39 results about "Phospholipase D" patented technology

The invention belongs to the field of enzymatic preparation of structural phospholipids, and particularly relates to a method for preparing phosphatidylserine through interfacial enzyme catalysis. A series of high-activity immobilized phospholipase are prepared by innovatively adopting bifunctional group modified hollow mesoporous silica microspheres as a carrier and adsorbing immobilized phospholipase D. The catalyst has the functions of a catalyst and an emulsifier, and a Pickering emulsion interfacial enzyme catalysis system can be efficiently constructed. In the system, the phosphatidylcholine and the serine are efficiently converted into the phosphatidylserine through a phosphatidyl transfer reaction, the conversion rate of the phosphatidylserine within 20 minutes can reach 85%-95%, and the catalytic efficiency reaches up to 372 mmol / (g.h). In addition, after the catalyst is recycled for 10 times, the conversion rate is still kept at 70.9%, and the catalyst shows excellent stability and reusability. The method has the remarkable advantages of being mild in reaction condition, high in catalytic efficiency, capable of being repeatedly used and the like, and a new technical approach is provided for industrial production of phosphatidylserine.

The invention discloses a novel nucleic acid delivery system based on phosphatidylmitoxantrone as well as a preparation method and application of the novel nucleic acid delivery system based on phosphatidylmitoxantrone, and phosphatidylmitoxantrone (PMA) is used as a novel cationic lipid to construct a novel lipidosome nano nucleic acid delivery system by utilizing the enzymatic response release property and ionizable cationic phospholipid characteristics of phosphatidylmitoxantrone (PMA). PMA not only can be used as a novel cationic lipid entrapped nucleic acid, but also can be used as an auxiliary lipid component to promote nucleic acid delivery, so that the synergistic anti-tumor effect of nucleic acid and mitoxantrone is realized. The system can respond to high expression of phospholipase D (PLD) in tumor tissues, promote hydrolysis of PMA and release chemotherapeutic drug mitoxantrone, dual functions of nucleic acid delivery and drug release are achieved, the purpose of double-target inhibition of tumor growth is achieved, and a new strategy is provided for cancer treatment.

The invention discloses a phospholipase D mutant with improved thermal stability and a preparation method thereof. The amino acid sequence of the phospholipase D mutant is as shown in SEQ ID NO.4; according to the phospholipase D mutant A426P with improved thermal stability, the mutant is incubated for 30 min at 55 DEG C and 60 DEG C respectively, the residual enzyme activity is 85% and 58% respectively, the activity of wild-type enzyme is only 56% and 7% under the same treatment condition, the thermal stability of the mutant enzyme is remarkably improved, and the phospholipase D mutant A426P can be used as a phospholipase D mutant. The problem that phospholipase D is not ideal in thermal stability in biological catalytic reaction is solved.

The invention provides a lipoprotein sub-component phospholipid detection kit, and belongs to the field of in-vitro diagnostic reagents, and in particular, the lipoprotein sub-component phospholipid detection kit provided by the invention comprises a reaction reagent, the reaction reagent comprises a buffer solution, a surfactant, magnesium chloride, 4-aminoantipyrine, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3, 3, 4-trimethyl-1, 3, 5-trimethyl-1, 3, 5-trimethyl-1, 3, 5-trimethyl-1, 3, 5-trimethyl-1, 3, 5-trimethyl-1, 3, 5-trimethyl-1, 3, 5-trimethyl-1, 3, 5- The preservative is prepared from 2, 5-dimethoxyaniline sodium salt, phospholipase D, choline oxidase, peroxidase, ascorbic acid oxidase, a protective agent and a third preservative. The lipoprotein subfraction phospholipid detection kit provided by the invention can be used for determining the content of lipoprotein subfraction phospholipid by using a VAP system, simultaneously realizes simultaneous detection of a plurality of lipoprotein subfraction phospholipid items for the first time, and has the advantages of high precision and high accuracy.

The invention relates to a phospholipase D mutant Lip-Gri / NQ, a plasmid, a recombinant bacterium and application of the phospholipase D mutant Lip-Gri / NQ and belongs to the field of enzyme engineering, the amino acid sequence of the phospholipase D mutant is shown as SEQ ID NO: 1, and the nucleotide sequence of a gene for coding the phospholipase D mutant Lip-Gri / NQ is shown as SEQ ID NO: 2. The invention also provides a recombinant plasmid and an engineering bacterium containing the gene of the recombinant plasmid phospholipase D mutant Lip-Gri / NQ, and application of the phospholipase D mutant Lip-Gri / NQ in catalyzing phospholipids to generate specific-configuration functional lipids including phosphatidylserine. According to the phospholipase D Lip-Gri disclosed by the invention, wild phospholipase D Lip-Gri from Streptomyces griseofuscus is transformed through site-directed mutagenesis, and the enzyme activity of the obtained mutant is remarkably improved and is 11 times that of the wild phospholipase D Lip-Gri.

Engineered phospholipase D mutants are described herein. Also described herein are methods of making engineered phospholipase D mutants. Additionally, methods of using engineered phospholipase D mutants are described.

The present invention relates to the technical field of plasmalogens, and discloses a preparation method and application of an amide plasmalogen compound, which comprises the following steps: (1) reacting compound 1 and compound 2 in HEPES buffer to obtain compound 3; (2) reacting compound 3 with phospholipase D in an ethyl acetate phase and an aqueous phase with the assistance of Ca<supgt;2+< / supgt; ions to obtain compound 4; (3) expressing and purifying the CarF protein derived from myxobacteria; (4) reacting compound 4 and the CarF protein in Tris-HCl buffer with the assistance of NADH and catalase to obtain compound 5. By using the purified recombinant myxobacterial CarF protein and the substrate for the reaction of chemically synthesized CarF protein, the present invention finally synthesizes an amide plasmalogen, which can show an effect similar to that of natural plasmalogens.

To obtain a novel phospholipase D against choline-type plasmalogen.SOLUTION: Prepared is a phospholipase D comprising any of the described polypeptides: (1-1) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1; (1-2) a polypeptide having at least 85% identity with the amino acid sequence set forth in SEQ ID NO: 1, and having a hydrolytic activity to a phosphate ester bond in a choline-type plasmalogen molecule in which oleic acid is ester-bonded to the sn-2 position of a glycerol skeleton; and (1-3) a polypeptide in which one or several amino acids are added, substituted, deleted and / or inserted in the amino acid sequence set forth in SEQ ID NO: 1, and having a hydrolytic activity to a phosphate ester bond in a choline-type plasmalogen molecule.SELECTED DRAWING: Figure 3

The present invention belongs to the field of enzyme genetic engineering technology and specifically relates to a phospholipase D mutant, its preparation, and use. The phospholipase D gene from Streptomyces antibioticus was molecularly modified to mutate histidine at position 443 to asparagine and serine at position 199 to histidine, resulting in the phospholipase D mutant of the present invention, the amino acid sequence of which is shown in SEQ ID NO. 3. The catalytic activity of this mutant is significantly enhanced compared to that of wild-type phospholipase D. The phospholipase D mutant obtained by the present invention is suitable for catalyzing the synthesis of glycerophospholipids and also provides a reference for the synthesis of other functional phospholipids.

The invention belongs to the technical field of phosphatidylserine production, and particularly relates to immobilized phospholipase D based on biological imprinting and a preparation method and application thereof. The preparation method comprises the following steps: S1, dissolving L-serine in an acetic acid buffer solution to obtain a serine solution, adding a phospholipase D solution into the serine solution, and stirring to obtain a mixed solution; s2, adding sodium alginate into the mixed solution, stirring and dissolving to obtain a biopolymer solution; s3, adjusting the pH value of the chitosan solution to be neutral, then adding CaCl2 into the chitosan solution, and stirring and dissolving to obtain a CaCl2-containing chitosan solution; and S4, dropwise adding the biopolymer solution into a chitosan solution containing CaCl2, complexing to form microspheres, collecting the microspheres, and washing and drying the microspheres to obtain the immobilized phospholipase D based on biological imprinting. According to the method, the biological imprinting technology and the enzyme immobilization technology are combined, the immobilized PLD with high selectivity and high stability is prepared, and the phosphatidyl transfer reaction efficiency is improved.

This invention discloses a phospholipase D and its application in the preparation of ω-3 PUFAs-PA, belonging to the field of functional enzyme technology. The amino acid sequence of the phospholipase D is shown in SEQ ID NO. 9. The application of phospholipase D in the preparation of ω-3 PUFAs-PA is also disclosed. This invention also discloses a recombinant engineered bacterium expressing OUC-nPLD. The phospholipase D of this invention—OUC-nPLD—is a recombinant enzyme obtained by functional domain recombination based on the Non-HKD phospholipase D of *Streptomyces aquamarine*, and its PLD hydrolytic activity is 221.73 U / mg. This invention constructs a recombinant engineered bacterium expressing OUC-nPLD. The OUC-nPLD of this invention can be used for the efficient preparation of ω-3 PUFAs-PA, which is of great significance for the large-scale production of ω-3 PUFAs-PA.

The invention discloses a preparation method of phosphatidylserine, which comprises the following steps: (1) taking crude phospholipid as a raw material, adding an acetate organic solvent, stirring and dissolving, then adding a phospholipase D aqueous solution containing L-serine and calcium chloride, adjusting the temperature, mixing an organic phase and an aqueous phase, and reacting; (2) after the reaction is finished, separating out an organic phase, washing the organic phase with deionized water, separating out the organic phase, crystallizing the organic phase at low temperature, filtering to obtain filtrate, adding macroporous resin into the filtrate, stirring, adsorbing, filtering, and evaporating the filtrate to dryness the organic solvent to obtain a reactant crude product; and (3) adding lower alcohol into the reactant crude product, then adding deionized water, stirring and dissolving at a constant temperature, and then placing at a low temperature for heat preservation to separate out a phosphatidylserine product. According to the method, crude phospholipids are used for preparation through an enzyme method, conditions are mild, the process is simple, cost and energy consumption are low, industrial production is easy to achieve, the content of the prepared phosphatidylserine is 75% or above, and the purity is high.

The invention discloses a method for producing phosphatidylserine through enzyme catalysis, and belongs to the technical field of biology, and the method specifically comprises the following steps: in a reaction system of a [BMIM] [BF4] aqueous solution, taking natural phospholipid containing phosphatidylcholine and cephalin as a raw material, adding L-serine, calcium salt and phospholipase D, and carrying out a trans-acylation reaction. By introducing cephalin (4%-10%, preferably 6%-8%) and sphingomyelin (5%-10%) in a specific proportion and combining with a [BMIM] [BF4] ionic liquid system, high efficiency and high purity (the purity gt, 79.6% and the yield gt, 76.4%) of PS production are realized, and the problem that the yield and the purity cannot be considered at the same time in the traditional process is thoroughly solved.

The present invention aims to provide a method for producing a protein-containing food in which an unpleasant texture is improved, and the like. A method for producing a modified protein-containing food, including treating a food ingredient containing a protein with phospholipase D. An enzyme preparation for modifying a protein-containing food, which preparation contains phospholipase D. A pickling liquid for meat processing, which contains phospholipase D, for modifying a protein in a processed meat food. A method for modifying a protein-containing food, including treating a food ingredient containing a protein with phospholipase D.

This invention provides a mutant phospholipase D for the efficient synthesis of phosphatidylserine, its products, and applications. The mutant is based on phospholipase D with the amino acid sequence shown in SEQ ID NO.1, obtained by mutating one or more of five sites at positions 87, 136, 189, 379, and 458 of this amino acid sequence. The amino acid mutation types at these sites include: L87E, M136G, I189R, E379L, and A458Y. This invention provides both a mutant phospholipase D and its applications. This mutant phospholipase D exhibits improved thermostability and catalytic activity, enabling the catalytic preparation of phosphatidylserine series products. The phosphatidylserine series products prepared using this enzyme can effectively alleviate metabolic syndrome induced by antipsychotic drugs.

The application relates to a preparation method of immobilized phospholipase D and a preparation method of phosphatidylserine thereof, and belongs to the technical field of biotechnology.The preparation method of the immobilized phospholipase D comprises the following steps: (1) mixing mesoporous molecular sieve SBA-15 and a sodium acetate buffer solution to obtain a mixed solution; (2) mixing the mixed solution with an enzyme solution, oscillating, centrifuging, taking the precipitate, cleaning and freeze-drying to obtain the immobilized phospholipase D.The application improves the catalytic performance and stability of PLD by fixing PLD through the immobilization technology, realizes the reusability of PLD, and provides technical support for the industrialized production of the immobilized enzyme of PS.The preparation of phosphatidylserine by the immobilized PLD is optimized, and the optimal process condition of the preparation of PS by the immobilized PLD is obtained.The application further discloses a potential molecular mechanism of PS in treating AD, and provides a theoretical basis for the further clinical application of PS.

The invention relates to the field of synthetic phospholipids, and discloses a method for preparing phosphatidyl ethanolamine synthetic phospholipids under catalysis of phospholipase D. The method comprises the following steps: A, completely dissolving PC in an organic solvent to obtain a PC solution; b, adding ethanolamine into the buffer solution, and adjusting the pH value in an ice bath; c, mixing a metal salt solution with phospholipase D to obtain a fermentation enzyme solution, pouring a buffer solution containing ethanolamine, and uniformly mixing to obtain a water phase; d, mixing the PC solution with a water phase to uniformly disperse PC, and carrying out hydrolysis reaction to obtain a PE crude product reaction solution; e, standing for layering to obtain a water layer, extracting the water layer, and concentrating the obtained organic layer to obtain a PE crude product concentrate; f, refining to obtain a PE crude product I; g, performing column chromatography separation and vacuum concentration to obtain a PE crude product II with the purity not lower than 98%; f, drying to obtain a PE finished product. The method is simple in process, good in enzyme specificity, mild in reaction condition, environment-friendly, high in yield and beneficial to large-scale production of enterprises.

The invention discloses immobilized phospholipase D. The immobilized phospholipase D is prepared by the following method: phospholipase D-Sp-256-A409C is loaded on D301 alkalescent styrene anion resin, and the enzyme loading ratio is 10-120 mg / g. According to the application of the immobilized phospholipase D in preparation of DHA-PE, DHA-phosphatidylcholine and ethanolamine hydrochloride are taken as substrates, and transesterification reaction is carried out under the catalytic action of the immobilized phospholipase D to synthesize DHA-PE. According to the method, the D301 alkalescent styrene anion resin is used as a carrier to adsorb phospholipase D-Sp-256-A409C, the protein adsorption capacity is high, the transesterification activity is high, the enzyme activity recovery rate is high, and the method can be used for efficiently preparing DHA-PE. The research of the invention has important significance for industrial preparation of DHA-PE.

The present invention belongs to the field of biological enzymes and specifically relates to phospholipase D mutants and their applications. The phospholipase D mutants have an amino acid sequence containing at least one of the following mutations: T346V, K653L, S697Y, T346H, S697P, T346L, and K653A, in the wild-type amino acid sequence shown in SEQ ID NO. 1. The mutants described in the present invention significantly improve enzyme stability and catalytic activity, broaden the range of reaction conditions, and reduce the difficulty of process control. They also significantly enhance substrate affinity and catalytic efficiency, reduce enzyme production and application costs, and are suitable for large-scale industrial production.

Described herein are benzothiazole-phenyl-sulfonyl-piperidine compounds for activating N-acyl-phosphatidylethanolamine hydrolyzing phospholipase D (NAPE-PLD).

The invention discloses a preparation method of phosphatidylserine, which comprises the following steps: 1) hydrolyzing crude phospholipid by using broad-spectrum hydrolytic active phospholipase D to generate phosphatidic acid; the invention discloses a method for preparing phosphatidylserine by using crude phospholipid, which comprises the following steps of: 1) preparing phosphatidylserine, 2) inactivating broad-spectrum hydrolysis activity phospholipase D, and 3) adding high transesterification activity phospholipase B and L-serine for esterification.According to the method, the phosphatidylserine can be prepared by directly using the crude phospholipid, the process can be simplified, the production efficiency can be improved, and the production cost can be reduced.

The invention belongs to the technical field of enzyme co-immobilization, and particularly relates to a phospholipase D-choline oxidase-catalase composite microcapsule as well as a preparation method and application thereof. The method comprises the following steps: S1, adding sodium alginate, choline oxidase and catalase into a phospholipase D solution, and uniformly mixing to obtain a mixed solution; s2, the pH value of the chitosan solution is adjusted to be neutral, then CaCl2 is added into the chitosan solution to be stirred and dissolved, and a chitosan solution containing CaCl2 is obtained; s3, dropwise adding the mixed solution prepared in the step S1 into the chitosan solution containing CaCl2 prepared in the step S2, complexing to form microspheres, collecting the microspheres, and washing and drying the microspheres to obtain the phospholipase D-choline oxidase-catalase composite microcapsule. The invention provides a three-enzyme co-immobilization method for improving the synthesis capacity of phosphatidylglycerol, which not only solves the problem of inhibition of a byproduct choline in phospholipid conversion, but also realizes process upgrading through oxygen internal circulation and enzyme reutilization.

A method for diagnosing facioscapulohumeral muscular dystrophy (FSHD) using one or more biomarkers is provided. More particularly, provided is a method for diagnosing FSHD in a subject, the method comprising detecting the presence of a biomarker or change in level of a biomarker in a biological sample from the subject, wherein the biomarker is wherein the biomarker is mannose binding lectin 2 (MBL2), junction plakoglobin (JUP), desmoplakin (DSP), tetranectin (CLEC3B), complement component 7 (C7), complement component 9 (C9), tenascin C (TNG), lumican (LUM), phosphatidylinositol-glycan-specific phospholipase D (GPLD1 ), complement component 4 binding protein, beta chain (C4BPB), carnosine dipeptidase (CNDP1), or immunoglobulin kappa variable 2-30 (IGKV2-30), or a combination of any two or more of MBL2, JUP, DSP, CLEC3B, C7, C9, TNG, LUM, GPLD1, C4BPB, CNDP1, or IGKV2-30. In some aspects, when the change in level of the biomarker is an increase in the level of MBL2, JUP, DSP, CLEC3B, C7, C9, TNG, and / or LUM, or a combination of any thereof, the subject is diagnosed as having FSHD. In some aspects, when the change in level of the biomarker is a decrease in the level of GPLD1, C4BPB, CNDP1, and / or IGKV2-30, or a combination of any thereof, the subject is diagnosed as having FSHD. Also provided is a method for determining efficacy of a therapeutic treatment for FSHD in a subject, the method comprising measuring the level of a biomarker in a biological sample from the subject before and after the treatment, and determining that the treatment is effective in treating FSHD if the level of the biomarker is decreased or increased relative to a reference level or to the level of the biomarker in the sample from the subject before treatment, wherein when the biomarker is mannose binding lectin 2 (MBL2), junction plakoglobin (JUP), desmoplakin (DSP), tetranectin (CLEC3B), complement component 7 (C7), complement component 9 (C9), tenascin C (TNG), or lumican (LUM), or a combination of any two or more of MBL2, JUP, DSP, CLEC3B, C7, C9, TNG, or LUM, the treatment is effective when the level of the biomarker decreases, or wherein when the biomarker is phosphatidylinositol-glycan-specific phospholipase D (GPLD1), complement component 4 binding protein, beta chain (C4BPB), carnosine dipeptidase (CNDP1), or immunoglobulin kappa variable 2-30 (IGKV2-30), or a combination of any two or more of GPLD1, C4BPB, CNDP1, or IGKV2-30, the treatment is effective when the level of the biomarker increases. In various aspects, the method further comprises treating the subject diagnosed with FSHD.

The application discloses an enzyme catalysis production method of phosphatidylserine, and belongs to the technical field of biology, and specifically comprises the following steps: in a reaction system of [BMIM][BF4] aqueous solution, taking natural phosphatide containing phosphatidylcholine and cephalin as raw materials, adding L-serine, calcium salt and phospholipase D to carry out a transacylation reaction. By introducing specific proportions of cephalin (4%-10%, preferably 6%-8%) and sphingomyelin (5%-10%), in combination with the [BMIM][BF4] ionic liquid system, the application realizes efficient and high-purity (purity>79.6%, yield>76.4%) production of PS, and completely solves the problem that yield and purity cannot be considered simultaneously in the traditional process.

To provide a new method for improving the taste of food products. [Solution] To provide a food flavor enhancer containing phospholipase D and γ-glutamyl peptide or a salt thereof.

To provide a method for more efficiently producing a phospholipid represented by a specific formula.SOLUTION: A method for producing a phospholipid represented by formula (1) according to reaction formula I includes mixing, in a flow path, an organic solvent solution containing a compound represented by formula (A) with an aqueous solvent solution containing phospholipase D and a compound represented by formula (B), and then allowing the mixture to react at an enzymatic reaction temperature of the phospholipase D.SELECTED DRAWING: Figure 1

The present disclosure relates to a synthesis method of β-nicotinamide mononucleotide (β-NMN) and an intermediate thereof. In the present disclosure, phospholipid metabolism enzymes phospholipase D (PLD) and phospholipase C (PLC) widely present in the biosphere are used as catalysts to prepare β-NMN through two-step enzymolysis or one-pot synthesis; and an intermediate, namely phosphatidyl nicotinamide riboside (PNR), is obtained during the two-step enzymolysis. The present disclosure has simple reaction steps, low production cost, and environmental friendliness, and is suitable for large-scale industrial production.

The invention belongs to the field of plant genetic engineering, and particularly relates to a rice phospholipase D coding gene OsNRF14 and application thereof in high yield of crops. The invention discloses a rice phospholipase D coding gene OsNRF14, and the genome nucleotide sequence of the OsNRF14 is as shown in Seq ID No.1. The invention also discloses a preparation method of the rice phospholipase D coding gene OsNRF14. The invention also provides application of the gene OsNRF14. The gene OsNRF14 in rice is knocked out by using a gene editing technology CRISPR-Cas9, so that the rice yield and the rice protein content can be increased; on the contrary, the OsNRF14 gene is over-expressed in the rice, so that the rice yield is reduced, and the protein content of the rice is not influenced.

The invention belongs to the field of biological enzymes, and particularly relates to a phospholipase D mutant and application thereof, and the phospholipase D mutant is an amino acid sequence with at least one mutation of T346V, K653L, S697Y, T346H, S697P, T346L and K653A in a wild amino acid sequence as shown in SEQ ID NO.1. The phospholipase D mutant is a phospholipase D mutant with at least one mutation of T346V, K653L, S697Y, T346H, S697P, T346L and K653A. According to the mutant disclosed by the invention, the stability and catalytic activity of enzyme are remarkably improved, the reaction condition range is widened, and the process control difficulty is reduced. The affinity and catalytic efficiency to a substrate are remarkably improved, the production and application cost of the enzyme is reduced, and the method is suitable for large-scale industrial production.

The application relates to a phospholipase D mutant Lip-Gri / NQ, a plasmid, a recombinant bacterium and application thereof, belongs to the field of enzyme engineering, the amino acid sequence of the phospholipase D mutant is shown as SEQ ID NO: 1, the gene for encoding the phospholipase D mutant Lip-Gri / NQ is shown as SEQ ID NO. 2. The application also provides a recombinant plasmid containing the recombinant plasmid phospholipase D mutant Lip-Gri / NQ gene and an engineering bacterium, and application of the phospholipase D mutant Lip-Gri / NQ in catalyzing phospholipid to generate specific configuration functional lipids including phosphatidylserine. The application is obtained by site-directed mutagenesis on wild-type phospholipase D Lip-Gri, the enzyme activity of the mutant is significantly improved, and is 11 times that of the wild type. Streptomyces griseofuscus The enzyme activity of the mutant is significantly improved, and is 11 times that of the wild type.