129results about "Racemaces/epimerases" patented technology

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.

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.

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 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.

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.

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.

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.

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.

Provided herein are genetically modified yeast cells capable of producing one or more human milk oligosaccharides (HMOs) and methods of making such cells. The yeast cells are engineered to comprise a heterologous nucleic acid encoding a transporter protein and one or more heterologous nucleic acids that encode enzymes of a HMO biosynthetic pathway. Also provided are fermentation compositions including the disclosed genetically modified yeast cells, and related methods of producing and recovering HMOs generated by the yeast cells.

An object of the present invention is to provide a microorganism having an ability to produce NeuAc and / or a NeuAc-containing carbohydrate, and a method for producing NeuAc and / or the NeuAc-containing carbohydrate more efficiently by the microorganism. The present invention relates to a microorganism which has an ability to produce NeuAc and / or a NeuAc-containing carbohydrate and whose enterobacterial common antigen (ECA) biosynthesis pathway is blocked, and a method for producing NeuAc and / or the NeuAc-containing carbohydrate using the microorganism.

The present disclosure provides a recombinant yeast host cell having a plurality of native and / or heterologous enzymes that function in an engineered metabolic pathway to convert fructose-6-phosphate to acetyl-coenzyme A. The recombinant yeast host cell can be used, for example, in a process for making acetyl-coenzyme A, acetone and / or isopropanol. The plurality of native and / or heterologous enzymes is activated, upregulated, or overexpressed. The plurality of native and / or heterologous enzymes comprises a phosphoketolase; and optionally an acetate kinase; and / or a phosphotransacetylase. The recombinant yeast host cell comprises at least one of: at least two copies of a heterologous nucleic acid molecule encoding the phosphoketolase; a native and / or heterologous enzyme that functions in an engineered non-oxidative pentose phosphate pathway, wherein the native and / or heterologous enzyme is activated, upregulated, or overexpressed; and / or a native and / or heterologous protein that functions in an engineered metabolic pathway to convert pantothenate into acetyl-coenzyme A, wherein the native and / or heterologous protein is activated, upregulated or overexpressed and comprises at least one of: FEN2 or CAB1.

The present invention discloses an L-alanine-producing genetically engineered strain, as well as a method of construction and use thereof, and pertains to the field of bioengineering. According to the present invention, through enhancing the glycolysis pathway or / and introducing a gene for thermostable alanine dehydrogenase, a genetically engineered strain capable of high-yield production of alanine under a high temperature condition of 42 °C to 55 °C can be constructed. Moreover, through knocking out alanine racemase genes, optical purity of L-alanine can be significantly increased. When the original strain possesses a lactate synthesis pathway, blocking this lactate synthesis pathway can augment the proportion of a pyruvate synthesis pathway, resulting in an additionally increased yield of L-alanine. The present invention overcomes the problems of fermentation at a low temperature, high cost and the like, which arise from the use of conventional L-alanine production techniques, enables production of L-alanine by fermentation at a high temperature of 42 °C to 55 °C with a yield of 95 g / L or higher, and is of high value to industrial application.

Provided herein are compositions and methods related to improved production of psicose by converting fructose to psicose using a psicose from Brachyspira suanatina, or a psicose-3-epimerase enzyme, in the presence of 50 or 100 mg / L of an alkali NaOH, and methods related to improved production of psicose by using a psicose from Brachyspira suanatina, or a psicose-3-epimerase enzyme from Brachyspira suanatina, or a psicose-3-epimerase enzyme from Brachyspira suanatina, or a psicose-3-epimerase enzyme from Brachyspira suanatina or a psicose-3-epimerase.

The present disclosure relates to cell immobilized beads and a method for preparing the same and, more specifically, to cell-immobilized beads wherein the conversion activity of cells contained in the immobilized beads is excellent and wherein the conversion activity is maintained even during distribution and storage processes, a method for preparing the cell-immobilized beads, and a use of the conversion activity of the beads.

Disclosed herein are methods of producing hexoses from saccharides by enzymatic processes. The methods utilize fructose 6-phosphate and at least one enzymatic step to convert it to a hexose.

The invention discloses a D-psicose-3-epimerase mutant, a host cell and application of the D-psicose-3-epimerase mutant in synthesis of psicose, and belongs to the technical field of genetic engineering. According to the invention, a series of D-psicose-3-epimerase mutants are obtained by simultaneously mutating any one or more of a plurality of sites such as a 47 site, a 72 site, a 114 site and a 221 site of wild type D-psicose-3-epimerase derived from ruminococcus sp. CAG55, and the catalytic activity and the stability of the D-psicose-3-epimerase mutants are obviously improved. Wherein the catalytic activity of mutants obtained by simultaneous mutation of the 47 site, the 72 site, the 114 site and the 221 site is the highest. The D-psicose-3-epimerase mutant provided by the invention has an important application value in the field of production of D-psicose.

The present invention discloses a variant D-allulose 3-epimerase (DAEase) that is distinguished from a wild-type DAEase derived from Spirochaetales bacterium (spDAEase) by at least one amino acid sequence variation comprising substitution of an 5 amino acid residue corresponding to position 69, 119, 138, 175, 203, 208, 229, 240, or 265 of SEQ ID NO. 1; or a variant DAEase that is distinguished from a wild-type DAEase derived from Clostridium cellulolyticum (ccDAEase) by an amino acid sequence variation comprising substitution of an amino acid at position 46 and / or a truncation of C-terminal end of in SEQ ID NO. 2.

The invention provides a genetic engineering modification method for improving acetic acid generation and application of the genetic engineering modification method. Specifically, provided herein is a recombinant prokaryotic cell that is genetically engineered directed to comprise a defect of one or more genes selected from the group consisting of galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl wherein the recombinant prokaryotic cell has reduced acetic acid production as compared to the prokaryotic cell before modification. Also provided herein is a method of making the recombinant prokaryotic cell comprising directed genetic engineering of the prokaryotic cell to deficit one or more genes therein selected from the group consisting of galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA and pgl wherein the recombinant prokaryotic cell has reduced acetic acid production compared to the prokaryotic cell before modification. The invention also provides a product containing the recombinant prokaryotic cell, an application of the product in metabolic engineering and a method for carrying out recombinant production by utilizing the recombinant prokaryotic cell.

Provided herein are compositions and methods relating to improved production of allulose by converting fructose to allulose using a allulose or psicose-3-epimerase from Brachyspira suanatina in the presence of base NaOH at 50 or 100mg / L.

The invention relates to a method for producing an aqueous solution containing L-psicose, wherein a first D-psicose is formed from D-fructose, which is present in a dissolved state in an aqueous solution, by treating the D-fructose with an epimerase in vitro, the first D-psicose is then reduced in order to form allitol by treating the D-psicose with a corresponding NAD(P)H-based oxidoreductase in vitro, and the allitol is treated with a corresponding NAD(P)+-based oxidoreductase in order to form L-psicose after a deactivation and / or ultrafiltration of the epimerase. The deactivated epimerase and the oxidoreductases are then removed.

The present application provides a recombinant E. coli for synthesizing heme and derivatives thereof, wherein the recombinant E. coli is knocked out of genes such as pyruvate oxidase poxB, pyruvate formate-lyase pflB and protoporphyrinogen peroxidase yfeX, and meanwhile, the genes such as phosphopyruvate carboxylase ppc, glycerol transporter glpF and 3-phosphoglycerate dehydrogenase glpD are enhanced, and the genes such as 5-aminovaleryl-CoA synthase hemA and ferrous chelatase hemH are introduced, so that the recombinant E. coli can synthesize heme and derivatives thereof by using glycerol as a raw material, and has application prospects in the field of biological manufacturing.

The invention belongs to the technical field of biological enzyme engineering, and particularly relates to a D-psicose 3-epimerase mutant derived from rumen clostridium and application of the D-psicose 3-epimerase mutant. The mutant disclosed by the invention is obtained through specific single-point mutation on the basis of a wild type amino acid sequence of D-psicose 3-epimerase, and specifically, the mutant is obtained through mutation of the following five sites: L159V, Q164E, N238T, E288G and C289I; wherein mutants containing all five single point mutations are named as XT mutants. The mutants significantly improve the thermal stability of the enzyme and the catalytic conversion rate under high substrate concentration, especially under the high substrate concentration of 300-700 g / L, the conversion rate of D-psicose of the XT mutant is improved by 2-3% compared with that of a wild type, and the thermal stability of the XT mutant is improved by 28 times compared with that of the wild type. The mutant is suitable for a high-temperature and high-substrate-concentration industrial conversion system, the production cost of D-psicose can be remarkably reduced, and the mutant has important industrial application value.

The present invention relates to the the cell-based production of bacterial nonulosonates and their biosynthetic precursors. Specifically, the present invention provides recombinant cells for the production of legionaminic acid and UDP-2,4-diacetamido-2,4,6-trideoxy-a-D-glucopyranose. Methods for producing the sugars are also provided.