WO2021134440A1 - Method for resisting viscosity during enzyme-catalyzed production of phosphatidylserine and method for producing phosphatidylserine using same - Google Patents

Method for resisting viscosity during enzyme-catalyzed production of phosphatidylserine and method for producing phosphatidylserine using same Download PDF

Info

Publication number
WO2021134440A1
WO2021134440A1 PCT/CN2019/130408 CN2019130408W WO2021134440A1 WO 2021134440 A1 WO2021134440 A1 WO 2021134440A1 CN 2019130408 W CN2019130408 W CN 2019130408W WO 2021134440 A1 WO2021134440 A1 WO 2021134440A1
Authority
WO
WIPO (PCT)
Prior art keywords
phosphatidylserine
ethanol
ethanol solution
catalyzed production
production
Prior art date
Application number
PCT/CN2019/130408
Other languages
French (fr)
Chinese (zh)
Inventor
张章
周志刚
张琦
Original Assignee
邦泰生物工程(深圳)有限公司
邦泰合盛生物科技（深圳）有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 邦泰生物工程(深圳)有限公司, 邦泰合盛生物科技（深圳）有限公司 filed Critical 邦泰生物工程(深圳)有限公司
Priority to CN201980042286.0A priority Critical patent/CN112384630B/en
Priority to PCT/CN2019/130408 priority patent/WO2021134440A1/en
Publication of WO2021134440A1 publication Critical patent/WO2021134440A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/09—Esters of phosphoric acids
    • C07F9/10—Phosphatides, e.g. lecithin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6481—Phosphoglycerides

Definitions

  • the invention relates to the technical field of biocatalysis, in particular to a method for anti-stickiness in phosphatidylserinase catalyzed production and a method for producing phosphatidylserine by using the same.
  • PS Phosphatidylserine
  • DHA blood-brain gold
  • the preparation methods of phosphatidylserine mainly include extraction method and biological enzyme catalysis method.
  • the extraction method is mainly extracted from the brains of plants (soybeans, sunflower seeds, etc.) and animals (cattle, sheep, horses).
  • the cost of this method is high, which is not conducive to Modern industrial production. Therefore, the current industry mainly uses biological enzyme catalysis to produce phosphatidylserine, which uses phospholipase D to catalyze the transphosphatidyl reaction between lecithin (from soybeans, sunflower seeds, eggs, etc.) and L-serine to generate phosphatidylserine. Because the source of the raw materials in this method is wide, and the content of the target substance in the raw materials is high, the production cost is greatly reduced compared with the extraction method, and it has been widely used in industrial production.
  • Chinese invention patent application CN103555783A discloses one A method for preparing phosphatidylserine, specifically: mixing phosphatidylcholine or natural phospholipids containing phosphatidylcholine with Tween-80, L-serine, calcium chloride, buffer solution, and phospholipase D, and then mixing phosphatidylcholine or phosphatidylcholine-containing natural phospholipids with Tween-80, L-serine, calcium chloride, buffer solution, and phospholipase D.
  • the reaction was stirred at 45°C for 8-10h; after the reaction, the crude reaction product precipitated and floated, the aqueous phase and the crude reaction product were separated by centrifugation, the separated crude reaction product was washed with water, and centrifuged filtered; the crude reaction product after centrifugal filtration was soaked in absolute ethanol, Stir, centrifuge and filter; vacuum dry the product after centrifugal filtration to obtain the phosphatidylserine product.
  • the method does not bring in other organic solvents except ethanol used in the purification process, reduces the toxicity of organic solvents, reduces the cost, and is environmentally friendly.
  • the surfactant Tween-80 is introduced in the process of biological enzyme catalyzed reaction, and the residual surfactant may cause irritation to the human body and even produce toxic side effects; on the other hand,
  • the raw materials used natural phospholipids often have relatively high viscosity, which causes the crude reaction products obtained by the enzyme-catalyzed reaction to severely stick and form agglomerates, and the subsequent purification treatment cannot be carried out smoothly;
  • the method controls the feeding amount of natural phospholipids below 20%, and at the same time uses a large amount of L-serine (the feeding amount is 2-3 times the mass of natural phospholipids, and the molar amount is over 14 times) to reduce Its stickiness.
  • the disadvantage of this approach is that a small amount of substrate feed will result in a low single-batch production capacity, and a high feed rate of L-serine will result in high residues in the product
  • the purpose of the present invention is to solve the problem of viscosity in the process of catalyzed production of phosphatidylserine by biological enzymes on the one hand, so as to provide a phosphatidylserinase catalyzed
  • the method of anti-stickiness in production on the other hand, it solves the technical problems of surfactant residue, high production cost and L-serine residue, so as to provide a solution that can not only solve the stickiness problem but also increase the single batch capacity and reduce production costs ,
  • the inventors conducted a large number of experimental studies and found that an ethanol solution with a specific pH value would improve the viscosity of the crude product obtained by the enzyme-catalyzed production of phosphatidylserine. Therefore, the inventor finally found a The simplest and most convenient method for anti-stickiness in the production of phosphatidylserine enzyme catalyzed production, including: adding a pH 7.5-9.5 ethanol solution to the crude reaction product obtained by enzyme-catalyzed production of phosphatidylserine, mixing uniformly, and then performing solid-liquid separation , Collect solids.
  • the above-mentioned anti-sticking method in phosphatidylserinase catalyzed production is suitable for processing any viscous natural phospholipids with unlimited content of phosphatidylcholine, especially for those with a higher viscosity of phosphatidylcholine with a content of 40%. %-65% of natural phospholipids have a significant anti-sticking effect.
  • the pH value of the ethanol solution is the key to solving the problem.
  • the inventors have found through experiments that the pH value below 7.5 cannot effectively improve the viscosity.
  • the purification treatment still cannot proceed smoothly, and the pH value higher than 9.5 will cause the target product in the crude reaction product to dissolve and reduce the yield.
  • the pH value of the ethanol solution is 8.0-9.0.
  • the concentration of ethanol in the ethanol solution is 85%-96%, which has the best impurity removal effect, and the final product obtained The highest content.
  • the volume of the ethanol solution is 4-6 times the weight of the crude product.
  • the ethanol solution can be a mixed solution of ethanol and any alkaline reagent capable of adjusting the pH of the solution.
  • the ethanol solution is an aqueous ethanol solution. Mixed solution with ammonia water.
  • the concentration of the ethanol aqueous solution is more than 93%
  • the concentration of the ammonia water is 25%
  • the volume ratio of the ethanol aqueous solution to the ammonia water is 96: 4 ⁇ 92: 8.
  • step (1) After the reaction of step (1) is completed, perform solid-liquid separation, collect the solids, and obtain the crude reaction product;
  • step (3) Add ethanol solution to the crude reaction product obtained in step (2), perform solid-liquid separation after uniform mixing, collect the solids, and dry to obtain the phosphatidylserine product, wherein the pH of the ethanol solution is 7.5-9.5.
  • the content of phosphatidylcholine in natural phospholipids is not limited, but from the perspective of market supply and feed cost, it is preferred to select a phosphatidylcholine content of 40%- 65% natural phospholipids.
  • the natural phospholipids are preferably soybean phospholipids and/or sunflower phospholipids, which are widely available in the market and are inexpensive.
  • the pH of the ethanol solution is 8.0-9.0.
  • the concentration of ethanol in the ethanol solution is 85%-96%.
  • step (3) of the method for producing phosphatidylserine catalyzed by the above-mentioned enzyme provided by the present invention the volume of the ethanol solution is 4-6 times the weight of the crude reaction product obtained in step (2).
  • the ethanol solution is a mixed solution of ethanol aqueous solution and ammonia water.
  • the concentration of the aqueous ethanol solution is 93% or more, the concentration of the ammonia water is 25%, and the volume ratio of the ethanol aqueous solution to the ammonia water is 96: 4 ⁇ 92: 8.
  • the method for producing phosphatidylserine catalyzed by the above-mentioned enzyme provided by the present invention further comprises the following step (4): cooling the filtrate obtained after the solid-liquid separation in step (2) to 4-10°C, and adding 1-2 times the volume to the filtrate.
  • the pre-cooled ethanol solution is mixed uniformly and then subjected to solid-liquid separation.
  • the solids are collected and dried to obtain recovered L-serine.
  • the pre-cooling temperature of the pre-cooled ethanol solution is 0-10°C, and in the present invention, the temperature of the fed ethanol solution is lowered in advance to 0-10°C helps to speed up the crystallization of L-serine.
  • the concentration of the pre-cooled ethanol solution is more than 90%.
  • step (4) of the method for producing phosphatidylserine catalyzed by the above-mentioned enzyme provided by the present invention L-serine is dried until the loss on drying is below 7.5%.
  • the present invention has the following advantages:
  • the anti-sticking method in the phosphatidylserinase catalyzed production provided by the present invention is extremely simple and easy to operate, but the effect achieved is extremely significant, and it can replace the technical means of reducing the viscosity by using a large amount of L-serine. And has no effect on the process of enzyme-catalyzed reaction;
  • the enzyme-catalyzed production method of phosphatidylserine provided by the present invention is simple and easy to operate, and has the following 5 advantages at the same time: 1) It solves the problem that the product cannot be purified smoothly due to the high viscosity of the product in the production process; 2 ) Increase the feed amount of natural phospholipids to 25%, thereby increasing the capacity of a single batch and reducing production costs; 3) Reduce the feed amount of L-serine to 1.3-1.5 times the feed amount of natural phospholipids, reducing the cost of feeding At the same time, it is also conducive to the full dispersion of phosphatidylcholine; 4) It reduces the residue of L-serine in the product, and at the same time increases the recycling process of L-serine, which further reduces the cost; 5) Avoids the use of surfactants.
  • Example 4 Collect the supernatant of the crude reaction product from Example 2 to Example 4 by centrifugation, cool to 7-8°C, stir and add 2 times the volume of 95% ethanol solution precooled to 4°C, flow for 1-2h After adding, continue to stir for 2h, centrifuge to collect the solid matter, and obtain the recovered L-serine after vacuum drying (drying loss 5.76%).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Molecular Biology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Provided is a method for resisting viscosity during enzyme-catalyzed production of phosphatidylserine, comprising: adding an ethanol solution with a pH value of 7.5-9.5 into a crude product obtained during enzyme-catalyzed production of phosphatidylserine, uniformly mixing, carrying out solid-liquid separation, and collecting the solid. The method can significantly reduce the viscosity of an enzyme reaction liquid. Also provided is a method for producing phosphatidylserine using the method for resisting viscosity, comprising: mixing natural phospholipids containing phosphatidylcholine with water, L-serine, calcium chloride, and phospholipase D, and stirring at 39-43℃ and reacting for 8-10 h; and collecting a solid after the reaction is finished, then adding an ethanol solution with a pH value of 7.5-9.5, uniformly mixing, collecting the solid, and drying to obtain the phosphatidylserine product. The method improves the single-batch production capacity, reduces the L-serine feeding amount, and reduces production costs.

Description

一种磷脂酰丝氨酸酶催化生产中抗黏性的方法以及用其生产磷脂酰丝氨酸的方法A method of phosphatidylserinase catalyzed production and anti-stickiness and a method for producing phosphatidylserine by using the same 技术领域Technical field
本发明涉及生物催化技术领域，特别涉及一种磷脂酰丝氨酸酶催化生产中抗黏性的方法以及用其生产磷脂酰丝氨酸的方法。The invention relates to the technical field of biocatalysis, in particular to a method for anti-stickiness in phosphatidylserinase catalyzed production and a method for producing phosphatidylserine by using the same.
背景技术Background technique
磷脂酰丝氨酸（Phosphatidylserine，英文简称PS）又名丝氨酸磷脂、二酰甘油酰磷酸丝氨酸、复合神经酸等，是一类普遍存在于细菌、酵母、植物和哺乳动物细胞中的一种重要的膜磷脂，通常位于细胞膜的内层，是细胞膜的活性物质，尤其存在于大脑细胞中。其功能主要是改善神经细胞功能，调节神经脉冲的传导，增进大脑记忆功能等。由于PS具有很强的亲脂性，吸收后能够迅速通过血脑屏障进入大脑，起到舒缓血管平滑肌细胞，增加脑部供血的作用，被誉为继胆碱和“脑黄金”DHA之后的一大新兴的“智能营养素”。Phosphatidylserine (PS), also known as serine phospholipid, diacylglyceryl phosphoserine, complex neuric acid, etc., is an important type of membrane phospholipid that is ubiquitous in bacteria, yeast, plants and mammalian cells , Usually located in the inner layer of the cell membrane, is the active substance of the cell membrane, especially in the brain cells. Its function is mainly to improve the function of nerve cells, regulate the conduction of nerve impulses, and enhance the memory function of the brain. Because PS has strong lipophilicity, it can quickly enter the brain through the blood-brain barrier after being absorbed, so as to soothe vascular smooth muscle cells and increase the blood supply to the brain. It is known as one of the major players after choline and "brain gold" DHA. Emerging "smart nutrients".
磷脂酰丝氨酸的制备方法主要包括提取法和生物酶催化法。提取法主要是从植物（大豆、葵花籽等）、动物（牛、羊、马）的大脑中提取，但由于原料中的PS含量较低且提取工艺复杂，导致该方法成本较高，不利于现代化工业生产。因此，目前工业上主要是采用生物酶催化法生产磷脂酰丝氨酸，其是利用磷脂酶D催化卵磷脂（来源于大豆、葵花籽、鸡蛋等）和L-丝氨酸发生转磷脂酰基反应生成磷脂酰丝氨酸，由于该方法中的原料来源广泛，且原料中的目标物质含量较高，所以其生产成本较提取法大大降低，已经被广泛应用于工业生产中。The preparation methods of phosphatidylserine mainly include extraction method and biological enzyme catalysis method. The extraction method is mainly extracted from the brains of plants (soybeans, sunflower seeds, etc.) and animals (cattle, sheep, horses). However, due to the low PS content in the raw materials and the complex extraction process, the cost of this method is high, which is not conducive to Modern industrial production. Therefore, the current industry mainly uses biological enzyme catalysis to produce phosphatidylserine, which uses phospholipase D to catalyze the transphosphatidyl reaction between lecithin (from soybeans, sunflower seeds, eggs, etc.) and L-serine to generate phosphatidylserine. Because the source of the raw materials in this method is wide, and the content of the target substance in the raw materials is high, the production cost is greatly reduced compared with the extraction method, and it has been widely used in industrial production.
在生物酶催化法生产磷脂酰丝氨酸的工艺开发过程中，为了避免有机溶剂的使用，人们开发了一种在单一水相体系中进行生物酶催化反应的方法，如中国发明专利申请CN103555783A公开了一种制备磷脂酰丝氨酸的方法，具体为：将磷脂酰胆碱或含有磷脂酰胆碱的天然磷脂与吐温-80、L-丝氨酸、氯化钙、缓冲溶液、磷脂酶D混合，在35-45℃搅拌反应8-10h ；反应结束后反应粗品析出并上浮，离心分离水相和反应粗品，将分离得到的反应粗品用水洗涤，并离心过滤；离心过滤后的反应粗品加入无水乙醇浸泡，搅拌，再离心过滤；对离心过滤后产物真空干燥，即得磷脂酰丝氨酸产品。该方法除纯化过程中使用乙醇外无其它有机溶剂带入，减少了有机溶剂的毒害，降低了成本且绿色环保。但是，该方法尚存在一定的问题：一方面，在生物酶催化反应过程中引入了表面活性剂吐温-80，而表面活性剂残留则可能会引起人体的刺激性反应甚至产生毒副作用；另一方面，在实际生产过程中，人们发现，所使用的原料天然磷脂往往具有比较大的黏性，从而导致酶催化反应得到的反应粗品严重粘结成块，而无法顺利进行后续的纯化处理；为解决这个问题，该方法将天然磷脂的投料量控制在20%以下，并同时使用了大量的L-丝氨酸（投料量为天然磷脂质量的2-3倍，摩尔量过量14倍以上）以降低其黏性。此种做法的弊端在于，底物投料量少则单批次产能低，L-丝氨酸投料量高则产品中残留高且增加生产成本。In the process of developing the process of producing phosphatidylserine by biological enzymatic catalysis, in order to avoid the use of organic solvents, people have developed a method for carrying out biological enzymatic reactions in a single aqueous phase system. For example, Chinese invention patent application CN103555783A discloses one A method for preparing phosphatidylserine, specifically: mixing phosphatidylcholine or natural phospholipids containing phosphatidylcholine with Tween-80, L-serine, calcium chloride, buffer solution, and phospholipase D, and then mixing phosphatidylcholine or phosphatidylcholine-containing natural phospholipids with Tween-80, L-serine, calcium chloride, buffer solution, and phospholipase D. The reaction was stirred at 45°C for 8-10h; after the reaction, the crude reaction product precipitated and floated, the aqueous phase and the crude reaction product were separated by centrifugation, the separated crude reaction product was washed with water, and centrifuged filtered; the crude reaction product after centrifugal filtration was soaked in absolute ethanol, Stir, centrifuge and filter; vacuum dry the product after centrifugal filtration to obtain the phosphatidylserine product. The method does not bring in other organic solvents except ethanol used in the purification process, reduces the toxicity of organic solvents, reduces the cost, and is environmentally friendly. However, this method still has certain problems: On the one hand, the surfactant Tween-80 is introduced in the process of biological enzyme catalyzed reaction, and the residual surfactant may cause irritation to the human body and even produce toxic side effects; on the other hand, On the one hand, in the actual production process, people found that the raw materials used natural phospholipids often have relatively high viscosity, which causes the crude reaction products obtained by the enzyme-catalyzed reaction to severely stick and form agglomerates, and the subsequent purification treatment cannot be carried out smoothly; In order to solve this problem, the method controls the feeding amount of natural phospholipids below 20%, and at the same time uses a large amount of L-serine (the feeding amount is 2-3 times the mass of natural phospholipids, and the molar amount is over 14 times) to reduce Its stickiness. The disadvantage of this approach is that a small amount of substrate feed will result in a low single-batch production capacity, and a high feed rate of L-serine will result in high residues in the product and increase production costs.
技术问题technical problem
鉴于上述背景技术中提到的现有技术存在的问题，本发明的目的在于，一方面解决生物酶催化生产磷脂酰丝氨酸的过程中所存在的黏性问题，从而提供一种磷脂酰丝氨酸酶催化生产中抗黏性的方法；另一方面解决表面活性剂残留、生产成本高以及L-丝氨酸残留的技术问题，从而提供一种既能解决黏性问题又能增加单批次产能、降低生产成本、减少L-丝氨酸残留的新的酶催化生产磷脂酰丝氨酸的方法。In view of the problems in the prior art mentioned in the above background art, the purpose of the present invention is to solve the problem of viscosity in the process of catalyzed production of phosphatidylserine by biological enzymes on the one hand, so as to provide a phosphatidylserinase catalyzed The method of anti-stickiness in production; on the other hand, it solves the technical problems of surfactant residue, high production cost and L-serine residue, so as to provide a solution that can not only solve the stickiness problem but also increase the single batch capacity and reduce production costs , A new method of producing phosphatidylserine catalyzed by a new enzyme that reduces the residue of L-serine.
技术解决方案Technical solutions
为实现上述目的，发明人进行了大量的实验研究，发现特定pH值的乙醇溶液会对酶催化生产磷脂酰丝氨酸得到的反应粗品的黏性起到改善作用，因此，发明人最终摸索出一种最为简单便捷的用于磷脂酰丝氨酸酶催化生产中抗黏性的方法，包括：向酶催化生产磷脂酰丝氨酸得到的反应粗品中加入pH7.5-9.5的乙醇溶液，混合均匀后进行固液分离，收集固体物。In order to achieve the above objectives, the inventors conducted a large number of experimental studies and found that an ethanol solution with a specific pH value would improve the viscosity of the crude product obtained by the enzyme-catalyzed production of phosphatidylserine. Therefore, the inventor finally found a The simplest and most convenient method for anti-stickiness in the production of phosphatidylserine enzyme catalyzed production, including: adding a pH 7.5-9.5 ethanol solution to the crude reaction product obtained by enzyme-catalyzed production of phosphatidylserine, mixing uniformly, and then performing solid-liquid separation , Collect solids.
本发明提供的上述磷脂酰丝氨酸酶催化生产中抗黏性的方法适用于处理磷脂酰胆碱不限含量的任何有黏性的天然磷脂，特别对黏性较高的磷脂酰胆碱含量在40%-65%的天然磷脂的抗黏性效果显著。The above-mentioned anti-sticking method in phosphatidylserinase catalyzed production provided by the present invention is suitable for processing any viscous natural phospholipids with unlimited content of phosphatidylcholine, especially for those with a higher viscosity of phosphatidylcholine with a content of 40%. %-65% of natural phospholipids have a significant anti-sticking effect.
本发明提供的上述磷脂酰丝氨酸酶催化生产中抗黏性的方法中，乙醇溶液的pH值是解决问题的关键所在，发明人实验发现，pH值低于7.5则不能有效改善黏性，后续的纯化处理仍然无法顺利进行，pH值高于9.5则会造成反应粗品中的目的产物发生溶解而降低收率。为了获得更好的处理效果，优选地，乙醇溶液的pH值为8.0-9.0。In the anti-sticking method in the phosphatidylserinase catalyzed production provided by the present invention, the pH value of the ethanol solution is the key to solving the problem. The inventors have found through experiments that the pH value below 7.5 cannot effectively improve the viscosity. The purification treatment still cannot proceed smoothly, and the pH value higher than 9.5 will cause the target product in the crude reaction product to dissolve and reduce the yield. In order to obtain a better treatment effect, preferably, the pH value of the ethanol solution is 8.0-9.0.
优选地，本发明提供的上述磷脂酰丝氨酸酶催化生产中抗黏性的方法中，乙醇溶液中乙醇的浓度为85%-96%，该浓度具有最好的除杂效果，得到的终产品的含量最高。Preferably, in the anti-sticking method in the phosphatidylserinase catalyzed production provided by the present invention, the concentration of ethanol in the ethanol solution is 85%-96%, which has the best impurity removal effect, and the final product obtained The highest content.
优选地，本发明提供的上述磷脂酰丝氨酸酶催化生产中抗黏性的方法中，乙醇溶液的体积用量为反应粗品重量的4-6倍。Preferably, in the anti-sticking method in the phosphatidylserinase catalyzed production provided by the present invention, the volume of the ethanol solution is 4-6 times the weight of the crude product.
乙醇溶液可以是乙醇与能够起到调节溶液pH值作用的任意碱性试剂的混合溶液，优选地，本发明提供的上述磷脂酰丝氨酸酶催化生产中抗黏性的方法中，乙醇溶液为乙醇水溶液与氨水的混合溶液。The ethanol solution can be a mixed solution of ethanol and any alkaline reagent capable of adjusting the pH of the solution. Preferably, in the anti-sticking method in the phosphatidylserinase catalyzed production provided by the present invention, the ethanol solution is an aqueous ethanol solution. Mixed solution with ammonia water.
更优选地，本发明提供的上述磷脂酰丝氨酸酶催化生产中抗黏性的方法中，乙醇水溶液的浓度为93%以上，氨水的浓度为25%，乙醇水溶液与氨水的体积比为96 : 4～92 : 8。More preferably, in the anti-sticking method in the phosphatidylserinase catalyzed production provided by the present invention, the concentration of the ethanol aqueous solution is more than 93%, the concentration of the ammonia water is 25%, and the volume ratio of the ethanol aqueous solution to the ammonia water is 96: 4 ~92: 8.
基于上述磷脂酰丝氨酸酶催化生产中抗黏性的方法，发明人进一步开发出了一种酶催化生产磷脂酰丝氨酸的方法，包括以下步骤：Based on the above-mentioned method of anti-stickiness in phosphatidylserinase-catalyzed production, the inventors have further developed a method for enzymatically catalyzed production of phosphatidylserine, which includes the following steps:
（1）将含有磷脂酰胆碱的天然磷脂与水、L-丝氨酸、氯化钙、磷脂酶D混合，在39-43℃下搅拌反应8-10h；其中，天然磷脂的加入量为200-250g/L水的体积，L-丝氨酸的加入量为250-320g/L水的体积，氯化钙的加入量为10-20g/L水的体积，磷脂酶D的加入量为25-40U/g天然磷脂；(1) Mix natural phospholipids containing phosphatidylcholine with water, L-serine, calcium chloride, and phospholipase D, and stir for 8-10 hours at 39-43°C; wherein the amount of natural phospholipids added is 200- The volume of 250g/L water, the addition amount of L-serine is 250-320g/L water volume, the addition amount of calcium chloride is 10-20g/L water volume, the addition amount of phospholipase D is 25-40U/L g natural phospholipids;
（2）待步骤（1）的反应结束后，进行固液分离，收集固体物，得反应粗品；(2) After the reaction of step (1) is completed, perform solid-liquid separation, collect the solids, and obtain the crude reaction product;
（3）向步骤（2）得到的反应粗品中加入乙醇溶液，混合均匀后进行固液分离，收集固体物，干燥后即得磷脂酰丝氨酸产品，其中乙醇溶液的pH值为7.5-9.5。(3) Add ethanol solution to the crude reaction product obtained in step (2), perform solid-liquid separation after uniform mixing, collect the solids, and dry to obtain the phosphatidylserine product, wherein the pH of the ethanol solution is 7.5-9.5.
本发明提供的上述酶催化生产磷脂酰丝氨酸的方法中，天然磷脂中磷脂酰胆碱的含量不限，但从市场供应量以及投料成本的层面考虑，优先选用磷脂酰胆碱含量在40%-65%的天然磷脂。In the method for producing phosphatidylserine catalyzed by the above-mentioned enzyme provided by the present invention, the content of phosphatidylcholine in natural phospholipids is not limited, but from the perspective of market supply and feed cost, it is preferred to select a phosphatidylcholine content of 40%- 65% natural phospholipids.
本发明提供的上述酶催化生产磷脂酰丝氨酸的方法中，天然磷脂优选采用市场来源广、价格低廉的大豆磷脂和/或葵花磷脂。In the method for producing phosphatidylserine catalyzed by the above-mentioned enzyme provided by the present invention, the natural phospholipids are preferably soybean phospholipids and/or sunflower phospholipids, which are widely available in the market and are inexpensive.
优选地，本发明提供的上述酶催化生产磷脂酰丝氨酸的方法的步骤（3）中，乙醇溶液的pH值为8.0-9.0。Preferably, in step (3) of the method for producing phosphatidylserine catalyzed by the above-mentioned enzyme provided by the present invention, the pH of the ethanol solution is 8.0-9.0.
优选地，本发明提供的上述酶催化生产磷脂酰丝氨酸的方法的步骤（3）中，乙醇溶液中乙醇的浓度为85%-96%。Preferably, in step (3) of the method for producing phosphatidylserine catalyzed by the above-mentioned enzyme provided by the present invention, the concentration of ethanol in the ethanol solution is 85%-96%.
优选地，本发明提供的上述酶催化生产磷脂酰丝氨酸的方法的步骤（3）中，乙醇溶液的体积用量为步骤（2）得到的反应粗品重量的4-6倍。Preferably, in step (3) of the method for producing phosphatidylserine catalyzed by the above-mentioned enzyme provided by the present invention, the volume of the ethanol solution is 4-6 times the weight of the crude reaction product obtained in step (2).
优选地，本发明提供的上述酶催化生产磷脂酰丝氨酸的方法的步骤（3）中，乙醇溶液为乙醇水溶液与氨水的混合溶液。Preferably, in step (3) of the method for producing phosphatidylserine catalyzed by the above-mentioned enzyme provided by the present invention, the ethanol solution is a mixed solution of ethanol aqueous solution and ammonia water.
更优选地，本发明提供的上述酶催化生产磷脂酰丝氨酸的方法的步骤（3）中，乙醇水溶液的浓度为93%以上，氨水的浓度为25%，乙醇水溶液与氨水的体积比为96 : 4～92 : 8。More preferably, in step (3) of the method for producing phosphatidylserine catalyzed by the enzyme provided by the present invention, the concentration of the aqueous ethanol solution is 93% or more, the concentration of the ammonia water is 25%, and the volume ratio of the ethanol aqueous solution to the ammonia water is 96: 4～92: 8.
优选地，本发明提供的上述酶催化生产磷脂酰丝氨酸的方法还包括如下步骤（4）：将步骤（2）固液分离后得到的滤液降温至4-10℃，流加1-2倍体积的预冷乙醇溶液，混合均匀后进行固液分离，收集固体物，干燥后即得回收后的L-丝氨酸。Preferably, the method for producing phosphatidylserine catalyzed by the above-mentioned enzyme provided by the present invention further comprises the following step (4): cooling the filtrate obtained after the solid-liquid separation in step (2) to 4-10°C, and adding 1-2 times the volume to the filtrate. The pre-cooled ethanol solution is mixed uniformly and then subjected to solid-liquid separation. The solids are collected and dried to obtain recovered L-serine.
更优选地，本发明提供的上述酶催化生产磷脂酰丝氨酸的方法的步骤（4）中，预冷乙醇溶液的预冷温度为0-10℃，本发明中将流加的乙醇溶液提前降温至0-10℃有助于加快L-丝氨酸结晶的速度。More preferably, in step (4) of the method for producing phosphatidylserine catalyzed by the above enzyme provided by the present invention, the pre-cooling temperature of the pre-cooled ethanol solution is 0-10°C, and in the present invention, the temperature of the fed ethanol solution is lowered in advance to 0-10°C helps to speed up the crystallization of L-serine.
更优选地，本发明提供的上述酶催化生产磷脂酰丝氨酸的方法的步骤（4）中，预冷乙醇溶液的浓度为90%以上。More preferably, in step (4) of the method for producing phosphatidylserine catalyzed by the above-mentioned enzyme provided by the present invention, the concentration of the pre-cooled ethanol solution is more than 90%.
更优选地，本发明提供的上述酶催化生产磷脂酰丝氨酸的方法的步骤（4）中，L-丝氨酸干燥至干燥失重在7.5%以下。More preferably, in step (4) of the method for producing phosphatidylserine catalyzed by the above-mentioned enzyme provided by the present invention, L-serine is dried until the loss on drying is below 7.5%.
有益效果Beneficial effect
与现有技术相比，本发明具有以下优点：Compared with the prior art, the present invention has the following advantages:
1、本发明提供的磷脂酰丝氨酸酶催化生产中抗黏性的方法极其简单易操作，但是所取得的效果却是极其显著的，可以取代通过大量使用L-丝氨酸来降低黏性的技术手段，且对酶催化反应过程无影响；1. The anti-sticking method in the phosphatidylserinase catalyzed production provided by the present invention is extremely simple and easy to operate, but the effect achieved is extremely significant, and it can replace the technical means of reducing the viscosity by using a large amount of L-serine. And has no effect on the process of enzyme-catalyzed reaction;
2、本发明提供的酶催化生产磷脂酰丝氨酸的方法工艺简单易操作，并且同时具备以下5方面优点，1）解决了生产过程中存在的因产品黏性太高而无法顺利纯化的问题；2）使原料天然磷脂的投料量提高至25%，从而提高单批次产能，降低生产成本；3）使L-丝氨酸的投料量降低至天然磷脂投料量的1.3-1.5倍，减少了投料成本的同时也有利于磷脂酰胆碱充分分散；4）减少了产品中L-丝氨酸的残留，同时增加了对L-丝氨酸的回收利用工艺，进一步降低了成本；5）避免了表面活性剂的使用。2. The enzyme-catalyzed production method of phosphatidylserine provided by the present invention is simple and easy to operate, and has the following 5 advantages at the same time: 1) It solves the problem that the product cannot be purified smoothly due to the high viscosity of the product in the production process; 2 ) Increase the feed amount of natural phospholipids to 25%, thereby increasing the capacity of a single batch and reducing production costs; 3) Reduce the feed amount of L-serine to 1.3-1.5 times the feed amount of natural phospholipids, reducing the cost of feeding At the same time, it is also conducive to the full dispersion of phosphatidylcholine; 4) It reduces the residue of L-serine in the product, and at the same time increases the recycling process of L-serine, which further reduces the cost; 5) Avoids the use of surfactants.
本发明的实施方式Embodiments of the present invention
下面结合具体实施例对本发明做进一步的详细说明，以下实施例是对本发明的解释，本发明并不局限于以下实施例。The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are an explanation of the present invention, and the present invention is not limited to the following embodiments.
实施例1Example 1
抗黏性对比实验Anti-sticking comparison test
在1L纯水中加入250g磷脂酰胆碱含量为62.2%的大豆磷脂，300g L-丝氨酸，10g氯化钙，500rpm搅拌均匀且升温至39-43℃，加入2mL 磷脂酶D（40U/g大豆磷脂），反应9h，检测转化率。离心收集反应粗品，分为5组，分别加入不同浓度的含氨乙醇，搅拌0.5h，观察物料的黏性状态，结果见下表。Add 250g of soy phospholipid with 62.2% phosphatidylcholine content in 1L of pure water, 300g of L-serine, 10g of calcium chloride, stir evenly at 500rpm and raise the temperature to 39-43℃, add 2mL of phospholipase D (40U/g soybean Phospholipids), react for 9h, and check the conversion rate. The crude reaction product was collected by centrifugation, divided into 5 groups, added different concentrations of ammonia-containing ethanol, stirred for 0.5h, and observed the viscosity state of the material. The results are shown in the following table.
pHpH 95%乙醇：25%氨水 (V/V)95% ethanol: 25% ammonia (V/V) 乙醇终浓度Final ethanol concentration 物料黏性状态Material viscosity state
无水乙醇Absolute ethanol \\ 100%100% 黏结成球，无法搅拌开Sticks into a ball and cannot be stirred
7.57.5 97:397:3 92%92% 搅拌下分散成均匀小块，少量黏附于搅拌桨及器壁Disperse into uniform small pieces under stirring, and a small amount adheres to the stirring blade and the wall of the vessel
8.08.0 96:496:4 91%91% 搅拌下分散成均匀小块，搅拌桨及器壁无黏附Disperse into uniform small pieces under stirring, without sticking to the stirring paddle and the wall
9.09.0 94:694:6 89%89% 搅拌下分散成均匀颗粒，搅拌桨及器壁无黏附Disperse into uniform particles under stirring, without sticking to the stirring paddle and the wall
9.59.5 92:892:8 87%87% 搅拌下分散成均匀颗粒，部分溶解，搅拌桨及器壁无黏附，底层有棕色膏状物Disperse into uniform particles under stirring, and partially dissolve. There is no adhesion between the stirring paddle and the wall, and there is brown paste on the bottom layer.
实施例2Example 2
酶催化生产磷脂酰丝氨酸Enzyme-catalyzed production of phosphatidylserine
在1L纯水中加入250g磷脂酰胆碱含量为53.6%的大豆磷脂，320g L-丝氨酸，20g氯化钙，500rpm搅拌均匀且升温至39-43℃，加入2mL 磷脂酶D（40U/g大豆磷脂），反应8h，检测转化率。离心收集反应粗品，称重，加入5倍体积的pH8.0的乙醇溶液（浓度95%的乙醇与浓度25%的氨水按体积比96:4配制而成），搅拌3h，离心收集固体物，真空干燥得到206.6g磷脂酰丝氨酸产品。Add 250g of soybean phospholipid with a phosphatidylcholine content of 53.6%, 320g of L-serine, 20g of calcium chloride in 1L of pure water, stir evenly at 500rpm and raise the temperature to 39-43℃, add 2mL of phospholipase D (40U/g soybean Phospholipids), react for 8h, and check the conversion rate. The crude reaction product was collected by centrifugation, weighed, and 5 times the volume of ethanol solution with pH 8.0 (concentration of 95% ethanol and 25% ammonia water was prepared in a volume ratio of 96:4), stirred for 3 hours, and centrifuged to collect the solids. Vacuum drying obtains 206.6 g of phosphatidylserine product.
实施例3Example 3
酶催化生产磷脂酰丝氨酸Enzyme-catalyzed production of phosphatidylserine
在1L纯水中加入200g磷脂酰胆碱含量为58.6%的大豆磷脂，274g 回收L-丝氨酸（干燥失重4.98%），20g氯化钙，500rpm搅拌均匀且升温至39-43℃，加入1mL 磷脂酶D（25U/g大豆磷脂），反应10h，检测转化率。离心收集反应粗品，称重，加入4倍体积的pH9.0的乙醇溶液（浓度93%的乙醇与浓度25%的氨水按体积比94:6配制而成），搅拌3h，离心收集固体物，真空干燥得到160.5g 磷脂酰丝氨酸产品。Add 200g of soybean phospholipid with 58.6% phosphatidylcholine content to 1L of pure water, 274g of recovered L-serine (dry weight loss 4.98%), 20g of calcium chloride, stir evenly at 500rpm and raise the temperature to 39-43℃, add 1mL of phospholipid Enzyme D (25U/g soybean phospholipid), react for 10 hours, and detect the conversion rate. The crude reaction product was collected by centrifugation, weighed, 4 times volume of ethanol solution of pH 9.0 (concentration of 93% ethanol and 25% ammonia water was prepared in a volume ratio of 94:6), stirred for 3 hours, and centrifuged to collect the solids. Vacuum drying obtains 160.5 g of phosphatidylserine product.
实施例4Example 4
酶催化生产磷脂酰丝氨酸Enzyme-catalyzed production of phosphatidylserine
在1L纯水中加入200g磷脂酰胆碱含量为43.1%的葵花磷脂，280g 回收L-丝氨酸（干燥失重6.99%），20g氯化钙，500rpm搅拌均匀且升温至39-43℃，加入1mL 磷脂酶D（25U/g葵花磷脂），反应8h，检测转化率。离心收集反应粗品，称重，加入6倍体积的pH9.5的乙醇溶液（浓度97%的乙醇与浓度25%的氨水按体积比92:8配制而成），搅拌3h，离心收集固体物，真空干燥得到146.7g PS磷脂酰丝氨酸产品。Add 200g of sunflower phospholipid with a phosphatidylcholine content of 43.1% in 1L of pure water, 280g of recovered L-serine (with a loss on drying of 6.99%), 20g of calcium chloride, stir evenly at 500rpm and raise the temperature to 39-43℃, add 1mL of phospholipid Enzyme D (25U/g sunflower phospholipid), react for 8h, and detect the conversion rate. The crude reaction product was collected by centrifugation, weighed, and 6 times the volume of the ethanol solution of pH 9.5 (concentration of 97% ethanol and 25% ammonia water was prepared in a volume ratio of 92:8), stirred for 3 hours, and centrifuged to collect the solids. After vacuum drying, 146.7 g of PS phosphatidylserine product was obtained.
实施例5Example 5
L-丝氨酸的回收Recovery of L-serine
收集实施例2至实施例4离心收集反应粗品后的上清液，降温至7-8℃，搅拌下流加2倍体积的浓度为95%的预冷至4℃的乙醇溶液，1-2h流加完，继续搅拌2h，离心收集固体物，真空干燥后得到回收后的L-丝氨酸（干燥失重5.76%）。Collect the supernatant of the crude reaction product from Example 2 to Example 4 by centrifugation, cool to 7-8°C, stir and add 2 times the volume of 95% ethanol solution precooled to 4°C, flow for 1-2h After adding, continue to stir for 2h, centrifuge to collect the solid matter, and obtain the recovered L-serine after vacuum drying (drying loss 5.76%).

Claims (15)

  1. 一种磷脂酰丝氨酸酶催化生产中抗黏性的方法，其特征在于所述方法包括：向酶催化生产磷脂酰丝氨酸得到的反应粗品中加入乙醇溶液，混合均匀后进行固液分离，收集固体物，所述乙醇溶液的pH值为7.5-9.5。A method for anti-stickiness in phosphatidylserine enzyme catalyzed production, characterized in that the method comprises: adding an ethanol solution to the crude reaction product obtained by enzymatically catalyzing the production of phosphatidylserine, and then performing solid-liquid separation after uniform mixing, and collecting solids , The pH of the ethanol solution is 7.5-9.5.
  2. 根据权利要求1所述的磷脂酰丝氨酸酶催化生产中抗黏性的方法，其特征在于：所述乙醇溶液的pH值为8.0-9.0。The method for phosphatidylserinase-catalyzed production of anti-sticking properties according to claim 1, wherein the pH of the ethanol solution is 8.0-9.0.
  3. 根据权利要求1或2所述的磷脂酰丝氨 酸酶催化生产中抗黏性的方法，其特征在于：所述乙醇溶液中乙醇的浓度为85%-96%。The anti-sticking method in phosphatidylserinase catalyzed production according to claim 1 or 2, characterized in that the concentration of ethanol in the ethanol solution is 85%-96%.
  4. 根据权利要求1或2所述的磷脂酰丝氨酸酶催化生产中抗黏性的方法，其特征在于：所述乙醇溶液的体积用量为所述反应粗品重量的4-6倍。The anti-sticking method in phosphatidylserinase catalyzed production according to claim 1 or 2, characterized in that the volume of the ethanol solution is 4-6 times the weight of the crude reaction product.
  5. 根据权利要求1或2所述的磷脂酰丝氨酸酶催化生产中抗黏性的方法，其特征在于：所述乙醇溶液为乙醇水溶液与氨水的混合溶液。The anti-sticking method in phosphatidylserinase catalyzed production according to claim 1 or 2, wherein the ethanol solution is a mixed solution of ethanol aqueous solution and ammonia water.
  6. 根据权利要求5所述的磷脂酰丝氨酸酶催化生产中抗黏性的方法，其特征在于：所述乙醇水溶液的浓度为93%以上，所述氨水的浓度为25%，所述乙醇水溶液与所述氨水的体积比为96 : 4～92 : 8。The anti-sticking method in the phosphatidylserinase catalyzed production according to claim 5, characterized in that: the concentration of the ethanol aqueous solution is 93% or more, the concentration of the ammonia water is 25%, and the ethanol aqueous solution The volume ratio of the ammonia water is 96:4～92:8.
  7. 一种酶催化生产磷脂酰丝氨酸的方法，其特征在于所述方法包括以下步骤：An enzyme-catalyzed method for producing phosphatidylserine, characterized in that the method comprises the following steps:
    （1）将含有磷脂酰胆碱的天然磷脂与水、L-丝氨酸、氯化钙、磷脂酶D混合，在39-43℃下搅拌反应8-10h；所述天然磷脂的加入量为200-250g/L水的体积，所述L-丝氨酸的加入量为250-320g/L水的体积，所述氯化钙的加入量为10-20g/L水的体积，所述磷脂酶D的加入量为25-40U/g天然磷脂；(1) Mix natural phospholipids containing phosphatidylcholine with water, L-serine, calcium chloride, and phospholipase D, and stir for 8-10 hours at 39-43°C; the addition amount of natural phospholipids is 200- The volume of 250g/L water, the addition of L-serine is 250-320g/L the volume of water, the addition of calcium chloride is 10-20g/L the volume of water, the addition of phospholipase D The amount is 25-40U/g natural phospholipids;
    （2）待步骤（1）的反应结束后，进行固液分离，收集固体物，得反应粗品；(2) After the reaction of step (1) is completed, perform solid-liquid separation, collect the solids, and obtain the crude reaction product;
    （3）向步骤（2）得到的反应粗品中加入乙醇溶液，混合均匀后进行固液分离，收集固体物，干燥后即得磷脂酰丝氨酸产品，所述乙醇溶液的pH值为7.5-9.5。(3) Add an ethanol solution to the crude reaction product obtained in step (2), perform solid-liquid separation after uniform mixing, collect the solids, and dry the phosphatidylserine product to obtain the phosphatidylserine product, and the ethanol solution has a pH value of 7.5-9.5.
  8. 根据权利要求7所述的酶催化生产磷脂酰丝氨酸的方法，其特征在于：所述天然磷脂为大豆磷脂和/或葵花磷脂。The method for enzymatically catalyzed production of phosphatidylserine according to claim 7, wherein the natural phospholipid is soybean phospholipid and/or sunflower phospholipid.
  9. 根据权利要求7所述的酶催化生产磷脂酰丝氨酸的方法，其特征在于：所述乙醇溶液的pH值为8.0-9.0。The method for enzymatically catalyzed production of phosphatidylserine according to claim 7, wherein the pH of the ethanol solution is 8.0-9.0.
  10. 根据权利要求7或9所述的酶催化生产磷脂酰丝氨酸的方法，其特征在于：所述乙醇溶液中乙醇的浓度为85%-96%。The method for enzymatically catalyzed production of phosphatidylserine according to claim 7 or 9, characterized in that the concentration of ethanol in the ethanol solution is 85%-96%.
  11. 根据权利要求7或9所述的酶催化生产磷脂酰丝氨酸的方法，其特征在于：所述乙醇溶液的体积用量为步骤（2）得到的反应粗品重量的4-6倍。The method for enzymatically catalyzed production of phosphatidylserine according to claim 7 or 9, characterized in that the volume of the ethanol solution is 4-6 times the weight of the crude reaction product obtained in step (2).
  12. 根据权利要求7或9所述的酶催化生产磷脂酰丝氨酸的方法，其特征在于：所述乙醇溶液为乙醇水溶液与氨水的混合溶液。The method for enzymatically catalyzed production of phosphatidylserine according to claim 7 or 9, characterized in that the ethanol solution is a mixed solution of ethanol aqueous solution and ammonia water.
  13. 根据权利要求12所述的酶催化生产磷脂酰丝氨酸的方法，其特征在于：所述步骤3）中，乙醇水溶液的浓度为93%以上，氨水的浓度为25%，乙醇水溶液与氨水的体积比为96 : 4～92 : 8。The method for enzymatically catalyzed production of phosphatidylserine according to claim 12, characterized in that: in the step 3), the concentration of the ethanol aqueous solution is 93% or more, the concentration of the ammonia water is 25%, and the volume ratio of the ethanol aqueous solution to the ammonia water It is 96: 4 to 92: 8.
  14. 根据权利要求7所述的酶催化生产磷脂酰丝氨酸的方法，其特征在于：所述方法还包括如下步骤（4）：将步骤（2）固液分离后得到的滤液降温至4-10℃，流加1-2倍体积的预冷乙醇溶液，混合均匀后进行固液分离，收集固体物，干燥后即得回收后的L-丝氨酸。The method for enzymatically catalyzed production of phosphatidylserine according to claim 7, characterized in that: the method further comprises the following step (4): cooling the filtrate obtained after step (2) solid-liquid separation to 4-10°C, Add 1-2 times the volume of the pre-cooled ethanol solution, mix well and perform solid-liquid separation, collect the solids, and dry the recovered L-serine to obtain the recovered L-serine.
  15. 根据权利要求14所述的酶催化生产磷脂酰丝氨酸的方法，其特征在于：所述步骤（4）中，所述预冷乙醇溶液的浓度为90%以上。The method for enzymatically catalyzed production of phosphatidylserine according to claim 14, characterized in that: in the step (4), the concentration of the pre-cooled ethanol solution is more than 90%.
PCT/CN2019/130408 2019-12-31 2019-12-31 Method for resisting viscosity during enzyme-catalyzed production of phosphatidylserine and method for producing phosphatidylserine using same WO2021134440A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201980042286.0A CN112384630B (en) 2019-12-31 2019-12-31 Anti-viscosity method for catalytic production of phosphatidylserine enzyme and method for producing phosphatidylserine by using same
PCT/CN2019/130408 WO2021134440A1 (en) 2019-12-31 2019-12-31 Method for resisting viscosity during enzyme-catalyzed production of phosphatidylserine and method for producing phosphatidylserine using same

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2019/130408 WO2021134440A1 (en) 2019-12-31 2019-12-31 Method for resisting viscosity during enzyme-catalyzed production of phosphatidylserine and method for producing phosphatidylserine using same

Publications (1)

Publication Number Publication Date
WO2021134440A1 true WO2021134440A1 (en) 2021-07-08

Family

ID=74586229

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/130408 WO2021134440A1 (en) 2019-12-31 2019-12-31 Method for resisting viscosity during enzyme-catalyzed production of phosphatidylserine and method for producing phosphatidylserine using same

Country Status (2)

Country Link
CN (1) CN112384630B (en)
WO (1) WO2021134440A1 (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1048738A1 (en) * 1999-04-28 2000-11-02 CHEMI S.p.A. A process for the preparation of phosphatidylserines
CN103555783A (en) * 2013-10-24 2014-02-05 陕西源邦生物技术有限公司 Method for preparing phosphatidyserine
CN104059949A (en) * 2014-05-30 2014-09-24 广东省食品工业研究所 Preparation method of phosphatidyl serine
CN105296560A (en) * 2015-12-07 2016-02-03 杏辉天力（杭州）药业有限公司 Preparation method of phosphatidylserine

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1048738A1 (en) * 1999-04-28 2000-11-02 CHEMI S.p.A. A process for the preparation of phosphatidylserines
CN103555783A (en) * 2013-10-24 2014-02-05 陕西源邦生物技术有限公司 Method for preparing phosphatidyserine
CN104059949A (en) * 2014-05-30 2014-09-24 广东省食品工业研究所 Preparation method of phosphatidyl serine
CN105296560A (en) * 2015-12-07 2016-02-03 杏辉天力（杭州）药业有限公司 Preparation method of phosphatidylserine

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HOU HAI-JUAN; GONG JIN-SONG; DONG YU-XIU; QIN JIUFU; LI HENG; LI HUI; LU ZHEN-MING; ZHANG XIAO-MEI; XU ZHENG-HONG; SHI JIN-SONG: "Phospholipase D engineering for improving the biocatalytic synthesis of phosphatidylserine", BIOPROCESS AND BIOSYSTEMS ENGINEERING, SPRINGER, DE, vol. 42, no. 7, 16 April 2019 (2019-04-16), DE, pages 1185 - 1194, XP036827427, ISSN: 1615-7591, DOI: 10.1007/s00449-019-02116-7 *
SUN MING-BO, LIU TIAN-WEI, WANG JI-WEN, ZHANG YI-XUAN: "Advanced in Phosphatidylserine Preparation", WEISHENGWUXUE ZAZHI - ACTA MICROBIOLOGICA SINICA, LIAONING SHENG WEISHENGWU YANJIUSUO, CN, vol. 32, no. 3, 1 May 2012 (2012-05-01), CN, pages 80 - 86, XP055827972, ISSN: 1005-7021 *

Also Published As

Publication number Publication date
CN112384630A (en) 2021-02-19
CN112384630B (en) 2022-04-01

Similar Documents

Publication Publication Date Title
CN104473120B (en) A kind of production technology of monosodium glutamate
CN107974480A (en) A kind of krill peptide preparation method
CN109554302B (en) Method for producing feed enzyme preparation by fermentation by using immobilized cell technology
CN108017535A (en) A kind of method that long-chain biatomic acid is extracted from zymotic fluid
JP2001025368A (en) Supplement for feed based on fermentation-treated liquid, its production, and use of l-lysin and/or salt thereof as supplement for feed
CN111087486B (en) Novel continuous preparation method of resistant dextrin dietary fiber
CN112778149A (en) Method for extracting and separating beta-alanine from fermentation liquor
CN104313105B (en) The method that a kind of utilization threonine fermentation liquid and waste liquor prepare 65% threonine
US3085049A (en) Process for producing vitamin b12 and antibiotics
WO2021134440A1 (en) Method for resisting viscosity during enzyme-catalyzed production of phosphatidylserine and method for producing phosphatidylserine using same
JPS61212249A (en) Composition for feed
CN105192352A (en) Process of using glutamic acid waste water to prepare feed
CN104830923B (en) A kind of method for producing L-Glutamine from Pidolidone biotransformation method using lactobacillus thermophilus
DE69829282T2 (en) PROCESS FOR THE PRODUCTION OF OPTICALLY ACTIVE ALCOHOLS
CN112998128A (en) Method for producing feed by using corn sugar residues
CN112790273A (en) Method for recovering L-lactic acid fermentation thallus and continuously producing fermented feed
CN106496075B (en) The preparation method and L-citrulline prepared therefrom of a kind of L-citrulline crude product
CN111363767A (en) Preparation method of phosphatidylserine for food
CN110483318A (en) A kind of extracting method of L-threonine
HU202406B (en) Process for producing fat mixture or fatty acid mixture containing eicosapentaenic acid ester or occasionally eicosapentaenic acid from rain-worms of lubriciade
CN109182407A (en) A kind of tryptophan preparation method and its fermentation medium and tryptophan that use fermentation special nutritional member
CN112772933A (en) Preparation method of high-protein sugar-free dietary fiber powder
RU2522006C1 (en) Method of growing yeast
CN109329804A (en) A kind of extracting method of jujube ingredient
CN104189007B (en) The preparation method of natural compound amino-acid raw material

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19958254

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 22.11.2022)

122 Ep: pct application non-entry in european phase

Ref document number: 19958254

Country of ref document: EP

Kind code of ref document: A1