516results about "Racemaces/epimerases" 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 gene engineering, and particularly relates to mutants of murA and ygeA genes and application of the mutants in production of human milk oligosaccharides. According to the invention, murA and ygeA genes of escherichia coli are edited by using a gene editing technology, so that a genetic engineering chassis strain carrying murA and ygeA mutants is obtained. Further, the chassis strain is applied to the construction process of production strains of LNT II, LNnT, LNT, 3 '-SL and 6'-SL, and the yield of the LNT II, the LNnT, the LNT, the 3 '-SL and the 6'-SL is further increased.

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 recombinant escherichia coli for producing D-psicose and a preparation method of the recombinant escherichia coli, and belongs to the field of genetic engineering. The technical problem to be solved in the invention is how to supply microorganisms for growth by using glycerol, acetic acid, fatty acid and the like as raw materials and prepare D-psicose by using glucose as a raw material. The invention provides recombinant Escherichia coli, which uses glycerol, acetic acid, fatty acid and the like as carbon sources to amplify (grow) the recombinant Escherichia coli, and uses D-glucose as a substrate to prepare D-psicose. According to the recombinant escherichia coli provided by the invention, glycerol, acetic acid, fatty acid and the like can be used as carbon sources for microbial growth, and D-psicose is synthesized by taking D-glucose as a raw material after the microbial growth reaches a certain biomass. Furthermore, a corresponding technical route for synthesizing the D-psicose is established.

The invention belongs to the technical field of chondroitin sulfate biosynthesis, and discloses a recombinant strain for high-yield chondroitin sulfate, application of the recombinant strain and a preparation method of the chondroitin sulfate. The recombinant strain provided by the invention can be used for simultaneously expressing UDP-glucosamine isomerase, UDP-glucose dehydrogenase, chondroitin polymerase, glucosamine synthetase, phosphoglucosamine mutase, UDP-N-acetylglucosamine pyrophosphorylase, glucose-6-phosphate isomerase and glucose phosphate mutase; according to the present invention, glucose-1-phosphate uridyl transferase, ATP sulfonase, sulfotransferase, pyruvate kinase and APS kinase are adopted, and the enzyme molecules are matched with the original carbon metabolism pathway in the recombinant strain cell to jointly form the enzyme-linked pathway having the efficient CSA synthesis ability, such that the high-yield and high-purity preparation of the CSA can be achieved; the method has the advantages of simple operation, high biological safety of the obtained product, easy realization of large-scale production and the like, and has a good application prospect.

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 invention provides a preparation method of immobilized D-psicose-3-epimerase, which comprises the following steps: firstly aminating inorganic silicon nanoflowers, then activating by using a cross-linking agent glutaraldehyde, finally adding enzyme liquid, reacting in a shaking table for a period of time, and centrifuging and washing the reactant to obtain the product, namely the immobilized D-psicose-3-epimerase. The enzymatic activity and the stability of the immobilized enzyme are obviously improved, the specific enzyme activity of the immobilized enzyme is 446.44-1033.09 U / g protein, the activity of the immobilized enzyme is 163.09% and 181.53% of that of free enzyme after the immobilized enzyme is incubated for 6 h under the condition that the temperature is 30 DEG C or the pH value is 5, 63.18-70.78% of the initial activity is still reserved after the immobilized enzyme is repeatedly used for 10 times, and 46.27-54.38% of the initial activity is still reserved after the immobilized enzyme is stored for 15 days. The immobilized enzyme can catalyze fructose to generate psicose, and a green and efficient preparation technology is provided for production of psicose.

The invention belongs to the field of gene engineering and biological treatment, and provides a hypoxia specific gene expression cassette, gram-negative bacteria containing the expression cassette and application of the gram-negative bacteria in preparation of tumor drugs. According to the invention, the forward hypoxia promoter and the reverse hyperxia promoter are reasonably designed, and appropriate survival essential genes are matched, so that the hypoxia specific gene expression cassette which is more efficient and safer is obtained. Gram-negative bacteria modified by the hypoxia specific gene expression cassette designed by the invention are low in overall mutation rate, and in a mouse in-vivo tumor model, more bacteria are distributed in tumors and the clearing speed in normal organs is higher.

The invention provides an engineering bacterium for producing 3 '-sialyllactose as well as a construction method and application thereof. The engineering bacterium has the capability of generating 3 '-sialic acid lactose, and an exogenous UDP-N-acetyl glucosamine-2-epimerase gene (neuC), a sialic acid synthase gene (neuB), an N-acetylneuraminic acid cytidyltransferase gene (css) and an alpha-2, 3-sialic acid transferase gene (ST) are introduced into a genome of the engineering bacterium or a recombinant plasmid carried by the engineering bacterium. The engineering bacterium has high capacity of producing 3 '-sialyllactose under the condition that antibiotics are not needed.

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 present invention relates to a recombinant yeast cell that produces UDP-D-glucuronic acid and / or UDP-N-acetyl-D-glucosamine compared to a wild type cell of the recombinant yeast cell; and optionally, the yield of 3 '-phosphate-adenylate sulfuric acid is increased. The invention also relates to a method for producing heparan sulfate, which comprises a step of culturing the recombinant yeast cell, further relates to an application of the non-invasive recombinant yeast cell for producing heparan sulfate, and finally relates to heparan sulfate obtained by applying the method.

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 provides a method for synthesizing D-psicose by using whole-cell transformed biomass hydrolysate, which comprises the following steps: by using a wet cell obtained from a culture solution obtained by inducing and culturing recombinant escherichia coli as a biocatalyst, directly using the biomass hydrolysate as a substrate in the presence of metal ions, and synthesizing D-psicose by using the whole-cell transformed biomass hydrolysate. A catalytic reaction is directly carried out without treatment to prepare D-psicose; the recombinant escherichia coli is the recombinant escherichia coli capable of simultaneously expressing glucose isomerase and D-psicose-3 epimerase. According to the method, the biomass hydrolysate can be converted into the high-concentration D-psicose solution in one step, whole cells are adopted as a catalyst, enzyme purification is not needed, the process steps are reduced, the inhibition effect of impurities in the hydrolysate on the enzyme is reduced, the production cost can be obviously reduced, industrial production is facilitated, and the method is suitable for industrial production. And the conversion rate of the D-psicose can reach more than 15%.

The invention discloses a high-temperature-resistant D-psicose-3-epimerase mutant and application of the high-temperature-resistant D-psicose-3-epimerase mutant in preparation of D-psicose. The mutant is obtained by performing single mutation or multiple mutations on the 17th site, the 118th site, the 121st site, the 165 site, the 197th site and the 249th site of an amino acid sequence shown in SEQ ID NO.2. The invention further discloses a preparation method of the high-temperature-resistant D-psicose-3-epimerase mutant. The mutant obtained through screening can tolerate the high temperature of 45-70 DEG C, the Tm value of the mutant enzyme is increased, the half-life period of the enzyme is prolonged, the affinity and conversion efficiency of the enzyme to a substrate D-psicose are improved, a relatively rare enzyme library is expanded, then the production cost is reduced, and the industrial application value of the D-psicose-3-epimerase is further improved.

The invention discloses application of D-mannose 2-epimerase in preparation of D-mannose, and belongs to the field of gene engineering and enzyme engineering. According to the present invention, the gene of the D-mannose 2-epimerase BbME derived from Bacteroidetes bacteria is expressed in E.coli BL21 (DE3), the D-mannose 2-epimerase is applied to a reaction system using D-glucose as a substrate, an enzyme reaction is performed at a pH value of 8.0 and a temperature of 50 DEG C, and the yield of the D-mannose after the reaction is performed for 48 h can achieve 141.05 g / L. The recombinant strain is subjected to high-density fermentation, crude enzyme liquid obtained after wall breaking of thallus cells is added into a 1.5 L large reaction system, the yield of D-mannose can reach 125.35 g / L after reaction is conducted for 48 h, and industrial production of D-mannose can be achieved.

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 D-psicose invertase mutant and application thereof. The mutant is obtained by performing single mutation or multiple mutation on the 13th site, the 49th site, the 54th site, the 100th site, the 235th site and the 242th site of an amino acid sequence shown in SEQ ID NO.2. The invention further discloses a preparation method of the D-psicose invertase mutant. The D-psicose invertase mutant can tolerate the high temperature of 40-80 DEG C, the half-life period of the mutant enzyme is prolonged, the substrate affinity and conversion efficiency of the enzyme to D-fructose used as a substrate for synthesizing D-psicose are improved, a relatively rare enzyme library is expanded, and the D-fructose is green, environment-friendly, low in toxicity and high in yield when being used for synthesizing the D-psicose invertase. The method overcomes the problem that three wastes are easily generated in a chemical synthesis method, and has important industrial application prospects.

The invention belongs to the technical field of biotechnology and biocatalysis, and particularly relates to a method for preparing D-tagatose by catalyzing isomerization of D-fructose. The invention provides a method for preparing D-tagatose by catalyzing isomerization of D-fructose, which comprises the following steps: by taking a wet cell obtained by inducible expression of a recombinant genetically engineered bacterium containing a D-tagatose-4-epimerase gene as a catalyst and D-fructose as a substrate, when the concentration of the substrate is higher than 300g / L, continuously supplementing the substrate and enzyme to prepare the D-tagatose by adopting a strategy of continuously supplementing the substrate and enzyme. The substrate inhibition is reduced, the conversion rate of the D-tagatose is increased, and the final conversion rate of the D-tagatose is increased from 19.5% to 24.2%.

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 field of gene engineering, and particularly relates to application of a willow uridine diphosphate glucose-4-epimerase gene SpUGE1 in plant cell wall component remodeling, organelle cadmium content reduction and cadmium resistance improvement. According to the invention, the SpUGE1 gene in willow is extracted, and is transferred into arabidopsis thaliana by constructing a recombinant plasmid, so that the biological activity, Cd < 2 + > concentration and the like of wild arabidopsis thaliana and transgenic arabidopsis thaliana are compared. The result of the embodiment shows that the transgenic arabidopsis thaliana has certain tolerance to cadmium stress, the cell wall of the transgenic arabidopsis thaliana is obviously thickened, and the content of cadmium in organelles is obviously reduced.

The invention provides a genetically engineered bacterium for producing lactose-N-neotetraose as well as a construction method and application of the genetically engineered bacterium. The genetically engineered bacterium meets the following conditions: (1) exogenous expression of an MFS transport protein, beta-1, 3-N-acetylglucosamine transferase and beta-1, 4-galactosyl transferase is carried out; and (2) over-expression of a galactoside osmose enzyme and a UDP-galactose-4-epimerase is carried out. According to the genetically engineered bacterium disclosed by the invention, the MFS transporter, the beta-1, 3-N-acetylglucosamine transferase and the beta-1, 4-galactosyl transferase are exogenously introduced, and the expression quantity of galactosidase and UDP-galactose-4-epimerase in a genome is enhanced, so that the residual quantity of LNT II in an LNnT synthesis route can be effectively reduced, and the fermentation yield of the LNnT is increased.

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 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 (1) above, 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 (1) above, 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 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.

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 discloses construction and application of a genetically engineered bacterium for synthesizing 2 '-fucosyllactose from glucose and xylose, and belongs to the field of microbial genetic engineering and the field of biosynthesis. According to the invention, escherichia coli is taken as an original strain, and the productivity of engineering bacteria is improved through strategies such as reconstruction of a glucose transport pathway, knockout of a competitive bypass, elimination of a byproduct pathway, up-regulation of key enzymes in a de novo synthesis pathway, enhancement of xylose utilization, overexpression of efficient alpha 1, 2-fucosyltransferase and the like; glucose and xylose are used as substrates to realize efficient synthesis of lactose and 2 '-fucosyllactose. In a shake flask fermentation experiment process, the yield of the 2 '-fucosyllactose can reach 6.53 g / L under the condition that no exogenous lactose is added. Under the condition of batch feeding, the yield of 2 '-fucosyllactose in a 5L fermentation tank reaches 27.53 g / L, and the method has industrial production potential.

The invention discloses a high-temperature-resistant cellobiose epimerase mutant and application thereof. The mutant is obtained by performing single mutation on the 242nd site of an amino acid sequence as shown in SEQ ID No.1. The invention also discloses a preparation method of the high-temperature-resistant cellobiose epimerase mutant. According to the invention, molecular modification is carried out on cellobiose epimerase (DiCE) from Dictyoglomus thermophilum, a mutant with improved thermal stability is screened out, the lactulose conversion efficiency of the obtained mutant is obviously improved, the mutant A242L can tolerate the temperature of 80 DEG C, the lactulose conversion rate is 64.41%, and the industrial application prospect is good.

The invention provides a method for producing sulfated polysaccharides by reacting a PAPS production / regeneration system utilizing the metabolic activity of a microorganism or a treated matter thereof with a microorganism expressing a sulfation enzyme or a treated matter or extract thereof upon mixing of inexpensive raw materials such as magnesium sulfate. The invention also provides a method for producing PAPS from inexpensive raw materials. The methods involve preparing a transformant (a) of a bacterium of the genus Corynebacterium, which contains a gene encoding an ATP sulfurylase and a gene encoding an APS kinase, which are expressible, and in which a cell plasma membrane of the transformant (a) is substance-permeable, or a treated matter of the transformant (a), and conducting a reaction for producing PAPS by using a reaction solution containing ATP or an ATP source, a sulfate ion source, and the transformant (a) or the treated matter thereof.

The invention belongs to the field of genetic engineering and biological treatment, and provides an eukaryotic expression vector, gram-negative bacteria containing the eukaryotic expression vector and application of the eukaryotic expression vector in preparation of tumor drugs. By selecting and combining a phage-derived promoter, a specific phage RNA polymerase gene, a membrane rupture protein gene and an eukaryotic expression drug protein gene, an eukaryotic expression vector is divided into three modules: (1) a phage-derived RNA polymerase gene transcription module; (2) a membrane rupture protein gene expression module; and (3) a eukaryotic drug protein gene expression module. Through organic combination of the three modules, the eukaryotic drug delivery system is constructed, and the system and method for stably and efficiently performing eukaryotic drug protein expression, drug delivery and drug killing are realized.