syrup are obtained. The enzyme combination provided by the present invention has a high catalytic rate relative to the substrate, significantly improving the conversion rate of D-allulose. At the same time, the present invention reduces the production costs of D-allulose by using inexpensive glucose as a raw material, and simultaneously produces fructoglucose syrup while producing D-allulose, thereby saving on the input of the fructoglucose syrup production line and significantly improving economic benefits.

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.

Disclosed herein is a genetically engineered yeast cell capable of producing xylitol, the genetically engineered yeast cell characterized by a genetic modification resulting in overexpression of a natural enzyme having xylitol-5-phosphate phosphatase (X5PP) activity and / or an exogenous polynucleotide sequence encoding an enzyme having xylitol-5-phosphate phosphatase (X5PP) activity. The genetically engineered yeast cell may additionally be engineered to overexpress a native RPE enzyme, to express an exogenous XPDH enzyme, to express an exogenous XKS enzyme, and / or to express an exogenous XDH enzyme.

A process for producing an aqueous solution containing allose by forming D-psicose in vitro from D-fructose, which is dissolved in an aqueous solution, by treating the dissolved D-fructose with an epimerase, whereupon the D-psicose is reduced in vitro to allitol by treating the D-psicose with an NAD(P)H-dependent oxidoreductase, resulting in the formation of oxidized cofactor NAD(P)+, and then the allitol is enzymatically oxidized to obtain allose.

The present disclosure provides recombinant Staphylococcus bacterium (e.g. S. epidermidis) that are dependent on D-alanine for growth. In one aspect, the disclosure features a recombinant Staphylococcus bacterium comprising two inactivated alanine racemase genes (Δalr1 Δalr2); and an inactivated D-alanine aminotransferase (dat) gene. In another aspect, the disclosure features a method of making the recombinant Staphylococcus bacterium. In another aspect, the disclosure features a method of treating or preventing a rash in a subject, comprising administering to the subject a population of the recombinant Staphylococcus bacterium of any one of the aspects or embodiments described herein, in an effective amount to treat or prevent the rash in the subject.

The present application relates to a fructose-4-epimerase variant exhibiting tagatose conversion activity and a method for preparing tagatose using the same.

Summary Disclosed are genetically engineered microbial cells for the production of oligosaccharides comprising a galactose-β1,4-glucose moiety at their reducing end, wherein said microbial cells are able to produce said oligosaccharides in the absence of exogenously added lactose, and a method of producing said oligosaccharides using said microbial cells.

The invention provides recombinant Escherichia coli for synthesizing heme and derivatives thereof, and the recombinant Escherichia coli is used for knocking out genes such as pyruvate oxidase poxB, pyruvate formate lyase pflB and porphyrinoperoxidase yfeX and enhancing genes such as phosphoenolpyruvate carboxylase ppc, glycerol transporter glpF and 3-phosphoglycerol dehydrogenase glpD, so that the heme and the derivatives thereof can be synthesized. After genes such as exogenous 5-aminolevulinic acid synthase hemA and ferrous chelating enzyme hemH are introduced, the heme and the derivatives thereof can be synthesized by taking glycerol as a raw material, and the heme has an application prospect in the field of biological manufacturing.

The present invention provides recombinant Corynebacterium glutamicum genetically engineered to introduce the N-acetylmannosamine (ManNAc) synthesis pathway, and genetically engineered to express an exogenous N-acetylneuraminic acid synthetase, an exogenous CMP-N-acetylneuraminic acid synthetase, an exogenous lactose transporter, and an exogenous sialyltransferase. The invention also provides a method for producing sialic acid lactose by using the recombinant corynebacterium glutamicum.

Provided are methods for producing polysaccharides in bacteria by expressing in a bacterium one or more coding sequences selected from the group consisting of a pglF dehydrogenase coding sequence, a wbpP UDP-N-acetyl-d-glucosamine C4 epimerase coding sequence, a wcfR aminotransferase coding sequence, and a wcfS phospho-glycosyltransferase coding sequence, a wcfQ glycosyltransferase coding sequence, a wcfO pyruvyltransferase coding sequence, a wcfP glycosyltransferase coding sequence, a wcfM UDP-galactopyranose mutase coding sequence, a wcfN glycosyltransferase coding sequence, a wza polysaccharide export protein coding sequence, a wzx fippase coding sequence, a wzy polymerase coding sequence, and a wzz coding sequence, wherein at least one of the coding sequences is heterologous to the bacterium. Also provided are expression cassettes with one or more of the disclosed coding sequences, recombinant bacteria that harbor one or more of the expression cassettes, and methods for producing immunogenic compositions using the polysaccharides produced by the recombinant bacteria.

The present invention relates to the technical field of genetic engineering and fermentation engineering, and specifically discloses a preparation method of an N-acetyl-D-amino acid. An L-amino acid and / or a D, L-amino acid are used as raw materials, the L-amino acid is converted into a D-amino acid under the action of an L-amino acid isomerase, and the D-amino acid is converted into an N-acetyl-D-amino acid under the action of an acyltransferase. General-use L-amino acid isomerase and acyltransferase are used to convert various amino acids into corresponding N-acetyl-D-amino acids. Raw materials used in the present method are low cost, toxic chemical racemization and high-cost chiral resolution steps are avoided, and the method is suitable for industrial processing and production. The prepared N-acetyl-D-amino acid can be further processed into a D-amino acid or a D-amino acid derivative.

To provide an immobilized enzyme composition for hexose production or an improved process for hexose production. [Solution] The present invention relates to an immobilized enzyme composition for the preparation of hexoses. Examples of hexoses include tagatose, psicose, fructose, allose, mannose, galactose, altrose, talose, sorbose, gross, idose, and inositol. The present invention also relates to an enzymatic process for preparing hexoses from sugars by contacting starch derivatives with the immobilized enzyme composition of the present invention.

The invention discloses an escherichia coli strain with high yield of riboflavin as well as a construction method and application of the escherichia coli strain. A series of Escherichia coli strains with high yield of riboflavin are constructed, the constructed Escherichia coli with high yield of riboflavin is safe and harmless, glucose is used as a substrate for fermentation under the aerobic condition of a shake flask, the yield of riboflavin is 1117.8 mg / L, 1330.1 mg / L, 1430.8 mg / L, 1508.7 mg / L and 1640.8 mg / L in sequence, and the yields are 112.5 mg / g of glucose, 134.1 mg / g of glucose, 146.3 mg / g of glucose, 158.8 mg / g of glucose and 168.1 mg / g of glucose in sequence; when the strain SN04M is subjected to fed-batch fermentation in a 1L fermentation tank, the yield of riboflavin reaches 8.52 g / L, and the total yield is 189.9 mg / g glucose. According to the invention, the fermentation yield of riboflavin and the substrate utilization rate of riboflavin to glucose can be obviously improved.

Provided is a heat-resistant D-allulose-3-epimerase. A polypeptide shown in any one of the following (1) to (3): (1) a polypeptide consisting of an amino acid sequence having one or two or more amino acid substitutions selected from the group consisting of A56E, N57H, R58G and F111W in the amino acid sequence shown in SEQ ID NO: 1; (2) a polypeptide consisting of the amino acid sequence having substitution, addition, insertion or deletion of one or several amino acid residues in the amino acid sequence described in the above (1), having D-allulose-3-epimerase activity and retaining one or two or more amino acid substitutions selected from the group consisting of A56E, N57H, R58G, and F111W; and (3) a polypeptide having a 90% or more sequence identity to the amino acid sequence described in the above (1), having D-allulose-3-epimerase activity and retaining one or two or more amino acid substitutions selected from the group consisting of A56E, N57H, R58G, and F111W.

The invention relates to the technical field of biology, in particular to epimerase for synthesizing D-tagatose and application. Epimerase producing mutation of 100th or 125th or 127th or 131 or 268th or 270th or 271th or 308th or 339th or 340th or 342th or 366th or 402th or 437th in amino acid sequence as shown in SEQ ID NO.7 mutation at the 440th site; through the mode, the epimerase shows excellent epimerization activity at the fourth carbon position of D-fructose, can efficiently convert D-fructose into D-tagatose, and is beneficial to improving the synthesis efficiency of D-tagatose.

Provided are mucin-active proteases. In certain embodiments, the mucin-active proteases are stably associated with a targeting moiety. According to some embodiments, the mucin-active protease is stably associated with the targeting moiety via fusion of a protein domain comprising the mucin-active protease and a protein domain comprising the targeting moiety. In other embodiments, the mucin-active protease is stably associated with the targeting moiety via conjugation. Also provided are methods of treating a mucin-associated condition in a subject in need thereof, such methods comprising administering to the subject an effective amount of a mucin-active protease of the present disclosure. Upon administration of the mucin-active protease to the subject, the targeting moiety targets the mucin-active protease to cell surface, extracellular and / or secreted mucins, and the mucin-active protease degrades the mucins.

The present invention relates to a fructose-6-phosphate epimerization enzyme protein, a nucleic acid molecule encoding the enzyme protein, a recombinant vector and transformed microorganism containing the nucleic acid molecule, and a composition for producing allulose using these.

Compositions and methods are described for inducing an immune response against extra-intestinal pathogenic Escherichia coli (ExPEC) to thereby provide immune protection against diseases associated with ExPEC. In particular, compositions are described comprising conjugates of E. coli polysaccharide antigen O1, 02, 04, 06, 08, 015, 016, 018, 025, and 075 and further comprising 0153 or 021 or both 0153 and 021 covalently bound to a carrier protein for the prevention of invasive ExPEC disease.

The invention discloses application of a BnGAE1 gene in regulation and control of sclerotinia rot resistance of brassica napus, and belongs to the technical field of plant genetic engineering. The gene BnGAE1 capable of enhancing the sclerotinia rot resistance of the brassica napus is separated and cloned from the brassica napus, and the nucleotide sequence of the gene is as shown in SEQ ID NO. 1. Experimental verification shows that the resistance of the BnGAE1 (KO) strain to sclerotinia sclerotiorum is remarkably enhanced, and the incidence rate is reduced by 25.46% after inoculation; physiological and molecular detection shows that the expression quantity of defense related genes (such as PR1 and PDF1.2) in a mutant strain is up-regulated, SA (salicylic acid) and JA / ET (jasmonic acid / ethylene) signal channels are activated, and meanwhile, excessive ROS (reactive oxygen species) is effectively removed, so that the redox balance is maintained. The invention provides a new gene target and a high-efficiency molecular breeding technical path for improvement of sclerotinia rot resistance of colza, and has an important application prospect.

The application belongs to the technical field of genetic engineering, and particularly relates to a Spytag / SpyCatcher cyclization modified D-allulose-3-epimerase mutant and application thereof. The mutant intermediate is obtained by simultaneously mutating an aspartic acid at the 109th position and a serine at the 160th position of a wild-type D-allulose-3-epimerase, and the mutant intermediate is cyclization modified by connecting a Spytag label and a SpyCatcher label at the N terminal and the C terminal of the mutant intermediate, respectively. When D-allulose is produced by using fructose as a raw material, the conversion rate reaches 35.7% when the host bacteria is Escherichia coli and the catalysis of 600 g / L fructose to produce D-allulose for 4 h, and the conversion rate reaches 36.5% when the host bacteria is Bacillus subtilis and the catalysis of 500 g / L fructose to produce D-allulose for 4 h, which is 13.5% higher than that of the wild-type D-allulose-3-epimerase strain of Bacillus subtilis.

The present invention discloses a feed medium and a feeding method for producing D-psicose-3 epimerase, and belongs to the field of microbial fermentation technology. The present invention adopts a specific proportion of glucose, corn steep liquor, soybean cake powder and trace elements to make a feed medium with a certain carbon-nitrogen ratio and a quick-acting nitrogen source, a slow-acting nitrogen source and trace elements, and uses Bacillus subtilis as a fermentation strain. During the fermentation process, feed is added in a fed-batch manner, and pH-dissolved oxygen-feed coupling regulation is simultaneously performed during the fermentation process to increase the cell concentration and the activity of D-psicose-3 epimerase as much as possible. The method of the present invention is simple to operate, reduces production costs, shortens the fermentation cycle, and significantly improves the enzyme activity of the target enzyme.

Animal free lactose synthesis using a phosphorylase The present invention relates to methods to produce lactose, more specifically without the need for isolation from mammalian milk. Furthermore, the present invention provides for methods to purify the produced lactose.