180 results about "Phospholipase D" patented technology

The invention relates to a method for preparing phosphatidylserine abundant in polyunsaturated fatty acid and belongs to the technical field of bioengineering. The method is characterized by comprising the following steps of: firstly, catalyzing ester exchange reaction between phosphatide and polyunsaturated fatty acid ester by utilizing one or a mixture of phosphatidase A and lipase to generate phosphatide abundant in polyunsaturated fatty acid; and then catalyzing phosphor-transfer esterification reaction between the phosphatide abundant in the polyunsaturated fatty acid and L-serine by utilizing phosphatidase D to generate the phosphatidylserine abundant in the polyunsaturated fatty acid. The method has the advantages of no discharge of waste water, good product quality, no solvent residue, safe process operation, few reaction byproducts, no waste generation, cost reduction, simple production process and easy realization of scale production because of utilizing two enzymes to perform sub-step catalysis and perform reaction in the same reactor, and completing the reaction process in a non-solvent system. Therefore, the invention provides a good and feasible method for preparing the phosphatidylserine abundant in the polyunsaturated fatty acid.

The invention relates to a method of preparing compounds, in particular to a method of preparing powder phosphatidylserine. The invention has to overcome the problems that the prior art inputs a lot of equipment and costs a lot, and the transformation ratio of the reaction is hard to be guaranteed. The invention has the technical proposal that a method of preparing powder phosphatidylserine takes natural lecithin as the reaction substrate; and after being separated by water, the reaction substrate can be added with an inorganic system prepared by L-serine, calcium salt and buffer solution to produce rough product under the effect of phospholipase D. Concentrate can be obtained by conducting the solvent extraction to the rough product, and then can be purified by solvent to obtain phosphatidylserine. The invention is characterized in that to separate the reaction substrate needs water three to ten times (w/v) of reaction substrate; and the separating temperature is ranged from 20 DEG C to 60 DEG C. In the system, the weight of the L-serine is two to ten times of the reaction substrate, and the weight of water used in the inorganic system is three to ten times(w/v) of reaction substrate. Ph value of the inorganic system can be adjusted between 4.5 and 6.0 by the buffer solution.

The invention provides an extraction process of phosphatidylcholine with antarctic krill as a source. The extraction process comprises the following steps: collecting heads of the antarctic krill, adding water for decoction, stirring, sending the krill head decoction liquid to a cold pressing machine for solid-liquid separation, to obtain an oily liquid rich in phospholipid and krill heads and other solids; adding absolute ethyl alcohol into the krill heads and other solids, carrying out high shearing extraction, carrying out centrifugal separation, and concentrating to obtain an oily liquid; merging the oily liquids, drying under a reduced pressure, then filtering through a plate frame filter to remove insoluble substances, followed by carrying out membrane separation and concentration, carrying out column chromatography separation and purification, carrying out freeze drying, and thus obtaining the phosphatidylcholine product. The invention also provides a preparation method for phosphatidylserine with the antarctic krill as the source. The preparation method comprises the steps of hydrolyzing the extracted phosphatidylcholine by phospholipase D, and extracting to obtain phosphatidylserine rich in DHA and EPA. The extraction process and the preparation method are environmentally friendly, and are low in cost; and the obtained products have high content and good activity, and can be industrially produced.

The invention relates to a method for preparing phosphatidylserine (2-DHA-PS) with docosahexaenoic acid at the sn-2 bit through high-activity phospholipase A2 and high-activity phospholipase D. Directed evolution is achieved through the overlapping PCR technology so that the high-activity phospholipase A2 and the high-activity phospholipase D can be obtained; the 2-DHA-PS is prepared through catalysis by means of the high-activity phospholipase A2 and the high-activity phospholipase D, phosphatidylserine is generated through phosphatidylcholine and serine under the catalysis of the high-activity phospholipase D first, and then the 2-DHA-PS is generated through the phosphatidylserine and the docosahexaenoic acid under the catalysis of the high-activity phospholipase A2. The relative content of the 2-DHA-PS in the product synthesized through the method is high, and the defects of an existing synthesizing method are effectively overcome.

The invention relates to a preparation method of phosphatidylserine. The preparation method comprises the steps of carrying out enzymic catalytic reaction on natural lecithin and L-serine in the presence of phospholipase D enzyme liquid, and carrying out a simple purification process after the reaction, so as to obtain phosphatidylserine with the yield exceeding 60%. The preparation method has the beneficial effects that the operation is simple and convenient, the yield is high, the cost is low, the product quality is stable, and the process is environmentally friendly.

The invention discloses a method for preparing phosphatidylserine under catalysis of immobilized phospholipase D. The method comprises the following steps: (1) with chitin as a vector and glutaraldehyde as a cross-linking reagent, immobilizing free phospholipase D to obtain immobilized enzyme of phospholipase D; (2) with the immobilized enzyme of the phospholipase D prepared in the step (1) as a catalyst, n-butyl acetate as an organic phase solvent, and 50mM of sodium acetate buffer solution of which the pH is 5.5 as a water phase solvent, reacting L-serine to react with soyabean lecithin at 20-60 DEG C according to the molar ratio of (10-100):1, thus obtaining phosphatidylserine; and at the end of reaction, filtering and recovering the phospholipase D for repeated use. By adopting the method disclosed by the invention, the ester transfer rate in the dual-phase catalytic system can be up to 78%, the immobilized enzyme can be repeatedly used, and the method is simple, easy to operate, and low in preparation cost.

The invention discloses a method for preparing phosphatidylserine. According to the method, phospholipid and L-serine are subjected to phospholipid translating acylation reaction under the action of phospholipase D to generate the phosphatidylserine, wherein the phospholipid translating acylation reaction is carried out in a homogeneous medium system and the yield of the phosphatidylserine reaches 80-98 percent. The method disclosed by the invention has the advantages that due to the adoption of a green reaction medium, a reaction system is a homogeneous medium system. No phosphatidic acid as a byproduct is generated, high catalytic efficiency of enzyme and favorable quality of products are obtained and the process is safe and environment-friendly.

A modified phospholipase D having an ability to synthesize phosphatidylinositol which contains: (A) an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO:2 by substitution of at least one of the amino acid residues at the 187-, 191- and 385-positions by other amino acid residue(s); or (B) an amino acid sequence of a polypeptide derived from the amino acid sequence as described in the above (A) by substitution, deletion, insertion and/or addition of at least one amino acid residue at a position different from the 187-, 191- and 385-positions and having an ability to synthesize phosphatidylinositol. By using this modified phospholipase D, phosphatidylinositol can be efficiently produced from a phospholipid. It is also intended to provide a method of screening a phospholipase D having an ability to synthesize an acylglycerophospholipid having glycol group.

The invention belongs to the technical field of plant molecular breeding and biology, and discloses application of a PLD (phospholipase D) epsilon gene in increasing nitrogen nutrition effective utilization and seed yield of crops. The nucleotide sequence of the PLD epsilon gene is shown as SEQ ID NO:1, or has a similarity more than or equal to 60 percent as the nucleotide sequence shown as SEQ ID NO:1. The invention also discloses a method for increasing nitrogen nutrition effective utilization and seed yield of rape. Tests prove that growth and occurrence of lateral roots of rape can be promoted due to overexpression of AtPLD epsilon either in nitrogen starvation or nitrogen sufficiency condition, increase of rape leaf tissue cell growth and biological yield can be reinforced, absorption transportation and assimilation metabolism processes of rape plants can be promoted, which are particularly indicated in that the expression quantities of genes NTR1.1 and NTR2.1 related to overexpressed plant N transportation are remarkably higher than that of a wild type, and the nitrogen absorption rate is remarkably higher than that of the wild type.

The invention relates to preparation of high-activity phospholipids enzyme D and cell surface display phospholipids enzyme D yeast whole cell catalysts, which belongs to a method for carrying out site directed mutagenesis wild phospholipids enzyme D by recombinant deoxyribonucleic acid (DNA) for improving the activity, connecting the mutated gene with yeast show carriers pPIC9K-Flo and efficiently displaying the mutated gene on the surface of pichia pastoris cells, and relates to a preparation method of the high-activity phospholipids enzyme D and cell surface display phospholipids enzyme D yeast whole cell catalysts. The method has the advantages that the activity of the wild phospholipids enzyme D is improved and is shown on the surfaces of the pastoris cells, the stability is improved,and the advantages of immobilized enzymes are realized. The method has the technical scheme that wild phospholipids enzyme D genes are separated from microbes, particularly streptomyces aureofuscus, the mutation is carried out on the amino acid residue of Glu69 and Ser285, through the efficient expression on the surfaces of the pichia pastoris cells, the enzyme activity of the high-activity phospholipids enzyme is improved by 11 percent than the wild type phospholipids enzyme D, and the enzyme activity of the recombination strains GS115/pPIC9K-Flo-pldm prepared through high-density fermentation is 120U/(g. stem cells).

The invention relates to a method for producing recombinant phospholipase D by genetic engineering bacteria fermentation, and particularly relates to a production method of recombinant phospholipase D. The production method is characterized by comprising the following steps of (1) building recombinant gene engineering bacteria; (2) preparing a shake flask seed culture medium and a fermentation medium; (3) activating the built recombinant genetic engineering bacteria for 16-20h on a plane plate, and inoculating into a shake flasks with the shake flask seed culture medium to obtain seed liquid; (4) inoculating into the fermentation medium for fermenting; adding an inducer until the final volume concentration in fermentation liquor is 1-5%, and continuing to ferment for 32-48h; and (5) obtaining the phospholipase D from culture of the fermentation liquor. Compared with the prior art, the production method disclosed by the invention has the following beneficial effects that carbon source dextrin is utilized as a carbon source at a slow speed; on one hand, the culture density of the cell is increased; and high-density fermentation is achieved; on the other hand, the expression speed of a PLD (programmable logic device) is reduced, and generation of an inclusion body is also reduced.

The invention relates to the technical field of serum phospholipid detection and particularly relates to a serum phospholipid detecting reagent. A reagent R1 comprises a buffer solution, phospholipase D, DAOS, ascorbic acid oxidase, bilirubin oxidase, polyethylene glycol 6000, cane sugar, xanthan gum, mannitol, trehalose, BSA, glycerol propoxylate-block-ethoxylate (Pluranic L64) and an aseptic. A reagent R2 comprises the buffer solution, 4-aminoantipyrine, peroxidase, choline oxidase, the polyethylene glycol 6000, the cane sugar, the xanthan gum, the mannitol, the trehalose, the BSA, the glycerol propoxylate-block-ethoxylate (Pluranic L64) and the aseptic. The HEPES buffer solutions and a novel Trinder reaction chromogen DAOS are adopted. A plurality of stabilizers are added to obviously improve stability of the detecting reagent. The bilirubin oxidase and the ascorbic acid oxidase are added, thus effectively avoiding interference caused by bilirubin and ascorbic acid and greatly improving interference-resisting capability of the detecting reagent. In addition, addition of the glycerol propoxylate-block-ethoxylate (Pluranic L64) which is an optional nonionic surfactant prevents reaction system turbidity, enhances substrate stability and improves the interference-resisting capability of the detecting reagent.

The present invention relates to a method of enhancing fruit ripening and stability and of delaying senescence in fruit and other plant tissues. This method consists of applying an effective amount of a lysophospholipid, such as lysophosphatidylethanolamine (18:1) (hereinafter referred to as LPE (18:1)) or lysophosphatidylinositol (hereinafter referred to as LPI) to the fruit and other plant tissues. Lysophospholipids such as LPE (18:1) and LPI were found to be superior to other lysophospholipids in delaying senescence and in inhibiting phospholipase D, a key enzyme in mediating membrane deterioration during of plant senescence. LPE (18:1) and LPI are naturally occurring and environmentally safe. Their use could replace many environmentally toxic compounds that are currently being used to retard senescence of flowers, fruits and leaves and to enhance fruit ripening.

A method for the preparation of the polyunsaturated fatty acids-containing phosphatidylserine, the method comprising: combining L-serine with a fish liver phosphatidylcholine having a polyunsaturated fatty acid to form a mixture; reacting the mixture with phospholipase D to effect transphosphatidylation of L-serine and the phosphatidylcholine having polyunsaturated fatty acids to produce the polyunsaturated fatty acids-containing phosphatidylserine.

The invention discloses a method for producing phospholipase D through recombinant escherichia coli. According to the method, a phospholipase D gene is in preciseness-type starting sub control, a pararea of pSC101 is inserted in an expression plasmid, an escherichia coli mutant strain recA is used for keeping the genetic stability of the plasmid, cells containing the plasmid at the growth periodare cultured to high density, and saturation induction is conducted at the induction period; meanwhile, the temperature is decreased, alkali metal salt stress is applied, the cytotoxicity of PLD to ahost is reduced, cell lysis is inhibited, the synthesis time of PLD is prolonged, and accordingly PLD expression is improved.