266 results about "Host bacterium" patented technology

The inventive method allows peptides or polypeptides to be exposed on the surface of gram-negative host bacteria using specific intimin-based anchor modules. Intimins with shortened carboxy terminals have been found to be particularly suitable anchor modules for passenger domains in the exterior E. coli cell membrane. According to the method, host bacteria are transformed using vectors, on which are located a fused nucleic acid sequence consisting of a sequence segment which codes for an intimin with a shortened carboxy terminal and a nucleic acid sequence segment which codes for the passenger peptide that is to be exposed. The invention permits a particularly large number of passenger domains to be exposed on the cell surface of the bacteria, without adversely affecting the viability of the bacteria.

The invention discloses a method for synthesizing L-theanine through an enzyme process, and belongs to the biotechnical field. The method is characterized in that a gamma-glutamyltranspeptidase gene is obtained through chemical synthesis, a gene engineering bacterium over-expressing gamma-glutamyltranspeptidase is constructed by treating Escherichia coli as a host bacterium, glutamine and ethylamine hydrochloride having different concentrations are acted by a recombinase, and theanine is efficiently produced at a temperature of 37-50DEG C under a pH value of 9.5-10.5. The enzyme source preparation process has the advantages of simplicity, low cost, and large enzyme amount, and the theanine production method has the advantages of simplicity, high conversion rate, high output, short time and the like, and is in favor of the industrialized amplification production.

The invention discloses a bacillus licheniformis host bacterium BL10 with multiple gene deletion, the accession number of which is CCTCCNO:M2013400. The host bacterium derives from a bacillus licheniformis WX-02 which partially or completely has deletion of ten genes. The ten genes comprises eight protease genes (mpr encoding a metalloprotease; vpr encoding a serine protease; aprX encoding an intracellular serine protease; epr encoding a minimal extracellular protease; bpr encoding a bacillus peptidase F; wprA encoding a protease combined with cell walls; aprE encoding an extracellular alkaline serine protease; and bprA encoding a bacillus peptidase F) and two extracellular secretory protein genes (hag encoding flagellin; and amyL encoding alpha-amylase). The BL10 has completely no extracellular protease activity and can reduce the degradation effect of a protease on a target protein. When the target protein is expressed by the expression host, the expression level is higher, and the host bacterium benefits protein expression enhancement.

The invention discloses gene recombination bacillus amyloliquefaciens and a microbial inoculum preparation method. A bacillus amyloliquefaciens FZB42 strain is taken as a host bacterium to construct the gene recombination bacillus amyloliquefaciens XLV09-1 with transparent vitreoscilla hemoglobin which is regulated and controlled by a P43 strong promoter by contrasting a gene recombination vector. The produced spore number of microbial inoculum prepared by fermenting the bacillus amyloliquefaciens can reach more than 100 hundred millions per milliliter, the overall yield of antibacterial lipopeptid reaches more than 1 gram per liter and prevention effect on various fungal plant diseases is improved by over 30 percent. The microbial inoculum is a green microecological preparation, of no toxic residue, safe to human and livestock, and favorable for protecting the ecological environment.

The invention relates to the field of aquaculture, and discloses a broad-spectrum bacteriophage preparation for aquaculture and a reparation method thereof. The bacteriophage preparation is prepared by fermenting a bacteriophage strain with a collection number of CGMCC No.9623 through a gene engineering escherichia coli DH5alpha serving as a host bacterium. The preparation method comprises the following steps: (1) adding the escherichia coli DH5alpha into a culture medium for fermental cultivation, thereby obtaining a host bacterium fermented solution; (2) feeding bacteriophage and MgCl2 mother liquid into the host bacterium fermented solution for uniform mixing, and then performing stewing and multiplication culture, thereby obtaining a fermented mixed solution; (3) centrifugating the fermented mixed solution, thereby obtaining the bacteriophage preparation. The broad-spectrum bacteriophage preparation disclosed by the invention has splitting action on various aquacultured pathogenicbacteria, and can be independently used or used with other microecological preparations. Furthermore, the host bacterium adopts a gene engineering bacterium. The preparation method is simple, convenient and safe to use, has no side effects and can be popularized and applied to prevention and treatment of bacterial diseases of aquaculture.

The invention discloses an engineering bacterium for producing a PL A2 and applications thereof. A preparation method of the PLA2 comprises the following steps: A, preparing a PLA2 gene: designing a primer PCR and amplifying a PLA2 gene fragment, and cloning a DNA fragment containing the PLA2 gene (a sequence is SEQ ID NO.2) with an amplified fragment plaT as a probe; B, constructing a recombinant plasmid: cloning a Bam H1 restricted fragment with the size of 3 kb which contains the PLA2 gene to a high-copy plasmid pHZ132 to obtain a recombinant plasmid pLH 001; and C, constructing the engineering bacterium: introducing the plasmid pLH001 into a streptomyces lividans 1326 of a host bacterium through protoplast transformation to obtain a streptomyces lividans LH001 of the engineering bacterium. The engineering bacterium has good stability and the introduced plasmid is not easy to lose. The secretory active PLA2 (an amino acid sequence is SEQ ID NO.1) can be generated through liquid fermentation, the enzymatic activity of a fermentation liquid is equal to or more than 2,000 U/mL, and the enzyme production capability is better than present levels. The PLA2 can be applied to fields of vegetable oil degumming, lysophospholipid preparation and the like.

The invention relates to a low-temperature alkaline protease gene, an engineering bacterium containing same, construction methods of the low-temperature alkaline protease gene and the engineering bacterium, and low-temperature alkaline protease. The sequence of the low-temperature alkaline protease gene is described as SEQ ID No. 2, and the sequence characteristic is 810bp, nucleic acid, single chain, linearity and DNA. The engineering bacterium producing the low-temperature alkaline protease is transformed from a host bacterium bacillussubtilis WB600 and has the characteristic of producing the low-temperature alkaline protease. The construction method of the engineering bacterium comprises the following steps of: obtaining an objective gene, reconstructing the objective gene directionally, constructing an expression carrier and constructing and sieving the engineering bacterium containing the low-temperature alkaline protease gene. The sequence of the mature peptide basic group of the low-temperature alkaline protease is disclosed in SEQ ID No. 2, the optimum operative temperature is 30 DEG C, and the optimum operative pH is 11.0. The optimum temperature of the alkaline protease produced by the engineering bacterium is 30 DEG C, the optimum pH is 11.0, and the invention provides a theoretical basis for the low-temperature alkaline protease added and used in a detergent and has important economic benefit and social benefit.

The invention discloses a preparation method of a genetically engineered bacterium for synthesizing glutathione. The preparation method comprises the following steps: (i) converting a gene for encoding gamma-glutamoyl cysteine synthetase-linker peptide-glutathione synthetase fusion protein into a host bacterium cell, and highly expressing the gene; (ii) converting a gene for encoding gamma-glutamoyl cysteine synthetase-linker peptide-glutathione synthetase into the host bacterium cell, and highly expressing the gene; (iii) knocking off the gamma-glutamyltranspeptidase of the host bacterium cell; (iv) knocking off an inositol-3-phosphatep synthase gene of the host bacterium cell; and completing the steps (i), (ii), (iii) and (iv) in any sequence, thereby obtaining the genetically engineered bacterium for synthesizing glutathione. The invention further relates to the prepared genetically engineered bacterium. The genetically engineered bacterium for synthesizing glutathione, which is disclosed by the invention, is not only high in efficiency in synthesizing glutathione, but also can greatly improve the conversion efficiency of exogenous cysteine, and can be applied to glutathione production.

This invention relates to a method for producing recombined human granular leukocyte colony stimulating factors including: fermenting, breaking bactrriums, extracting occlusion bodies, chromatographing with molecular sieves, renaturating, exchanging anions, exchanging cations to get the raw fluid, in which, BL21 is selected as the host bacterium and the engineering bacterium type is got in high expression volume, quick reproduction and stable passage, several purification steps greatly reduce the residural toxin in the bacterium and the specific activity is increased greatly, a dialysis method is applied for the renaturation to reduce the concentration of the denaturalization agent steadily so the renaturation rate is at high level.

The invention discloses a recombinant plasmid for expressing vitreoscilla hemoglobin in a cell and a construction method of the recombinant plasmid, a genetic engineering bacterium of Gluconobacter oxydans (G.oxydans) and a construction method and application of the genetic engineering bacterium. The recombinant plasmid comprises a vitreoscilla hemoglobin gene vgb, a PtufB promoter of the Gluconobacter oxydans (G.oxydans) and a suitable carrier, wherein the genetic engineering bacterium is obtained by transforming recombinant plasmid into a host bacterium. The genetic engineering bacterium can be used for expressing the vitreoscilla hemoglobin in the cell, can be used for improving the yield of biomass and catalysate 1, 3-dihydroxyacetone under the condition of not changing the existing equipment and energy consumption pressure and provides an effective path for solving the problem of in sufficient oxygen supply in the cell cultivation and catalytic process of the Gluconobacter oxydans (G.oxydans).

The invention belongs to the field of environmental science, and discloses a construction method of a host bacterium for expressing a PETase enzyme and a MHTEase enzyme, a composite strain composed ofthe host bacterium, pseudomonas putida and rhodococcus and a method for degrading the polyethylene terephthalate (PET). The composite strain is formed by mixing the host bacterium expressing the PETase enzyme and the MHTEase enzyme, the pseudomonas putida and the rhodococcus, and adopted to degrade the PET to solve the problem of the inhibition effect of a PET degradation product TPA and the toxic effects of the degradation product TPA and ethylene glycol (EG) on the environment, thereby achieving green degradation of PET.

The invention relates to a Fenneropenaeus chinensis antimicrobial protein gene and the recombination expression and the application. It has the amino acid sequence of SEQ ID No.2 and basic group sequence of SEQ ID No.1 in sequence table. It selects the carrier of the antimicrobial protein pCR T7/NT TOPO TA and host bacterium BL21(DE3)pLysS that could high efficiently express recombination protein. Aimed at the feature of plural disulfide bonds, using the oxidation reduction system made of oxidized form of glutathione and glutathione in separating and purification and protein recombination process, the recombination protein with antimicrobial ability would be gained. The protein has strong inhibition to the growth of gram positivebacteria, and some inhibition to gram negativebacteria.

The invention discloses a method for knocking out pckA to promote synthesis of acetylglucosamine through bacillus subtilis and belongs to the field of genetic engineering. The method comprises the steps that BSGNK-PxylA-glmS-P43-GNA1 serves as original strains, phosphoenolpyruvate carboxylase (pckA) genes are knocked out through homologous recombination, the reaction that enolphosphopyruvate is converted into oxaloacetic acid in host bacterium cells is blocked, the carbon flux flowing to a tricarboxylic acid cycle is reduced, and accumulation of acetylglucosamine is promoted. In the process that a composite culture medium is used for fermentation, the acetylglucosamine yield of recombination bacillus subtilis with pckA knocked out reaches 29.27 g/L and is increased by 28.1% compared with that of control strains. The method lays a foundation for transforming bacillus subtilis to produce acetylglucosamine in metabolic engineering.

The invention discloses provides a method of increasing heterologous expression quantity of hemicellulase, cellulase or lignocellulose hydrolysis assistance zymoprotein in pichia pastoris. An xylanase (XynC) gene from Aspergillus niger is subjected to codon optimization according to pichia pastoris codon preference characteristics, and an optimized nucleotide sequence (AxynC) is artificially synthesized, and constructs and recombines expression vectors pPIC9K-AxynC and pPICZ alpha A-AxynC. Recombined pichia pastoris of high yield xylanase is constructed by a two-step shock conversion method. That is to say, linearized pPIC9K-AxynC converts the pichia pastoris GS115 to obtain a strain with better enzyme producing ability. The high enzyme activity is subjected to second shock conversion as a host bacterium, and the linearized pPICZ alpha A-AxynC is shocked to convert the strain to obtain double-plasmid recombined pichia pastoris of the high yield xylanase. Tests show that the codon optimized xylanase gene can be expressed successfully in the pichia pastoris, the high yield xylanase recombinant bacterium constructed by a two-step shock conversion method can stably and efficiently produce the xylanase, the flask level enzyme activity reaches 410U/mL, and a protein content in a fermentated supernate reaches 0.37mg/mL.

The invention discloses a bile salt hydrolase (BSH) mutant and a use thereof, and belongs to the technical field of gene engineering. Through comparison analysis of a BSH amino acid sequence and homology modeling and overlay analysis of a 3-D structure, a BSH substrate specific amino acid mutation site is determined, through a site-specific mutagenesis technology, the substrate specific amino acid mutates into a nonpolar amino acid, and the mutant is transferred into a host bacterium so that a mutant strain is obtained. The BSH produced by the mutant is active enzyme. Compared with a wild-type bacterium, the mutant has a reduced glycine-combined bile salt hydrolysis capability and an improved taurine-combined bile salt hydrolysis capability. The BSH mutant lays a foundation for improving a taurine-combined bile salt hydrolysis capability of BSH and uncovering a substrate combination mechanism of BSH.

The invention discloses a method for separating purified bdellovibrio from active sludge. By virtue of key techniques of host bacteria screening and active sludge pretreatment and technical steps such as preparation of a culture medium and a culture solution and optimization of culture conditions and the like, efficient bdellovibrio is separated and purified from a homologous sludge sample by optimization of a series of key steps by virtue of gram negative bacteria which are obtained in active sludge as host bacteria for separating and purifying bdellovibrio. The invention not only puts forwards a method for successfully separating the efficient bdellovibrio from the active sludge of a municipal wastewater treatment plant for the first time, but also the culture period of the method is remarkably shortened, so that the defects that the existing conventional method is unstable in bdellovibrio separating effect, long in culture period, tedious in operation and the like.

The invention discloses a colibacillus engineering bacterium taking glucose as a substrate for synthesizing muconic acid, and belongs to the field of biotechnology. 2,3-dihydroxy benzoic acid (2,3-DHB) is led to colibacillus during the metabolic pathway of muconic acid (MA); the metabolic pathway of colibacillus from glucose to 2,3-DHB is reinforced. The colibacillus engineering bacterium has the advantages that less foreign genes are required; the foreign gene expression load of a host bacterium is reduced; the incompatibility problem of the foreign genes is solved. Moreover, the host bacteria are not auxotroph; the foreign addition of expensive shikimic acid or other aromatic amino acid is avoided; the flask shaking fermentation yield of muconic acid is 1.15 g/L.

The invention relates to the field of veterinary medicine, in particular to an animal recombinant protein vaccine. A recombinant protein contains an epitope of an antigen to a porcine circovirus (PCV) and an epitope of an antigen to a porcine reproductive and respiratory syndrome virus (PRRSV), and supports the vaccine after being emulsified. According to the recombinant protein, a target fragment shown as SEQ ID NO:6 is synthesized, the target fragment and a PGEX-4T-1 vector form a recombinant vector, a recombinant plasmid is converted into a host bacterium induced expression, and the recombinant protein is extracted. The recombinant protein content for swine immunization is a product of 4-hour host bacterium induced expression, and the recombinant protein at concentration of over 80 percent is purified by processes of inclusion body recovery, dissolution, concentration and the like. The prepared recombinant protein has a stable property, does not have the characteristics of virus transmission and the like, and is greatly advanced or plays an irreplaceable role in the researches and development of PRRSV and PCV2 prevention and control vaccines.

The invention provides a bacteriophage genome DNA (deoxyribonucleic acid) extraction kit and method. The kit comprises a reagent A (chloroform), a reagent B (DNase I), a reagent C (RNase A), a reagent D (precipitation buffer solution), a reagent E (pyrolysis buffer solution), a reagent F (primary pyrolysis main solution), a reagent G (secondary pyrolysis solution), a reagent H (impurity removal solution), a reagent I (DNA combination buffer solution), a reagent J (rinsing buffer solution) and a reagent K (DNA eluate). The extraction method comprises the following steps: removal of bacterium cells and fragments, degradation of bacterium nucleic acid, bacteriophage particle precipitation, bacteriophage capsid protein structure damage and hydrolysis, impurity removal, DNA liquid separation, DNA adsorption, cleaning of adsorbed DNA and elution of adsorbed DNA. The kit and method can be widely used for extracting the bacteriophage genome DNA, and has the advantages of high extraction speed, high extracted DNA yield and high purity, and the extracted DNA can not be polluted by the host bacterium genome DNA.