12 results about "Phospholipase D" patented technology

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.

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.

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 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.

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 present invention relates to novel compounds as phospholipase D (PLD) inhibitors and use thereof, more specifically to novel compounds that exhibit excellent phospholipase D inhibitory activity and can be utilized as therapeutic agents for cancer and degenerative neurological diseases.

The invention provides a phospholipase D mutant for efficiently synthesizing phosphatidylserine as well as a product and application of the phospholipase D. The mutant is a mutant phospholipase D obtained by mutating any one or more of the 87th site, the 136th site, the 189th site, the 379th site and the 458th site of an amino acid sequence on the basis of phospholipase D with the amino acid sequence as shown in SEQ ID NO.1. The amino acid mutation types of the mutation site comprise: L87E, M136G, I189R, E379L, and A458Y. On one hand, the invention provides the mutant phospholipase D, and on the other hand, the invention provides an application of the mutant phospholipase D. The mutant phospholipase D has heat-resistant stability, the catalytic capability is improved, and phosphatidylserine series products can be prepared through catalysis. The metabolic syndrome induced by antipsychotics can be effectively relieved.

The application relates to the field of biotechnology and discloses a single-point mutation method for improving the PS synthesis activity of phospholipase D. The activity center of ZET4 is determined through literature comparison, then PLD (2ZE4) is subjected to molecular docking with substrates PC and L-ser through AutoDock Vina, the binding sites of the substrate molecules and the enzyme molecules are determined, protein mutation design is carried out, primers are designed, and 14 mutant strains are constructed. The phospholipase D expressed by eight mutant strains improves the PS synthesis activity, the PS yield of T194A and T194L is increased from 27.46% to 53.52% and 49.23% respectively, the PS conversion rate of T194I, T194V, T194M, T194F, T194C and T194P is increased to different degrees, and the PS yield of T194I phospholipase D reaches 58.32%.