38 results about "Linoleate isomerase" patented technology

The invention is a method of extracting active substance-sea Erigeron acris seed oil from natural plant sea Erigeron acris, adding water to the dried and crushed or pulped whole plants or seeds of sea Erigeron acris to blend, heat and cook, then cooling to 40 deg.C-60 deg.C, adding enzyme preparation to regulate the pH value to 2-10 so as to make enzymolysis for 1-20 hours, heating and filtering deposit, taking enzymolysis solution to extract mixed oil of sea Erigeron acris seeds by organic solvent; or using linolic acid isomerizing enzyme to convert the linolic acid in the mixed oil to conjugated linolic acid. The mixed oil extraction ratio is up to 29%-35%, and the ratio of linolic acid to sea Erigeron acris oil >=74%.

The invention discloses a method to produce conjugate linolic acid with linolic acid isomerase, which comprises the following steps: reacting linolic acid and linolic acid isomerase in hypercritical carbon dioxide for 1-3h; setting pressure of the reaction at 25-35MPa and temperature at 32-44 deg. c; getting conjugate linolic acid. This invention can decrease the cost and increase the conversion rate of conjugate linolic acid.

The invention relates to a recombinant yeast strain capable of producing conjugated linoleic acid and an application thereof. In the invention, codon optimization is carried out on linoleate isomerase in propionibacterium acnes (PAI) gene to obtain an optimized gene opai, recombinant expression plasmid and recombinant Yarrowia lipolytica bacterium which contain the opai gene are prepared, and therecombinant Yarrowia lipolytica bacterium which expresses the opai gene is utilized to produce conjugated linoleic acid.

The present application is directed to a process for the production of trans-10, cis-12 conjugated linoleic acid in a transgenic microorganism comprising the steps of: (a) introducing into said microorganism at least one nucleic acid molecule encoding a trans-10, cis-12 conjugated linoleic acid isomerase, (b) culturing the transgenic microorganism obtained under (a), (c) inducing the production of trans-10, cis-12 conjugated linoleic acid by adding linoleic acid to the culture, (d) incubating the induced culture for at least 12 hours, and (e) isolating the conjugated linoleic acid from the culture media and / or transgenic microorganism.

The present invention is plant lactobacillus in the preservation number of CCTCC No. M206033 and the biological process of preparing conjugate linoleic acid with the lactobacillus, and belongs to the field of bioengineering technology. The plant lactobacillus can grow well in aerobic condition, and during fermentation with linoleic acid substrate contacting with the lactobacillus cell, linoleic acid will be converted into conjugate linoleic acid under the action of linoleic acid isomerase combined with membrane. The bioconversion process includes the first culturing of the lactobacillus strain in MRS culture medium containing 0.5mg / ml LA and the subsequent inoculation of the culture to MRS culture medium containing free linoleic acid in pH 6.5 at 37 deg.c with or without potassium phosphate buffering solution for culturing to produce conjugate linoleic acid.

The invention discloses a method for catalytically synthesizing conjugated linoleic acid by saccharomyces cerevisiae display linoleic acid isomerase, which comprises the following steps of: inoculating saccharomyces cerevisiae K601 containing recombinant plasmid into a yeast peptone glucose (YPG) culture medium, inducing and culturing, collecting thalli, and washing to obtain the saccharomyces cerevisiae display linoleic acid isomerase; adding the obtained saccharomyces cerevisiae display linoleic acid isomerase into a buffer solution containing linoleic acid, and performing oscillating reaction. The linoleic acid isomerase is displayed and expressed on the outer surface of the cell of the saccharomyces cerevisiae, and is directly contacted with the linoleic acid substrate, so that the conversion efficiency of the conjugated linoleic acid is improved and the production cost is reduced.

Propionibacterium acnes linoleate isomerase and its application. An amino acid sequence is as shown in SEQ ID NO.1. Through concerted catalysis of compound enzyme, conjugated linoleic acid can be directly prepared from plant oil containing high content of linoleic acid, thus overcoming the defect that conjugated linoleic acid must be produced by using expensive free linoleic acid as a substrate and saving costs greatly. Compound enzyme is prepared by the utilization of recombinant bacteria, thus raising enzyme activity and increasing enzyme output. There is no need to purchase commercial enzyme, and cost is saved effectively. The isomerization product conjugated linoleic acid contains single component and has a plurality of physiological activities. The scheme has good practicality. Good economic benefit can be produced. The propionibacterium acnes linoleate isomerase has a good industrial prospect.

The invention discloses dual purposes of linoleate isomerase in aspects of dehydrogenation and isomerism. A bifunctional enzyme PAI is used as delta-12 fattyaciddehydrogenase and linoleate isomerase or delta-12 fattyaciddehydrogenase; and the bifunctional enzyme PAI is protein a) or protein b), wherein the protein a) has amino acid sequences shown as a sequence 2 in a sequence table; and the protein b) has an amino acid residue sequence which is derived from the sequence 2 by substituting and / or losing and / or adding one or more amino acid residues, and has the activity of the delta-12 fattyaciddehydrogenase and linoleate isomerase or the delta-12 fattyaciddehydrogenase. The invention provides a basis and a method for further researching pai gene functions and performing related applicability research on the biosynthesis of t10c12-CLA (conjugated linoleic acid) by using the bifunctional enzyme PAI.

The invention discloses a biological extraction method of high-unsaturated-activity salicornia bigelovii seed oil, and belongs to the field of food biotechnology. The method comprises the following steps: firstly, shelling salicornia bigelovii seeds, drying, grinding, adding water for mixing, grinding until the particle size of pulp is 2-50 microns by a colloid grinder, compounding pectinase NCB-PE40 with cellulase AE80, and enzymatically hydrolyzing for breaking walls; secondly, adding 2.4 L of alcalase for enzymolysis, and centrifuging to obtain supernatant liquor to obtain salicornia bigelovii seed oil; finally, converting linoleic acid in the salicornia bigelovii seed oil into conjugated linoleic acid by linoleate isomerase to obtain the high-unsaturated-activity salicornia bigelovii seed oil. The method has the characteristics that the salicornia bigelovii seed oil is extracted by a bio-enzyme method, the process is simple, the conditions are mild, and the obtained salicornia bigelovii seed oil is high in unsaturated fatty acid content, high in activity, free of organic solvent residues, and environment-friendly.

The invention discloses a method for synchronously purifying and immobilizing linoleate isomerase. The method comprises the steps of: treating attapulgite by using hydrochloric acid, adjusting the pH value by using solution of sodium hydroxide, stirring uniformly, filtering, washing, and drying to obtain acid modified attapulgite; mixing the acid modified attapulgite and solution of magnesium sulfate, stirring, filtering, and drying to obtain attapulgite treated by the magnesium sulfate; mixing the attapulgite treated by the magnesium sulfate, gamma-(methacryloyloxy)propyltrimethoxysilane, water and dimethylbenzene and stirring, filtering, washing, and drying to obtain modified attapulgite; mixing enzyme fluid of linoleate isomerase produced from Lactobacillus delbrueckii subsp bulgaricus and the modified attapulgite, stirring, filtering, and drying to obtain the purified and immobilized linoleate isomerase. The method for synchronously purifying and immobilizing the linoleate isomerase is simple and convenient, simple in steps, low in cost, good in purification effect, and high in immobilization efficiency; the activity recovery rate of enzyme reaches over 60 percent during the purification; and the immobilized enzyme has better pH adaptability, thermal stability and operation stability.

The invention provides a method of preparing linoleate isomerase micro-capsules on the basis of a polyelectrolyte layer-by-layer self-assembly technology and can solve a problem that free linoleate isomerase is poor in tolerance, is poor in stability and cannot be recycled in industrial production. The method of the invention includes the following steps: with an amorphous calcium carbonate crystal, which is obtained through co-precipitation of calcium carbonate and the linoleate isomerase, as a template and with polyallylamine hydrochloride and sodium polystyrenesulfonate as coating materials, preparing the linoleate isomerase micro-capsules through the polyelectrolyte layer-by-layer self-assembly technology. The micro-capsule is high in activity, excellent in stability and long in half life.

The invention provides recombinant mortierella alpina oPAI for heterologous expression of linoleic acid isomerase gene from propionibacterium acnes and provides a method for constructing a recombinant mortierella alpina strain for heterologous expression of linoleic acid isomerase gene from propionibacterium acnes, and the invention further provides a use of the recombinant mortierella alpina strain for producing fatty acid, namely biological synthesizing conjugated linoleic acid. With mortierella alpina uracil auxotroph strain MAU1 as a material, a recombinant strain for heterologous expression of linoleic acid isomerase gene from propionibacterium acnes is successfully constructed, and a target product conjugated linoleic acid is detected in the recombinant strain added with long-chain acyl-coenzyme A for synthesizing an enzyme inhibitor.

The invention discloses food-grade recombined lactococcal lactis capable of producing conjugated linoleic acid as well as a construction method and an application thereof. The method for constructing the recombined lactococcal lactis comprises the step of introducing an encoding gene of a protein PAI (Plasminogen Activator Inhibitor) into lactococcal lactis serving as host bacteria to obtain the recombined lactococcal lactis, the conversion rate of the trans-10, cis-12 conjugated linoleic acid of which is higher than that of the host bacteria, wherein the protain PAI is a protein (a) composed of amino acid sequences shown in SEQ IN No. 2 or a protein (b) which is formed by replacing the amino acid sequences shown in SEQ IN No. 2 with one or several amino acid residues and / or deleting and / or adding the amino acid sequences shown in SEQ IN No. 2 and has the activity of linoleate isomerase, or is derived from the protein (1) and has the activity of linoleate isomerase. The food-grade recombined lactococcal lactis disclosed by the invention can produce t10c12-CLA, and the content of the t10c12-CLA accounts for 15.19-23.91% of the total quantity of thallus fatty acids.

The invention discloses a fermentation method of conjugated linoleic acid and a strain used in method, and relates to a method in which linoleate isomerase is expressed by using a saccharomyces cerevisiae surface display system, and capable of catalytic synthesis of t10, c12-conjugated linoleic acid by using the linoleate isomerase. An expression vector containing a propionibacterium acnes derived linoleate isomerase gene (pai) is constructed and a saccharomyces cerevisiae strain containing the expression vector is obtained. After the induction culture, the linoleate isomerase is displayed and expressed in saccharomyces cerevisiae, and the linoleate isomerase is added into a buffer solution containing substrate linoleic acid for catalytic reaction to generate the t10, c12-conjugated linoleic acid. According to the method provided by the invention, the linoleate isomerase is linked to cytoderm of the saccharomyces cerevisiae by the microorganism surface display system, so that extracellular substrates and the linoleate isomerase can be fully contacted, the catalysis efficiency is improved, a single isomer with physiological activity can be obtained, separation and purification steps are simplified and the production cost is reduced.

The invention discloses a method for obviously improving soluble expression quantity of linoleate isomerase in recombinant escherichia coli, and belongs to the field of genetic engineering. Accordingto the method, recombinant escherichia coli E.coli BL21(DE3)(pET-22b(+) / pai) is used as a starting strain; through co-expression of molecular chaperone proteins and assistance of the linoleate isomerase folding, soluble expression level of the linoleate isomerase is improved, so that enzyme activity of the linoleate isomerase is improved. Finally, a molecular chaperone system GroEL-GroES is preferably obtained, so that the enzyme activity of the linoleate isomerase is improved by 56 percent.

The invention discloses recombinant Escherichia coli capable of realizing soluble expression of linoleate isomerase and application of the recombinant Escherichia coli, and belongs to the technical field of bioengineering. The recombinant Escherichia coli is constructed by the following steps: inserting genes mal E of a coded maltose-binding protein MBP into the upstream of genes pai of linoleateisomerase PAI, and constructing the recombinant Escherichia coli containing an MBP-PAI fusion expression vector. The expression quantity of the PAI of the obtained recombinant Escherichia coli BL21 (DE3) (pET24a-Mpai) is far higher than that of other strains recorded in the prior art, and most of the recombinant proteins MBP-PAI exist in a soluble protein form. Therefore, the recombinant Escherichia coli disclosed by the invention is capable of realizing high-efficiency soluble expression of the MBP-PAI, and the problem of excessive inclusion bodies is solved.

The invention discloses genetically-engineered pichia pastoris capable of producing conjugated linoleic acid and application of genetically-engineered pichia pastoris, belonging to the technical fields of genetic engineering and microbial engineering. The genetically-engineered pichia pastoris can be used for producing conjugated linoleic acid; linoleate isomerase-containing cell disruption supernatant obtained by fermentation of the genetically-engineered pichia pastoris is added into a reaction system containing 0.9 g / L of free linoleic acid for a reaction; and only after the reaction is carried out for 3 h, the yield of conjugated linoleic acid in reaction liquid can reach 0.855 g / L, a conversion rate reaches 95%, all the conjugated linoleic acid in the reaction is cis9,trans11-CLA, and no other conjugated linoleic acid isomers exist.

The invention relates to a method for expressing a linoleic acid isomerase by using a Saccharomyces cerevisiae surface display system and synthesizing t10, c12-conjugated linoleic acid through the catalytic action of the enzyme. The invention performs codon optimization on a linoleic acid isomerase gene (pai) derived from Propionibacterium acnes to obtain a brand new linoleic acid isomerase gene (copai), construct an expression vector containing the copai and obtain a Saccharomyces cerevisiae strain containing the expression vector. According to the invention, a microbial surface display system is used to express the linoleic acid isomerase (coPAI) on the surface of a Saccharomyces cerevisiae cell, so that an extracellular substrate can be in full contact with the enzyme, thereby improving the catalytic efficiency; and the single isomer having physiological activity can be obtained, the separation and purification step can be omitted, and the production cost can be lowered.

The invention discloses lactobacillus reuteri HI120 of high-expression LAI (linoleic acid isomerase). The strain is collected in Guangdong Microbiological Culture Collection Center in November 21st of2016, and the collection number is GDMCC No: 60119. The lactobacillus reuteri HI120 is higher than lactobacillus reuteri standard strains and other probiotics in LAI expression level, and linoleic acid can be converted into conjugated linoleic acid in vivo and in vitro. The invention further discloses a microbiological viable bacterium preparation containing the lactobacillus reuteri HI120, and application of the microbiological viable bacterium preparation in preparation of food additives, food or drugs with the effect of preventing and / or adjunctively treating obese diabetics and colorectalcancer.

The invention discloses a Lactobacillus plantarum display linoleic acid isomerase, and a preparation method and application thereof. The method comprises the following steps: transforming a recombinant plasmid composed of Usp45 signal peptide gene, linoleic acid isomerase gene, M6 ankyrin gene and vector pMG36e into Lactobacillus plantarum CGMCC No.3782, inoculating in a culture medium containing erythromycin MRS (Methicillin-resistant Staphylococcus), culturing for 5-12 hours, centrifugalizing to collect Lactobacillus plantarum, flushing to obtain Lactobacillus plantarum display linoleic acid isomerase, adding the collected Lactobacillus plantarum display linoleic acid isomerase into a buffer solution containing linoleic acid, carrying out shake culture, and catalyzing to synthesize conjugated linoleic acid. In the invention, the Lactobacillus plantarum cell is subjected to gene engineering modification, so that the linoleic acid isomerase is expressed and secreted to the outside of the cell; and meanwhile, the M6 ankyrin cell is utilized to fix the linoleic acid isomerase to the cell surface, and the linoleic acid isomerase is utilized to carry out catalytic conversion to obtain the conjugated linoleic acid, thereby increasing the yield of the conjugated linoleic acid.

The invention provides a method for improving activity of lipase. The method comprises the following steps: connecting a lipase gene (Genebank number: AF229435) to the N terminal of a Saccharomyces cerevisiae cell wall component alpha lectin sequence (Genebank number: M28164); then inserting into the C terminal of a downstream MFalpha 1 signal peptide sequence of a Saccharomyces cerevisiae expression vector GAL1 promoter (Genebank number: AY428072) so as to construct an expression frame, wherein from the N end to the C end, the expression frame successively comprises: a GAL1 promoter, a MFalpha signal peptide sequence, a lipase gene and an alpha lectin sequence; and transforming yeast plasmid containing the expression frame into a Saccharomyces cerevisiae cell. The method has the advantages that linoleate isomerase is revealed and expressed on the outer surface of a Saccharomyces cerevisiae cell wall, and the complete contact of the linoleate isomerase and an extracellular substrate is achieved, thereby obviously improving the activity of the linoleate isomerase.

The invention discloses recombinant saccharomyces cerevisiae and application thereof in production of conjugated linoleic acid, belonging to the technical field of genetic engineering and microbial engineering. The recombinant saccharomyces cerevisiae can be used for producing conjugated linoleic acid, a linoleate isomerase-containing cell disruption supernatant obtained by fermentation of the recombinant saccharomyces cerevisiae is added into a reaction system containing 0.9 g / L of free linoleic acid for reaction, and the conversion rate of conjugated linoleic acid in a reaction solution can reach 8% only after the reaction is performed for 3 h; and moreover, all the conjugated linoleic acid in the reaction solution is cis9, trans11-CLA, and no other conjugated linoleic acid isomers exist.

The invention discloses a phage Phi X174 lytic cell protein E and application thereof and belongs to the field of genetic engineering. The method in the invention comprises the following steps: takingrecombinant Escherichia coli E. coli (PAI / GroELS) as an original strain, introducing a phage Phi X174 lytic cell protein E, producing holes in the surfaces of recombinant Escherichia coli cells, andreleasing linoleate isomerase from the cells by utilizing osmotic pressure differences inside and outside the cells. Finally, by optimizing the release conditions, 81% of linoleate isomerase in the cells is released out of the recombinant Escherichia coli cells.

Disclosed is linoleic acid isomerases and their application in production of conjugated linoleic acid, which belongs to the technical fields of protein engineering and microbial engineering. The linoleic acid isomerase derived from Bifidobacterium is used to produce the conjugated linoleic acid. The recombinant E. coli containing the linoleic acid isomerase of the invention is added into a reaction system containing linoleic acid and react for 3 h to produce conjugated linoleic acids. The conversion rate of the conjugated linoleic acid of the invented method ranges from 12.1% to 42.1%, and the percentage of cis9, trans11-CLA in the conjugated linoleic acid can reach 84.3% to 89.1%. The invention provides a method for using microorganisms to produce conjugated linoleic acids with high safety and yield where cis9, trans11-CLA isomer is the major form in the conjugated linoleic acid products.

A method for raising linoleate isomerase activity of the present invention comprises steps of: connecting a linoleate isomerase gene (Genbank No: DQ22732) to an N end of an alpha agglutinin sequence (Genbank No: M28164) of a saccharomyces cerevisiae cell wall component; inserting into a C end of a downstream MFalpha1 signal peptide sequence (Genbank No: M17301) of a saccharomyces cerevisiae expression vector GAL1 promoter (Genbank No: AY428072) to construct an expression frame of, from an N end to a C end, GAL1 promoter + MFalpha1 signal peptide sequence + linoleate isomerase gene + alpha agglutinin sequence; and conversing a yeast plasmid containing the expression frame to a saccharomyces cerevisiae cell. The invention has the advantage that the linoleate isomerase is expressed in exposure on an external surface of the saccharomyces cerevisiae cell wall to realize complete contact between the enzyme and an extracellular substrate, so as to substantially raise linoleate isomerase activity.

The invention discloses a method to produce conjugate linolic acid with linolic acid isomerase, which comprises the following steps: reacting linolic acid and linolic acid isomerase in hypercritical carbon dioxide for 1-3h; setting pressure of the reaction at 25-35 Mpa and temperature at 32-44 deg. c; getting conjugate linolic acid. This invention use hypercritical fluid, hypercritical carbon dioxide as media in enzyme catalytic reaction, thereby decreasing the cost and increasing the conversion rate of conjugate linolic acid.

The invention discloses a method for catalytically synthesizing conjugated linoleic acid by saccharomyces cerevisiae display linoleic acid isomerase, which comprises the following steps of: inoculating saccharomyces cerevisiae K601 containing recombinant plasmid into a yeast peptone glucose (YPG) culture medium, inducing and culturing, collecting thalli, and washing to obtain the saccharomyces cerevisiae display linoleic acid isomerase; adding the obtained saccharomyces cerevisiae display linoleic acid isomerase into a buffer solution containing linoleic acid, and performing oscillating reaction. The linoleic acid isomerase is displayed and expressed on the outer surface of the cell of the saccharomyces cerevisiae, and is directly contacted with the linoleic acid substrate, so that the conversion efficiency of the conjugated linoleic acid is improved and the production cost is reduced.