1117 results about "Recombinant escherichia coli" patented technology

The invention discloses a mixture of poly-pneumococcal capsular polysaccharide-protein conjugates. The mixture contains 13 pneumococcal capsular polysaccharide-protein conjugates and an immunity-enhancement adjuvant, wherein each pneumococcal capsular polysaccharide-protein conjugate is formed by combining corresponding serum-type pneumococcal capsular polysaccharide with a same protein carrier through a covalent bond; the 13 pneumococcal capsular polysaccharides are obtained by purifying and extracting 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F bacteria; the protein carrier is A chain of a diphtherin mutant CRM197 obtained from expression of genetic recombinant escherichia coli. Meanwhile, the invention discloses a preparation method of the mixture. The mixture, compared to conventional bacterial fermentation total-chain diphtherin mutant CRM197, is easy to purify, high in yield and low in cost; experiments prove that the mixture disclosed by the invention is immunogenic, and is applicable to clinical inoculation.

A scalable alkaline lysis process, including procedures and devices for the isolation of large quantities (grams and kilograms) of plasmid DNA from recombinant E. coli cells. Effective, controllable, and economical operation, and consistent low level of host chromosomal DNA in the final plasmid product. Involves a series of new unit operations and devices for cell resuspension, cell lysis, and neutralization.

This invention relates to a method for the production of D-(-)-3-hydroxybutyric acid, comprising the step of culturing a recombinant strain containing genes phbA, phbB, ptb and buk by fermentation. Preferably, the recombinant strain is a strain of E. coli. The method of the invention is simple, avoiding the technique of degrading polymer to produce D-(-)-3-hydroxybutyric acid. The present method also provides improved efficiency, lowers the complicated requirement for facilities as used in traditional chemical synthesis, simplifies the complicated technique flow, and omits the complicated chiral separation step. Therefore, the present method greatly reduces the costs associated with D-(-)-3-hydroxybutyric acid production. Also, with this invention, the problems such as environmental pollution of chemical synthesis and chiral separation are overcome.

The invention relates to a method for biosynthesis preparation of human glucagon-like peptide-1 (GLP-1) and an analogue thereof. With adopting of a gene engineering technology, a recombinant escherichia coli expressed GLP-1 fusion protein is constructed, and a protein enzyme digestion site is designed in the fusion protein; a fusion gene has a gene sequence with a form of A-B-C structure, wherein A is a chaperonin gene, B is a nucleotide sequence encoding a connection peptide containing the enzyme digestion site, and C is a gene encoding the GLP-1 or the analogue thereof. After recombinant engineering bacteria is subjected to induced expression, the fusion protein is purified and subjected to enzyme digestion, and then the GLP-1 and the analogue thereof are obtained and are detected to have biological activity. The preparation method of the GLP-1 and the analogue thereof provided by the invention is simple and quick, the production conditions are mild, the product is convenient to separate and extract, the process is simple, and the industrialization prospect is good.

The invention discloses an E. coli BL21(pETDuet-ydjLnox) containing a 2R,3R-butanediol dehydrogenase gene ydjL and an NADH oxidase gene nox, wherein the strain is preserved in the 'China Center for Type Culture Collection' on December 23 in 2008, and the preservation number is CCTCC NO: M 208259. The invention also discloses application of a gene recombination bacterium in producing chiral S-AC by catalyzing meso-BD, producing chiral R-AC by catalyzing 2R,3R-BD and producing chiral pure 2S,3S-BD by splitting a 2,3-BD mixture. The concentration of a chiral AC prepared from the recombinant E. coli can reach over 6 grams per liter (the ee value is more than or equal to 96 percent); and the ee value of the chiral pure 2S,3S-BD is more than or equal to 98 percent, and the regeneration of a cofactor is achieved, thus the gene recombination bacterium has great industrial application prospect.

The invention discloses novel N-acetylneuraminic acid-producing escherichia coli engineering bacteria as well as a construction method and application thereof. The engineering bacterial is constructed by introducing an encoded 6-glucosamine phosphate acetylase gene, an N-acetyl glucosamine-2-isomerase gene and an N-acetylneuraminic acid synthetase gene into escherichia coli to express, carrying out strengthened expression on 6-glucosamine phosphate deaminase gene contained in the escherichia coli per se and knocking off genes, for decomposing and utilizing enzyme in metabolic pathways, of the N-acetylneuraminic acid in the engineering bacteria. The engineering bacteria disclosed by the invention can be used for fermentation culture to synthesize the N-acetylneuraminic acid by using glucose or glycerinum as a substrate.

The invention discloses recombinant escherichia coli using glucose for producing hydroxytyrosol as well as a building method and application. The method comprises the following steps that SEQ ID NO.1 and 2 are used as primers; the escherichia coli BW25113 genome DNA (deoxyribonucleic acid) is used as a template; HpaBC gene PCR (polymerase chain reaction) augmentation is carried out; vectors pTrcHisB are connected; middle plasmids are obtained; the middle plasmids are used as templates; the SEQ ID NO.3 and 2 are used as primers; PCR augmentation is carried out, and plasmids pTrcHisB-ARO10 are connected; expression vectors pTrcHisB-ARO10-HpaBC are obtained; in addition, the vectors and the plasmids pBb3 are simultaneously transferred into escherichia coli strains BMGA to obtain the recombinant escherichia coli. The recombinant escherichia coli contains ARO10 genes from saccharomyces cerevisiae, and HpaBC genes from the escherichia coli endogenesis. The yield of the hydroxytyrosol can be obviously improved.

The invention discloses a high-yield N-acetylglucosamine metabolic engineering bacterium, as well a construction method and applications thereof. The engineering bacterium is a recombinant Escherichia coli by leading a coded UDP-N-acetylglucosamine epimerase gene and a coded 6-glucosamine phosphate synthetase gene into Escherichia coli for expression, and knocking out the gene N-acetylglucosamine in the Escherichia coli to decompose and utilize metabolic pathway enzyme; and the constructed engineering bacterium strain utilizes glucose as a substrate for fermenting and culturing and synthesizing the N-acetylglucosamine. The engineering bacterium is high in the fermenting level of synthesizing the N-acetylglucosamine by utilizing glucose, the accumulation of side products is less, and industrial production potential capability can be achieved.

The invention discloses aminotransferase, a mutant and application to Sitagliptin preparation. According to the application, wet thalli obtained by performing fermentation culture on recombinant escherichia coli containing aminotransferase encoding genes are used as biocatalysts; Sitagliptin precursor ketone is used as a substrate; dimethyl sulfoxide is used as a latent solvent; phosphopyridoxal is used as a coenzyme; isopropylamine is used as an auxiliary substrate; a trolamine buffer solution with the pH being 8 to 9 is used as a reaction medium; a reaction system is formed; the biocatalytic reaction is performed under the conditions of the temperature being 30 to 45 DEG C and the stirring speed being 100 to 250 r / min; after the reaction is completed, the reaction liquid is separated and purified; the Sitagliptin is obtained. The aminotransferase and the mutant are used as biocatalysts; the latent carbonyl compound of Sitagliptin precursor ketone is directly used as the substrate; meanwhile, biocatalytic reaction is performed by using isopropylamine as the auxiliary substrate and using the pyridoxal phosphate as the coenzyme; the separation and purification is performed; Sitagliptin with high optical purity is prepared. The method has the advantages that the total yield is 76 percent; the product e.e. value reaches 99 percent.

The invention discloses a method for biosynthesizing 2'-fucosyllactose by constructing recombinant colibacillus. The name of colibacillus genetic engineering bacteria is E.coli-XYY-1, a synthetic route of 2'-fucosyllactose is achieved, and meanwhile a construction method is disclosed. According to the method for biosynthesizing 2'-fucosyllactose by constructing recombinant colibacillus, existing technical problems are solved, and an efficient gene knockout scheme is obtained; after original strains are subjected to genetic reconstruction, 2'-fucosyllactose is produced, other characteristics of the strains are not changed, and fermentation production is not affected; plasmid adopted by the strains is mature colibacillus plasmid, and therefore growth and normal metabolism of bacteria are not affected in the metabolic process. The constructed recombinant colibacillus has a very large application prospect, and a new idea is provided for generating 2'-fucosyllactose through a biological method.

The invention provides a recombinant escherichia coli for high-yield tetrahydropyrimidine and a method for preparing tetrahydropyrimidine by using the recombinant escherichia coli. The recombinant escherichia coli provided by the invention is prepared by importing Halomonas elongate EctABC containing recombinant plasmids into escherichia coli. The recombinant escherichia coli disclosed by the invention realizes the soluble expression of three key enzymes synthesized by tetrahydropyrimidine under the adjustment and control of an arabinose promoter. Thalli subjected to induced expression implements the efficient secretory expression of tetrahydropyrimidine by taking sodium aspartate as a precursor through a bioconversion method. Thalli per gram can synthesize 1.1 grams of tetrahydropyrimidine, and more than 90% of tetrahydropyrimidine is secreted to extracellular receptors. The method for preparing tetrahydropyrimidine by using the recombinant escherichia coli provided by the invention facilitates the downstream purification and separation of products, and has great significance on the industrial production and large-scale application of tetrahydropyrimidine.

The invention discloses nitrilase mutant and application thereof. The mutant is obtained by carrying out mutation one or more of the sits including 201 site, 339 site and 343 site of an amino acid sequence as shown in SEQ ID No.2. By modification of protein molecules, the thermal stability of nitrilase AcN pure enzyme is improved by 14 times to a maximum extent, recombinant escherichia coli which contains the nitrilase mutant is used for hydrolyzing 1-cyanocyclohexane acetonitrile at the high temperature (45 DEG C), and the reaction time is shortened to be one third of the original reaction time. Therefore, the acquired mutant has good application prospect in efficient catalysis of the 1-cyanocyclohexane acetonitrile to synthesize 1-cyanocyclohexane acetic acid as a gabapentin intermediate.

The invention discloses a bacterial laccase mutant protein, which is characterized in that the mutant protein amino acid sequence is obtained by deletion mutation of the 323rd glycine residue to the 332rd glycine residue in the bacterial laccase amino acid sequence shown as SEQ ID No.1. Through a genetic engineering reconstruction method, a stability improved bacterial laccase protein coding gene, its expression plasmid and engineered bacteria can be obtained, and after large-scale fermentation and induced expression of the engineered bacteria, the stability improved bacterial laccase protein can be obtained. According to the invention, the marine uncultured microorganism source bacterial laccase Lac15 is taken as the foundation, and by means of genetic engineering reconstruction, mutant gene can be obtained. At the same time, a recombinant escherichia coli is employed to conduct high-density culture for high-efficiency expression of the bacterial laccase mutant protein. According to the invention, the stability and yield of the bacterial laccase are greatly improved.

This invention relates to a method for utilizing less purified starch in fermentation processes. One example is a recombinant E. coli containing a exogenous extracellular isoamylase activity that is capable of utilizing small oligomers containing (1,6) linkages (including but not limited to isomaltose and panose) in fermentations to produce useful products. The invention is useful in large-scale industrial biofermentations by reducing the cost of the substrate carbohydrate.

A scalable alkaline lysis process, including procedures and devices for the isolation of large quantities (grams and kilograms) of plasmid DNA from recombinant E. coli cell. Effective, contgrollable, and economical operation, and consistent low level of host chromosomal DNA in the final plasmid product. Involves a series of new unit operations and devices for cell resuspension, cell lysis, and nuetralization.

A process for producing recombinant anti-botulinum toxin antibody comprising the steps of fermenting recombinant E. Coli cells in broth, concentrating the cells by removing the broth, crushing the concentrated cells, separating a permeate derived from the crushed cells from cell debris, purifying a recombinant antibotulinum antibody (Fab) from said permeate, and separating said Fab from impurities by diafiltration.

The invention relates to an expression plasmid containing streptococcus zooepidemicus hyaluronidase genes and the application thereof and aims at providing a streptococcus zooepidemicus hyaluronidase expression vector, a recombinant Escherichia coil cell, a method for generating the hyaluronidase by using the cell and a method for preparing a hyaluronic acid oligose. By using a PCR method, the expression plasmid containing coding genes of the streptococcus zooepidemicus hyaluronidase is formed; a recombinant Escherichia coil strain is obtained by transforming the expression plasmid; and the hyaluronidase is generated by expressing the engineering bacterium. The hyaluronidase expression vector is an Escherichia coil expression vector containing streptococcus zooepidemicus hyaluronidase genes. The method for expressing the zooepidemicus hyaluronidase comprises the following steps of: culturing the recombinant Escherichia coil cells; expressing the hyaluronidase by controlling the temperature or IPTG induction; and using the hyaluronidase to prepare the HA oligose. The method for preparing the hyaluronidase has the characteristics of high expression level, easily expanded production and low cost. The hyaluronidase of the invention can be applied to degrading a hyaluronic acid.

The invention discloses escherichia coli genetically engineered bacteria co-expressed by three enzymes. The escherichia coli genetically engineered bacteria are characterized in that an L-amino-acid oxidase gene, a (D / L)-alpha-hydroxyl carboxylic acid dehydrogenase gene and a gene capable of reducing NAD(P) are imported. The invention also discloses a construction method and an application of the recombinant escherichia coli. The method is used to biologically synthesize optically pure alpha-hydroxyl carboxylic acid, has the characteristics of simple operation, low cost, high product synthesizing efficiency and high optical purity, and has a good industrial prospect.

The invention discloses an immune colloidal gold indicator paper and making method to test cow tuberculosis antibody, which comprises the following parts: sample pad, connecting pad, cellulose nitrate film, absorbent pad and PVC backing liner, wherein the sample pad, connecting pad, cellulose nitrate film and absorbent pad are attached on the PVC backing liner; the connecting pad clads MPB83 protein-colloidal gold marker; the purified MPB70 protein is cladded on the cellulose nitrate film, which constitutes detecting line and quality control line formed by pure rabbit-anti-MPB83 protein lgG. The invention also provides the making method of cow mycobacterium astopic antigen MPB83, which possesses the recombinant Escherichia coli BL21 / pET28a-MPB83 in the CCTCC with reserving number at CCTCC NO:M206142.

The invention discloses recombinant Escherichia coli producing salidroside, a construction method and applications thereof. The construction method comprises: (1) using Escherichia coli [delta]A as a starting strain, and carrying out gene knockdown or gene silencing to make the galE gene, the galT gene and the ugd gene on the Escherichia coli chromosome be not expressed to obtain an Escherichia coli strain BMGU; and (2) introducing genes such as ARO10, UGT73B6MK, pgm, galU and T7Polymerase to over-express the genes such as ARO10, UGT73B6MK, pgm, galU and T7Polymerase so as to achieve the heterologous synthesis of salidroside, such that the recombinant Escherichia coli producing salidroside is obtained. According to the present invention, the efficient UDP-glucosyltransferase mutant UGT73B6MK is introduced, and the glucose metabolism pathway is optimized, such that the yield of salidroside is significantly improved.

The invention discloses a recombinant escherichia coli which is called recombinant escherichia coli DALA. The recombinant escherichia coli DALA is prepared through the following steps: constructing a co-expression vector p-hemA<M>-hemL containing hemA<M> gene and hemL gene; constructing an expression vector p-rhtA containing rhtA gene; and converting constructed recombinant plasmids of p-hemA<M>-hemL and p-rhtA to the escherichia coli to obtain the recombinant escherichia coli DALA which can overexpress the hemA<M> gene, the hemL gene and the rhtA gene. The invention also discloses an application of the recombinant escherichia coli in production of the ALA. A fermentation result shows that the output of the ALA produced by the recombinant escherichia coli achieves 4.13 g / L, the conversionrate of the ALA to glucose is 0.168 g / g and the escherichia coli has good industrial development and application prospects.

An Escherichia coli recombinant strain producing shikimic acid, and a construction method and application thereof belong to the technical field of microbial gene engineering. The invention firstly utilizes a molecular biology technique to delete shikimic acid kinase I gene (aroK) and shikimic acid kinase II gene (aroL) of Escherichia coli CICIMB0013, and a gene (ptsG) of a key protein EIIBC<Glc> and a quinin acid / shikimic acid dehydrogenase gene (ydiB) of a glucose phosphotransferase system to obtain an Escherichia coli mutant strain CICIMB0013.SA4 (delta aroK, delta aroL, delta ptsG, delta ydiB). The invention also constructs a recombinant expression plasmid pTHGAA containing key genes comprising aroG*,ppsA and tktA in a metabolic pathway of shikimic acid; and the recombinant expression plasmid pTHGAA is transferred into the recombinant strain CICIMB0013.SA4 to obtain a recombinant Escherichia coli B0013 (SA4 / pTHGAA) capable of producing shikimic acid efficiently. The Escherichia coli recombinant strain provided by the invention can realize efficient accumulation of shikimic acid in a fermentation process.

The invention belongs to the field of molecular biology, and discloses a porcine circovirus type 2 recombinant cap protein and a subunit vaccine. The porcine circovirus type 2 cap protein expressed by recombinant Escherichia coli is obtained by steps of cloning a porcine circovirus type 2 cap protein in a nuclear localization signal area of which the N terminal is cut and which is rich in arginine into a prokaryotic expression vector to obtain a recombinant expression vector, transfecting the recombinant expression vector into Escherichia coli BL21(DE3), and expressing by using the recombinant Escherichia coli BL21(DE3). Tests prove that the constructed recombinant strain expresses a foreign protein stably. When the subunit vaccine is prepared from the expressed recombinant protein, an antigen has high purity and safety, does not have pathogenicity on animals such as pigs and the like, and passes safety evaluation easily.

The invention discloses a heparanase II fusion protein, and a coding gene and an expression method thereof. The fusion protein (named MBP-HepB) is a protein of a) or b), wherein a) is a protein consisting of an amino acid sequences shown from the 1st position to 1,118th position of a sequence 2 in a sequence table; and b) is a protein in which one or more amino acid is substituted, and / or deleted and / or added in the amino acid sequence of a sequence 2 in a sequence table and which has heparanase activity and derives from the a). The coding gene of the protein is also in the protection scope of the invention. The gene is introduced into the protein heparanase expressed in the recombinant escherichia coli, the enzyme activity reaches 118.4IU / L fermentation liquor, the expression quantity reaches 179mg / L fermentation liquor, and the specific enzyme activity reaches 0.66IU / mg. The one-step purification of the fusion protein can also be realized by affinity separation.

The invention discloses a high-density fermentation method of engineering bacteria containing nitrilase. The method adopts a novel recombinant Escherichia coli fermentation and supplementary material culture medium formula, and uses a method of DO-STAT coupling step-by-step induction and gradual increase of the speed stage by stage. The method not only meets the need for mixing power in the culture process with biomass increase, effectively controls the growth rate of thalli, and prevents disadvantages of produced acid accumulation caused by fast growth of thalli, feedback inhibition and culture medium waste. In addition, the induced mode of batch-by-batch lactose supplement addition avoids the high viscosity of the fermentation broth caused by high concentration of lactose, resulting in resistance of oxygen transfer and mass transfer, but also improves the induction strength. The high-density fermentation technology of the invention increase the biomass of recombinant Escherichia coli from 10.35g DCW / L to 70g DCW / L, volume enzyme activity from 18205U / L to 103530U / L, and the fermentation level by 4.7 times.

The invention belongs to the technical field of animal-borne disease subunit vaccine preparation and relates to preparation and application of the immune protective antigen of the haemophilus parasuis. Outer membrane protein Hbp B genes of the haemophilus parasuis are cloned, a nucleotide sequence is indicated as SEQID NO:1, and the sequence of gene code is indicated as SEQ ID NO:2. Recombination Escherichia coli BL21 / Pet-28a-Hbp B (preservation number is CCTCC NO:M2011228) is built and comprises genes in a sequence table SEQ ID NO:1. Antigen protein of the haemophilus parasuis is obtained and expressed through gene transformation Escherichia coli in the SEQ ID NO:1. The invention further discloses a preparation method and application of the recombination Escherichia coli. The haemophilus parasuis subunit vaccine has good safety, and an immune protection effect reaches 83%.

The invention discloses a heparanase III fusion protein and a coding gene and an expression method thereof. The fusion protein of the invention (named as MBP-HepC) is a protein in a) or b): a) a protein consisting of amino acid sequences shown by the 1-1028th site of a sequence 2 in a sequence table; and b) a protein derived from a), provided with heparanase III activity and obtained by substituting and / or deleting and / or adding one or more amino acids in an amino acid sequence of the sequence 2 in the sequence table. The coding gene of the protein is also within the protection range of the invention. By introducing the gene into protein heparanase expressed in recombinant escherichia coli, the enzymatic activity can reach 1,776.3 IU per liter of fermentation liquid, the expression amount can reach 240 mg per liter of fermentation liquid, and the specific enzymatic activity can reach 7.39 IU / mg. The invention also can realize further purification of the fusion protein through affinity separation.

The invention relates to a production method for synthesizing C8:0 / C10:0 / C12:0 / C14:0 medium-chain fatty acid and ethyl ester thereof by using engineering Escherichia coli. The production method comprises the following steps of: cloning thioesterase genes with different specificities chFatB / ucFatB / ccFatB in cuphea / laurel / camphorwood into an expression vector pETDuet-1 to obtain a corresponding recombinant plasmid; transferring the recombinant plasmid into Escherichia coli BL21, performing isopropyl-beta-d-thiogalactoside (IPDT) induction expression, and specifically hydrolyzing acyl carrier protein (ACP) with specific chain length by using the expressed thioesterase to obtain medium-chain fatty acid; cloning acyltransferase gene (atfA) in acinetobacter into expression vectors pETDuet-chFatB / pETDuet-ucFatB / pETDuet-ccFatB to obtain corresponding recombinant plasmids; and transferring the recombinant plasmids into the Escherichia coli BL21, performing IPTG induction expression, performing acyl transfer reaction on exogenously fed ethanol and acyl-CoA formed by the medium-chain fatty acid intracellularly by using the co-expressed acyltransferase to obtain fatty acid ethyl ester.

The invention provides malic acid-production gene engineering bacteria Escherichiacoli BA043. The malic acid-production gene engineering bacteria is named as BA043 (Escherichiacoli BA043) and has a preservation registration number CCTCC NO: M2014034. The invention also provides a construction method of the malic acid-production gene engineering bacteria and a method for fermentation production of malic acid. Through knockout or inactivation of fumarase and fumarate reductase and through over coexpression of exogenous pyruvate carboxylase and nicotinic acid phosphoribosyl transferase, recombinant escherichia coli can reduce by-product pyruvic acid generation by glucose metabolism growth so that a malic acid yield and production intensity are greatly improved.

Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.