WO2019011194A1 - 一种催化合成磷脂酰丝氨酸的生物酶及其制备方法和应用 - Google Patents

一种催化合成磷脂酰丝氨酸的生物酶及其制备方法和应用 Download PDF

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WO2019011194A1
WO2019011194A1 PCT/CN2018/094840 CN2018094840W WO2019011194A1 WO 2019011194 A1 WO2019011194 A1 WO 2019011194A1 CN 2018094840 W CN2018094840 W CN 2018094840W WO 2019011194 A1 WO2019011194 A1 WO 2019011194A1
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phosphatidylserine
enzyme
synthesis
bio
biological enzyme
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祝俊
黄科学
余玉奎
吴锋
张超
徐飞
孙锦龙
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江苏诚信药业有限公司
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids

Definitions

  • the invention relates to a method for synthesizing phosphatidylserine, in particular to a phosphatidylserine synthesis method using high-efficiency biological enzyme catalysis and modern separation technology, in particular to a biological enzyme for catalyzing the synthesis of phosphatidylserine and preparation method and application thereof .
  • Phosphatidylserine also known as serine phospholipid, diacylglycerol phosphate serine, is a ubiquitous phospholipid, usually located in the inner layer of the cell membrane, phosphoglycerides in phospholipid chemicals, is a cell membrane component One, related to a range of membrane functions. Especially in the human nervous system, it is one of the important components of the brain's cell membrane, and at the same time plays an important role in regulating various functions of the brain (especially the stability of the brain's memory and emotions), such as it can affect the cell membrane. It is fluid, permeable, and activates the metabolism and synthesis of many enzymes.
  • the preparation methods of industrially produced phosphatidylserine mainly include extraction method and enzymatic conversion method, wherein the extraction method mainly extracts PS from plant cells and animal lecithin, and the PS content in plants is less, so the extraction method is to take the eggs of the animal.
  • the main extract of phospholipids is mainly from the animal's brain and internal organs.
  • most of the foreign countries use the animal brains of poultry such as cattle, sheep, rabbits, horses and donkeys as raw materials to extract PS.
  • due to animal diseases such as mad cow disease the method of extracting PS by animal cells has been suspected of the safety of the extracted products, and is now on the verge of elimination.
  • a first object of the present invention is to provide a biological enzyme which catalyzes the synthesis of phosphatidylserine.
  • a second object of the present invention is to provide a nucleotide sequence encoding the biological enzyme which catalyzes the synthesis of phosphatidylserine.
  • a third object of the present invention is to provide a method for preparing a biological enzyme which catalyzes the synthesis of phosphatidylserine
  • the biosynthesis of phosphatidylserine adopts bioenzymatic catalysis to synthesize phosphatidylserine in one step; the invention adopts enzyme to efficiently convert maltose to produce phosphatidylserine, and adopts modern advanced separation technology to make industrial production cost of phosphatidylserine greatly reduce.
  • a fourth object of the present invention is to provide a method for the biocatalytic synthesis of phosphatidylserine.
  • a biological enzyme catalyzing the synthesis of phosphatidylserine which is the amino acid sequence shown in SEQ ID NO: 2.
  • the biological enzyme for catalytically synthesizing phosphatidylserine, is derived from a genetically engineered strain recombinantly constructed in vitro; and the genetically engineered strain is Escherichia coli, Pichia pastoris, Bacillus subtilis.
  • a nucleotide sequence encoding the above-described biological enzyme which is represented by SEQ ID NO: 1.
  • a method for preparing the above biological enzyme comprising the steps of:
  • the alkaline solution described in the step 3) is one of sodium hydroxide, sodium carbonate, sodium hydrogencarbonate, potassium hydroxide, potassium carbonate, potassium hydrogencarbonate or aqueous ammonia.
  • the binary mixed solvent described in the step 7) is one of ethyl acetate and methanol, n-hexane and isopropanol, cyclohexane and ethanol, and n-hexane and ethanol.
  • the crystallization solvent is one of methanol, ethanol, isopropanol, and acetone.
  • the beating solvent is one of methanol, ethanol, isopropanol, and acetone.
  • the present invention adopts the biological enzyme technology, and the enzyme conversion method mainly uses natural lecithin as a matrix, adds serine, and utilizes the high specificity of phosphatidylserine synthetase to catalyze the preparation of phosphatidylserine from lecithin and serine. It is expected to change the current problems in the production of phosphatidylserine and lay the foundation for the industrial production of high-purity phospholipids.
  • a genetically engineered strain of the biological enzyme was constructed, and the whole enzyme-synthesized biological enzyme gene fragment was linearized with a restriction endonuclease Sal I.
  • the positive clones were picked into the YPD liquid medium and transferred to BMGY liquid medium.
  • 1% methanol was induced and induced for 72 hours to express the biological enzyme.
  • the catalytic synthesis of phospholipids was obtained.
  • the enzyme of ylserine utilizes the high specificity of phosphatidylserine synthetase to catalyze the preparation of phosphatidylserine from lecithin and serine with a conversion rate of 80%, which effectively solves the problem that high-purity phospholipids cannot be obtained by current extraction and chemical synthesis.
  • the problem of acylosine, and the process is stable and reliable, high efficiency, suitable for industrial production, and has great application potential in the fields of medicine and food health products.
  • the enzymatic synthesis is safe, environmentally and sustainable, and the enzymatic reaction rate is high, reaching more than 95%, and the concentration of phosphatidylserine in the reaction solution is as high as 100 g/L.
  • composition of the LB liquid medium used in the present invention yeast powder 5g, peptone 10g, sodium chloride 10g, adjust the pH to 7.0; add water to a volume of 1L;
  • composition of YPD medium yeast powder 10g, peptone 20g, glucose 20g, add water to a volume of 1L;
  • BMGY liquid medium yeast powder 10g, peptone 10g, YNB 13.4g, glycerol 10g, biotin 0.004g, pH was adjusted to 6.0 with phosphate buffer (0.1M), and made up to 1L with water.
  • the whole gene-synthesized biological enzyme fragment (sequence as shown in SEQ ID NO: 1, synthesized by Changzhou Keyu Biotechnology Co., Ltd.), via restriction enzymes EcoR I and Not I (purchased from New England Biolabs, according to The instructions were manipulated and recombined into the yeast expression vector pPIC9k (invitrogen), transformed into E. coli Top10 competent state (purchased from Beijing Quanjin Biotechnology Co., Ltd.), and E. coli Top10 was placed in LB liquid medium. The recombinant plasmid was extracted by shaking at 37 ° C and 160 rpm overnight. The recombinant plasmid was linearized using restriction endonuclease Sal I (purchased from New England Biolabs, Inc., operating according to the instructions).
  • Pichia pastoris GS115 competent cells invitrogen: Pichia pastoris GS115 single colony was picked into YPD medium for activation, and activated Pichia GS115 was added to 50 ml of YPD medium at 30% inoculation at 0.5% inoculum. In several stages, the cells obtained by centrifugation were washed twice with 20 ml of sterile water, washed twice with 20 ml of sterile 1 M sorbitol, and the cells were resuspended by adding 1 ml of 1 M sorbitol solution to obtain P. pastoris GS115 competent cells.
  • the linearized fragment was added to 80 ⁇ l of Pichia pastoris GS115 competent cells for 5 minutes in an ice bath, and after electrotransformation, 800 ⁇ l of sorbitol was added to wash the cells into a 1.5 ml sterile centrifuge tube, and after incubation at 25 ° C for 2 hours, The MD plate was centrifuged and cultured at 30 ° C until the bacteria grew out, and single colonies were separated by scribing. A single colony was picked into sterile water and added with appropriate amount of Lyticase (purchased from sigma company). The cell wall was digested at 37 ° C for 1 hour, and a part of the digestion product was added to the PCR system to detect positive clones.
  • the positive clones were picked into YPD liquid medium and transferred to BMGY liquid medium. When cultured until the OD was 1.0, 1% methanol was induced and induced for 72 hours. The methanol was added once every 24 hours to express the organism of the present invention. Enzyme.
  • the original strains were streaked in YPD, and cultured at 30 ° C overnight.
  • the fermentation medium After the fermentation medium is configured per liter, pour into the fermenter (30L), sterilize at 121 °C for 30 min; control the temperature at 30 °C after cooling, and adjust the pH to 5.0 using ammonia water. Inoculate the long seed solution into the tank.
  • the inoculation amount is 5%.
  • the dissolved oxygen is controlled to be more than 30%.
  • the dissolved oxygen is increased.
  • the wet weight is about 140g/L
  • the feed is started at 50% (w/v)
  • the feed rate is about 15ml/L fermentation broth/hour
  • the feed rate is controlled to dissolve.
  • Oxygen is maintained at more than 30%; the wet weight of the strain is about 180g/L, the glycerin is stopped, and 100% methanol is added at a flow rate of 7.2ml/L fermentation broth/hour. After 10 hours of induction, the pH is adjusted to 6.0. After induction for 24 hours, the pH was adjusted to 7.0, the feed rate remained unchanged, and the dissolved oxygen was maintained at 30% or more according to the dissolved oxygen to adjust the rotation speed and ventilation. The wet weight of the cells was induced to be about 340 g/L in 96 hours. After centrifugation, the supernatant was collected, concentrated by ultrafiltration, and lyophilized to obtain a lyophilized powder of the synthetic phosphatidylserine.
  • a method for preparing the above biological enzyme comprising the steps of:
  • the alkaline solution described in the step 3) is one of sodium hydroxide, sodium carbonate, sodium hydrogencarbonate, potassium hydroxide, potassium carbonate, potassium hydrogencarbonate or ammonia.
  • the binary mixed solvent described in the step 7) is one of ethyl acetate and methanol, n-hexane and isopropanol, cyclohexane with ethanol, n-hexane and ethanol.
  • the crystallization solvent may be one selected from the group consisting of methanol, ethanol, isopropanol and acetone.
  • the beating solvent may be selected from one of methanol, ethanol, isopropanol and acetone.
  • the invention adopts the enzymatic synthesis to be safe, environmentally and sustainable, and has a high enzymatic reaction rate, which can reach more than 95%, and the concentration of phosphatidylserine in the reaction solution reaches 100 g/L.

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Abstract

一种催化合成磷脂酰丝氨酸的生物酶及其制备方法和应用，所述生物酶为SEQ ID NO：2所示的氨基酸序列。所述生物酶的制备，首先构建生物酶的基因工程菌株，将全基因合成的生物酶基因片段，利用限制性内切酶SalⅠ线性化重组质粒，用于表达生物酶，经过进一步发酵获得催化合成磷脂酰丝氨酸的生物酶，利用磷脂酰丝氨酸合成酶高度专一性，催化卵磷脂和丝氨酸制备磷脂酰丝氨酸，转化率达到80％，有效地解决了目前浸提法和化学合成法无法获取高纯度磷脂酰丝氨酸的问题，而且工艺稳定可靠，效率高，适合工业化生产，在医药及食品保健品领域具有很大的应用潜力。

Description

一种催化合成磷脂酰丝氨酸的生物酶及其制备方法和应用 【技术领域】
本发明涉及一种合成磷脂酰丝氨酸的方法，尤其是采用高效的生物酶催化和现代化的分离技术的磷脂酰丝氨酸合成方法，具体是一种催化合成磷脂酰丝氨酸的生物酶及其制备方法和应用。
【背景技术】
磷脂酰丝氨酸(phosphatidylserine简称PS)，又称丝氨酸磷脂，二酰甘油酰磷酸丝氨酸，是一类普遍存在的磷脂，通常位于细胞膜的内层，磷脂化学物中的磷酸甘油酯类，是细胞膜组分之一，与一系列的膜功能有关。尤其在人体的神经系统，是大脑的细胞膜的重要组成成分之一，同时对大脑的各种功能(尤其是对大脑的记忆力和情绪的稳定)起到重要的调节作用，如它能影响着细胞膜的流动性、通透性，并且能激活多种酶类的代谢和合成。具有重要的功能与作用：延缓衰老、维持机体健康年轻、强化脑部功能、增强记忆力、运动员等的营养必需品、治疗多动症儿童、促进血液循环、分解过高的血脂和过高的胆固醇、清扫血管，使血管顺畅，从而减轻脂肪肝的发病率，有“血管清道夫”的美誉。
目前工业化生产磷脂酰丝氨酸的制备方法主要有提取法和酶转化法，其中提取法主要是从植物细胞、动物的卵磷脂中提取PS，植物中的PS含量较少，所以提取法以动物的卵磷脂中提取为主，从动物中提取主要是动物的大脑及内脏，目前国外大部分以牛、羊、兔、马、驴等家禽的动物脑为原料来提取PS。近年来，由于疯牛病的等 动物疾病的原因，利用动物细胞提取PS的方法，其提取的产品的安全性收到人们的怀疑，现在已处于淘汰边缘。
【发明内容】
为了解决以上问题，本发明的第一个目的在于提供一种催化合成磷脂酰丝氨酸的生物酶。
本发明的第二个目的是提供一种编码所述催化合成磷脂酰丝氨酸的生物酶的核苷酸序列。
本发明的第三个目的是提供一种催化合成磷脂酰丝氨酸的生物酶的制备方法
本发明提供的磷脂酰丝氨酸的生物合成采用生物酶法催化一步合成磷脂酰丝氨酸；本发明采用酶高效转化麦芽糖生成磷脂酰丝氨酸，并采用现代化的先进分离技术，使磷脂酰丝氨酸的工业化生产成本大大降低。
本发明的第四个目的是提供一种生物酶催化合成磷脂酰丝氨酸的方法。
本发明的技术方案如下：
一种催化合成磷脂酰丝氨酸的生物酶，所述生物酶为SEQ ID NO：2所示的氨基酸序列。
根据上述催化合成磷脂酰丝氨酸的生物酶，所述生物酶来源于体外重组构建的基因工程菌株；所述基因工程菌株为大肠杆菌、毕氏酵母、枯草芽孢杆菌。
一种编码上述的生物酶的核苷酸序列，所述核苷酸序列如SEQ  ID NO：1所示。
一种上述的生物酶的制备方法，包括如下步骤：
(1)首先构建磷脂酰丝氨酸酶的基因工程菌株，将权利要求2所述的核苷酸序列经酶切重组到表达载体，转化到宿主细胞中；
(2)将阳性克隆挑入YPD液体培养基中活化后转入BMGY液体培养基中，培养，最后经发酵培养，得到催化合成磷脂酰丝氨酸的生物酶。
一种权利要求1所述的生物酶催化磷脂酰丝氨酸的合成方法，包括如下步骤：
(1)向反应釜中加入大豆卵磷脂，L-丝氨酸，醋酸钠，氯化钙，纯化水搅拌溶解；其中大豆卵磷脂的投料量为50～500g/L，L-丝氨酸的投料量为10～500g/L，醋酸钠的投料量为10～100g/L，氯化钙的投料量为1～100g/L；
(2)采用抽真空氮气置换的方法置换反应体系中的空气，置换完毕氮气保护整个反应体系；
(3)在25～65℃、氮气保护下，用碱性溶液调节pH值至5.0～8.0；
(4)加入上述的生物酶，氮气保护下、在25～65℃搅拌反应6～48h；
(5)反应完毕，放料，料液离心，收集滤饼；
(6)将得到的滤饼用去离子水打浆离心；
(7)滤饼用二元混合溶剂溶解后过滤除去不溶性杂质；
(8)减压浓缩滤液，浓缩至物料粘稠流动性较差时，加入析晶溶剂，产品析出；
(9)离心过滤后，用打浆溶剂打浆；
离心过滤，真空干燥得产品。
作为优选，步骤3)中所述的碱性溶液为氢氧化钠、碳酸钠、碳酸氢钠、氢氧化钾、碳酸钾、碳酸氢钾、氨水中的一种。
作为优选，步骤7)中所述的二元混合溶剂为乙酸乙酯同甲醇、正己烷同异丙醇、环己烷同乙醇、正己烷同乙醇中的一种。
作为优选，所述的析晶溶剂为甲醇、乙醇、异丙醇、丙酮中的一种。
作为优选，所述的打浆溶剂为甲醇、乙醇、异丙醇、丙酮中的一种。
有益的效果：本发明采用生物酶技术，通过酶转化法主要是以天然的卵磷脂为基质，加入丝氨酸，利用磷脂酰丝氨酸合成酶的高度专一性，催化卵磷脂和丝氨酸制备磷脂酰丝氨酸，有望改变目前在生产磷脂酰丝氨酸上存在的问题，为工业化定向生产高纯度磷脂类物质奠定基础。
构建生物酶的基因工程菌株，将全基因合成的生物酶基因片段，利用限制性内切酶Sal Ⅰ线性化重组质粒。将阳性克隆挑入YPD液体培养基活化后转入BMGY液体培养基中，培养至OD为1.0时接入1％的甲醇诱导，诱导72小时，用于表达生物酶，经过进一步发酵获得催化合成磷脂酰丝氨酸的生物酶，利用磷脂酰丝氨酸合成酶高度专 一性，催化卵磷脂和丝氨酸制备磷脂酰丝氨酸，转化率达到80％，有效地解决了目前浸提法和化学合成法无法获取高纯度磷脂酰丝氨酸的问题，而且工艺稳定可靠，效率高，适合工业化生产，在医药及食品保健品领域具有很大的应用潜力。采用酶法合成安全环保可持续，酶反应生成率高，可到达95％以上，反应液中磷脂酰丝氨酸浓度高达到100g/L。
【具体实施方式】
以下结合具体实施例，对本发明作进一步说明。应理解，以下实施例仅由于说明本发明而非用于限定本发明的范围。
实施例1：生物酶的基因工程菌株构建
本发明所应用LB液体培养基的组成：酵母粉5g，蛋白胨10g，氯化钠10g，调节pH到7.0；加水定容至1L；
YPD培养基的组成：酵母粉10g，蛋白胨20g，葡萄糖20g，加水定容至1L；
BMGY液体培养基的组成：酵母粉10g，蛋白胨10g，YNB 13.4g，甘油10g，生物素0.004g，用磷酸盐缓冲液(0.1M)调节pH至6.0，加水定容至1L。
将全基因合成的生物酶片段(序列如SEQ ID NO：1所示，由常州基宇生物科技有限公司合成)，经限制性内切酶EcoR Ⅰ和Not Ⅰ(购自New England Biolabs公司，根据说明书进行操作)酶切后重组到酵母表达载体pPIC9k(invitrogen公司)，转化到E.coli Top10感受态(购自北京全式金生物技术有限公司)，将E.coli Top10置于LB液体 培养基中，37℃、160rpm转振荡培养过夜，提取重组质粒。利用限制性内切酶Sal Ⅰ(购自New England Biolabs公司，根据说明书进行操作)线性化重组质粒。
巴斯德毕赤酵母GS115感受态细胞(invitrogen公司)：将Pichia Pastoris GS115单菌落挑入YPD培养基中活化，活化的Pichia GS115按0.5％的接种量接入50mlYPD培养基中30℃培养至对数期，离心获得的菌体用20ml无菌水洗2次，再用20ml无菌1M山梨醇洗2次，加入1ml1M山梨醇溶液重悬菌体获得巴斯德毕赤酵母GS115感受态细胞。
将线性化片段加入到80μl巴斯德毕赤酵母GS115感受态细胞中冰浴5分钟，电转化后加入800μl山梨醇将细胞洗至1.5ml无菌离心管中，25℃温育2小时后，离心涂MD平板，30℃培养至长出菌落后，划线分离出单菌落。将单菌落挑入无菌水中加入适量Lyticase(购自sigma公司)后37℃温育1小时消化细胞壁，取部分消化产物加入PCR体系检测阳性克隆。
将阳性克隆挑入YPD液体培养基后转接入BMGY液体培养基中，培养至OD为1.0时接入1％甲醇诱导，诱导72小时，每24小时补加一次甲醇用于表达本发明的生物酶。
实施例2：生物酶的发酵制备
取原种菌种在YPD划线，30℃，倒置培养过夜。在平板上挑取单菌落(直径1mm)至50mlYPD液体培养基(酵母粉10g，蛋白胨10g，葡萄糖10g，加水定容至1L)中，30℃，200rpm振荡培养过夜 (24h)，OD600长至4-5。以10％的接种量接种到300mlYPD液体培养基中(1L三角瓶)摇瓶中，30℃、200rpm摇床振荡培养，约24小时后OD600长到12左右。将发酵培养基按每升料配置好后，倒入发酵罐(30L)，121℃灭菌30min；降温后控制温度30℃，使用氨水调节pH值至5.0.将长好的种子液接种进罐，接种量5％。根据溶氧调节转速和通气，控制溶氧在30％以上。培养24小时左右，待溶氧突变上升，此时湿重约为140g/L，开始补料50％(w/v)，补料速度约为15ml/L发酵液/小时，补料速度控制溶氧保持在30％以上；待菌种湿重长至180g/L左右，停止补加甘油，以7.2ml/L发酵液/小时流速流加100％甲醇，诱导10小时后，pH值调节为6.0，诱导24小时，pH调节为7.0，补料速度保持不变，根据溶氧调节转速和通气，保持溶氧在30％以上。诱导96小时菌体湿重约为340g/L左右放罐。离心，收集清液，超滤浓缩后冻干，得到合成磷脂酰丝氨酸用生物酶冻干粉。
实施例3：生物酶催化合成磷酯酰丝氨酸
一种上述的生物酶的制备方法，包括如下步骤：
1)首先构建磷脂酰丝氨酸酶的基因工程菌株，将权利要求2所述的核苷酸序列经酶切重组到表达载体，转化到宿主细胞中；
2)将阳性克隆挑入YPD液体培养基中活化后转入BMGY液体培养基中，培养，最后经发酵培养，得到催化合成磷脂酰丝氨酸的生物酶。
实施例4
一种权利要求1所述的生物酶催化磷脂酰丝氨酸的合成方法，包 括如下步骤：
(1)向反应釜中加入大豆卵磷脂，L-丝氨酸，醋酸钠，氯化钙，纯化水搅拌溶解；其中大豆卵磷脂的投料量为50～500g/L，L-丝氨酸的投料量为10～500g/L，醋酸钠的投料量为10～100g/L，氯化钙的投料量为1～100g/L；
(2)采用抽真空氮气置换的方法置换反应体系中的空气，置换完毕氮气保护整个反应体系；
(3)在25～65℃、氮气保护下，用碱性溶液调节pH值至5.0～8.0；
(4)加入权利要求4所述的生物酶，氮气保护下、在25～65℃搅拌反应6～48h；
(5)反应完毕，放料，料液离心，收集滤饼；
(6)将得到的滤饼用去离子水打浆离心；
(7)滤饼用二元混合溶剂溶解后过滤除去不溶性杂质；
(8)减压浓缩滤液，浓缩至物料粘稠流动性较差时，加入析晶溶剂，产品析出；
(9)离心过滤后，用打浆溶剂打浆；
(10)离心过滤，真空干燥得产品。
步骤3)中所述的碱性溶液为氢氧化钠、碳酸钠、碳酸氢钠、氢氧化钾、碳酸钾、碳酸氢钾、氨水中的一种。
步骤7)中所述的二元混合溶剂为乙酸乙酯同甲醇、正己烷同异丙醇、环己烷同乙醇、正己烷同乙醇中的一种。
所述的析晶溶剂可选用甲醇、乙醇、异丙醇、丙酮中的一种。
所述的打浆溶剂可选用甲醇、乙醇、异丙醇、丙酮中的一种。
本发明采用酶法合成安全环保可持续，酶反应生成率高，可到达95％以上，反应液中磷脂酰丝氨酸浓度高达到100g/L。

Claims (9)

  1. 一种催化合成磷脂酰丝氨酸的生物酶，其中，所述生物酶为SEQ ID NO：2所示的氨基酸序列。
  2. 根据权利要求1所述催化合成磷脂酰丝氨酸的生物酶，其中，所述生物酶来源于体外重组构建的基因工程菌株；所述基因工程菌株为大肠杆菌、毕氏酵母、枯草芽孢杆菌。
  3. 一种编码权利要求1所述的生物酶的核苷酸序列，其中，所述核苷酸序列如SEQ ID NO：1所示。
  4. 一种权利要求1所述的生物酶的制备方法，其中，包括如下步骤：
    (1)首先构建磷脂酰丝氨酸酶的基因工程菌株，将权利要求2所述的核苷酸序列经酶切重组到表达载体，转化到宿主细胞中；
    (2)将阳性克隆挑入YPD液体培养基中活化后转入BMGY液体培养基中，培养，最后经发酵培养，得到催化合成磷脂酰丝氨酸的生物酶。
  5. 一种权利要求1所述的生物酶催化合成磷脂酰丝氨酸的方法，其中，包括如下步骤：
    (1)向反应釜中加入大豆卵磷脂，L-丝氨酸，醋酸钠，氯化钙，纯化水搅拌溶解；其中大豆卵磷脂的投料量为50～500g/L，L-丝氨酸的投料量为10～500g/L，醋酸钠的投料量为10～100g/L，氯化钙的投料量为1～100g/L；
    (2)采用抽真空氮气置换的方法置换反应体系中的空气，置换完毕氮气保护整个反应体系；
    (3)在25～65℃、氮气保护下，用碱性溶液调节pH值至5.0～8.0；
    (4)加入权利要求4所述的生物酶，氮气保护下、在25～65℃搅拌反应6～48h；
    (5)反应完毕，放料，料液离心，收集滤饼；
    (6)将得到的滤饼用去离子水打浆离心；
    (7)滤饼用二元混合溶剂溶解后过滤除去不溶性杂质；
    (8)减压浓缩滤液，浓缩至物料粘稠流动性较差时，加入析晶溶剂，产品析出；
    (9)离心过滤后，用打浆溶剂打浆；
    (10)离心过滤，真空干燥得产品。
  6. 根据权利要求5所述的生物酶催化合成磷脂酰丝氨酸的方法，其中，步骤(3)中所述的碱性溶液为氢氧化钠、碳酸钠、碳酸氢钠、氢氧化钾、碳酸钾、碳酸氢钾、氨水中的一种。
  7. 根据权利要求5所述的生物酶催化合成磷脂酰丝氨酸的方法，其中，步骤(7)中所述的二元混合溶剂为乙酸乙酯同甲醇、正己烷同异丙醇、环己烷同乙醇、正己烷同乙醇中的一种。
  8. 根据权利要求5所述的生物酶催化合成磷脂酰丝氨酸的方法，其中，所述的析晶溶剂为甲醇、乙醇、异丙醇、丙酮中的一种。
  9. 根据权利要求5所述的生物酶催化合成磷脂酰丝氨酸的方法， 其中，所述的打浆溶剂为甲醇、乙醇、异丙醇、丙酮中的一种。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020155558A1 (en) * 2001-02-09 2002-10-24 Guenter Kirschner Procedure for the preparation of pure phosphatides and their use in the cosmetic, pharmaceutical and alimentary fields
CN101818179A (zh) * 2010-04-28 2010-09-01 大连理工大学 一种制备富含多不饱和脂肪酸的磷脂酰丝氨酸的方法
US20110124061A1 (en) * 2004-01-21 2011-05-26 Su Chen Method for preparation of polyunsaturated fatty acid-containing phosphatidylserine
CN106085984A (zh) * 2016-06-02 2016-11-09 天津科技大学 一种新型磷脂酶d及其制备磷脂酸、磷脂酰丝氨酸的方法
CN107312756A (zh) * 2017-07-10 2017-11-03 江苏诚信药业有限公司 一种催化合成磷脂酰丝氨酸的生物酶及其制备方法和应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104480075B (zh) * 2014-11-20 2017-04-19 江苏诚信药业有限公司 一种催化合成丙谷二肽的生物酶及其制备方法和应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020155558A1 (en) * 2001-02-09 2002-10-24 Guenter Kirschner Procedure for the preparation of pure phosphatides and their use in the cosmetic, pharmaceutical and alimentary fields
US20110124061A1 (en) * 2004-01-21 2011-05-26 Su Chen Method for preparation of polyunsaturated fatty acid-containing phosphatidylserine
CN101818179A (zh) * 2010-04-28 2010-09-01 大连理工大学 一种制备富含多不饱和脂肪酸的磷脂酰丝氨酸的方法
CN106085984A (zh) * 2016-06-02 2016-11-09 天津科技大学 一种新型磷脂酶d及其制备磷脂酸、磷脂酰丝氨酸的方法
CN107312756A (zh) * 2017-07-10 2017-11-03 江苏诚信药业有限公司 一种催化合成磷脂酰丝氨酸的生物酶及其制备方法和应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE GenBank [O] 28 February 2017 (2017-02-28), "phospholipase [Streptomyces sp. fd1-xmd]", XP055569926, Database accession no. AQT70868.1 *
HU , FEI ET AL.: "Advance in Enzymatic Method for Phosphatidylserine Preparation", CHINA OILS AND FATS, vol. 37, no. 06, 20 June 2012 (2012-06-20), pages 54 - 57 *
IWASAKI, Y. ET AL.: "An Aqueous Suspension System for Phospholipase D-mediated Synthesis of PS without Toxic Organic Solvent", JAOCS, vol. 80, no. 7, 31 July 2003 (2003-07-31), pages 653 - 657, XP055569940 *

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