WO2020133314A1 - 一种磷脂酶d突变体、其应用及其制备磷酯酰丝氨酸的方法 - Google Patents

一种磷脂酶d突变体、其应用及其制备磷酯酰丝氨酸的方法 Download PDF

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WO2020133314A1
WO2020133314A1 PCT/CN2018/125222 CN2018125222W WO2020133314A1 WO 2020133314 A1 WO2020133314 A1 WO 2020133314A1 CN 2018125222 W CN2018125222 W CN 2018125222W WO 2020133314 A1 WO2020133314 A1 WO 2020133314A1
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phospholipase
mutant
phosphatidylserine
wild
phospholipids
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PCT/CN2018/125222
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French (fr)
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傅荣昭
李振伟
刘文山
汤小欣
朱志强
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邦泰生物工程(深圳)有限公司
江西邦泰绿色生物合成生态产业园发展有限公司
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Priority to CN201880037979.6A priority Critical patent/CN111051505B/zh
Priority to PCT/CN2018/125222 priority patent/WO2020133314A1/zh
Publication of WO2020133314A1 publication Critical patent/WO2020133314A1/zh

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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12Y301/04004—Phospholipase D (3.1.4.4)

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  • the invention relates to the technical field of biological enzyme catalysis, in particular to a phospholipase D mutant artificially obtained by a method of genetic site-directed mutation and a method for catalytically preparing phosphatidylserine.
  • Phospholipase D belongs to the class of phosphatidyl diester synthase, which can catalyze the transesterification reaction of the phospholipids in the substrate phase and the nucleophilic donor in the water phase similar to the ester condensation, based on The transesterification activity of PLD can modify phospholipids to prepare high-purity single phospholipids, rare phospholipids and a series of functional phospholipids. It has become a synthesis and modification of rare phospholipids such as phosphatidylserine and phosphatidylinositol. Important tool enzyme.
  • Phosphatidylserine also known as serine phospholipid, diacylglycerylphosphoserine, compound nerve acid, etc.
  • PS is an important membrane phospholipid commonly found in bacteria, yeast, plants and mammalian cells .
  • the active substance of the cell membrane especially in brain cells. Its function is mainly to improve the function of nerve cells, regulate the transmission of nerve impulses, and improve the memory function of the brain.
  • PS is very lipophilic, it can quickly enter the brain through the blood-brain barrier after absorption, play a role in soothing vascular smooth muscle cells and increasing blood supply to the brain. It is known as a major after choline and "brain gold" DHA The emerging "smart nutrients”.
  • the preparation method of phosphatidylserine mainly includes extraction method and enzyme conversion method.
  • the extraction method is mainly extracted from the brains of plants (soybeans, sunflower seeds, etc.) and animals (cattle, sheep, horses), but due to the low PS content in the raw materials and the complicated extraction process, the method has a high cost and is not conducive to Modern industrial production.
  • the industry mainly uses enzyme conversion to produce phosphatidylserine, which uses phospholipase D to catalyze the conversion of lecithin (from soybeans, sunflower seeds, eggs, etc.) and L-serine to produce phosphatidylserine due to this method.
  • the raw materials in the are widely sourced, and the content of target substances in the raw materials is high, so its production cost is greatly reduced compared with the extraction method, and it has been widely used in industrial production.
  • the purpose of the present invention is to reduce the effect of phosphatidylcholine hydrolysis activity of phospholipase D on the yield of phosphatidylserine prepared by enzyme conversion method, and to develop a phospholipase D whose enzyme activity is more biased towards phosphatidyl transfer activity, thereby enhancing the enzyme The yield of phosphatidylserine prepared by transformation method.
  • the inventors used recombinant DNA technology to conduct site-directed mutation screening of wild-type phospholipase D, and screened for some mutants with greatly improved PS synthesis activity, thus providing a mutant of phospholipase D, such as SEQ ID Compared with the amino acid sequence of wild-type phospholipase D shown in NO: 2, the phospholipase D mutants of the present invention are at the 93rd, 105th, 215th, 216th, 228th and 486th positions. There is a mutation at at least one site.
  • the phospholipase D mutant of the present invention has at least one mutation in K93A, V105M, G215S, G216S, Q228A and N486Y compared to the amino acid sequence of wild-type phospholipase D shown in SEQ ID NO:2. That is, the mutation of the phospholipase D mutant of the present invention is any one of K93A, V105M, G215S, G216S, Q228A, and N486Y, or any two or two of K93A, V105M, G215S, G216S, Q228A, and N486Y Combination of the above.
  • the mutation of the phospholipase D mutant of the present invention is K93A, V105M, Q228A, N486Y, G215S/K93A or G216S/ compared to the amino acid sequence of the wild-type phospholipase D shown in SEQ ID NO: 2.
  • Q228A is K93A, V105M, Q228A, N486Y, G215S/K93A or G216S/ compared to the amino acid sequence of the wild-type phospholipase D shown in SEQ ID NO: 2.
  • the present invention also provides a gene sequence encoding the above-mentioned phospholipase D mutant provided by the present invention.
  • the present invention also provides a biological material, including a recombinant vector, a recombinant cell or a recombinant microorganism, the biological material containing the above-mentioned gene sequence provided by the present invention. That is, the biological material provided by the present invention is a recombinant vector containing the gene sequence provided by the present invention, a recombinant cell containing the gene sequence provided by the present invention, or a recombinant microorganism containing the gene sequence provided by the present invention.
  • the present invention also provides the use of the above-mentioned phospholipase D mutant of the present invention.
  • the use is to apply the phospholipase D of the present invention to the preparation of phospholipids and phospholipid derivatives.
  • the above-mentioned application of the present invention refers to the use of the above-mentioned phospholipase D mutant of the present invention for catalyzing the transesterification reaction of phospholipids and nucleophilic donors.
  • the above-mentioned phospholipid derivative is phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylglucose or phosphatidylethanolamine.
  • the present invention also provides a method for preparing phosphatidylserine, which comprises catalyzing the conversion of lecithin and serine to phosphatidylserine using the above-mentioned phospholipase D mutant of the present invention.
  • the method for preparing phosphatidylserine of the present invention includes: dissolving lecithin in diethyl ether, then adding an aqueous solution of serine, and using the above-mentioned phospholipase D mutant of the present invention in the presence of calcium chloride and a buffer Catalytic preparation of phosphatidylserine.
  • the phospholipase D mutant provided by the present invention significantly improves the enzyme activity while greatly reducing the ratio of its hydrolytic activity to phosphatidyl transfer activity, making the enzyme activity more biased towards phospholipid Acyl transfer activity.
  • the yield of phosphatidylserine can be increased from 71.5% to 81.0%-94.5 %, reducing production costs.
  • the PCR amplification reaction system and PCR amplification reaction procedure are as follows:
  • the mutations of the phospholipase D mutant prepared in this section are K93A, V105M, Q228A, N486Y, G215S/K93A or G216S/Q228A .
  • Reaction solution 0.5% phosphatidylcholine PC, 0.1% Triton X-100, 40mM Tris-HCl (pH7.4), 10uL enzyme solution. Incubate at 37°C for 10 minutes, add 50uL of stop solution (50mM EDTA and 100mM Tris-HCl, pH 7.4), cool to room temperature, and then add 500uL color reaction solution (20mM potassium phosphate buffer containing pH 7.6 containing 21mM phenol) , 0.59mM 4-aminoantipyrine, 170ug choline oxidase and 1ug horseradish peroxidase), developed at 37°C for 5 minutes, and read the absorbance at 505nm.
  • the hydrolysis activity of phospholipase D is defined by the amount of choline released, and 1 unit of enzyme activity is defined as the amount of enzyme required to release 1 umol of choline per minute.
  • Dissolve lecithin in ether and prepare a 5 mM solution Take 1 mL and add 0.5 mL of 2 M serine aqueous solution, add 1 mL of 0.1 M Tris-HCl buffer (pH 7.4), 0.1 mL of 15 mM CaCl 2 solution and 10 uL of phospholipase D Enzyme liquid. The reaction was shaken at 300 rpm for 10 min at 37°C, 50 uL of stop solution (50 mM EDTA and 100 mM Tris-HCl, pH 7.4) was added, cooled to room temperature, and the content of phosphatidylserine was detected by HPLC.
  • One unit of enzyme activity is defined as the amount of enzyme required to produce 1 umol phosphatidylserine per minute.

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Abstract

一种磷脂酶D突变体、其应用及其制备磷酯酰丝氨酸的方法，涉及生物酶催化技术领域，提供一种酶活力更偏向于磷脂酰转移活性的磷脂酶D突变体，与野生型磷脂酶D的氨基酸序列相比，该磷脂酶D突变体在第93位、第105位、第215位、第216位、第228位和第486位的至少一个位点处有突变。

Description

一种磷脂酶D突变体、其应用及其制备磷酯酰丝氨酸的方法 技术领域
本发明涉及生物酶催化技术领域，特别涉及一种通过基因定点突变的方法人工获得的磷脂酶D突变体及其用于催化制备磷酯酰丝氨酸的方法。
背景技术
磷脂酶D(Phospholipase D，英文简称PLD)属于磷脂酰二酯合成酶类，可以催化底物相中的磷脂类物质和水相中的亲核供体发生类似于酯缩合的转酯反应，基于PLD所具有的这种转酯活性可对磷脂进行改性，制备高纯度的单一磷脂、稀有磷脂以及系列功能性磷脂，现已成为合成和改造磷酯酰丝氨酸、磷脂酰肌醇等稀有磷脂的重要工具酶。
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”。
磷脂酰丝氨酸的制备方法主要包括提取法和酶转化法。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。目前工业上主要采用酶转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。
但是，天然的磷脂酶D除了具备磷脂酰转移活性—可将卵磷脂和丝氨 酸转化成磷脂酰丝氨酸(PS)以外，还具备磷脂酰胆碱水解活性，在有水存在的环境中，磷脂酶D会催化水解卵磷脂产生磷脂酸(phosphatidic acid，PA)和胆碱。而PS的酶转化制备方法的反应体系中不可避免会有水的参与，这就导致部分卵磷脂不能成功转化为PS，从而在很大程度上降低了PS的产率，增加了制备成本。
发明内容
本发明的目的在于降低磷脂酶D的磷脂酰胆碱水解活性对酶转化法制备磷脂酰丝氨酸的产率的影响，开发一种酶活力更偏向于磷脂酰转移活性的磷脂酶D，从而提高酶转化法制备磷脂酰丝氨酸的产率。
为实现上述目的，发明人应用重组DNA技术对野生型磷脂酶D进行定点突变筛选，筛选到一些PS合成活性大幅度提高的突变体，从而提供了一种磷脂酶D突变体，与如SEQ ID NO：2所示的野生型磷脂酶D的氨基酸序列相比，本发明的磷脂酶D突变体在第93位、第105位、第215位、第216位、第228位和第486位的至少一个位点处有突变。
优选地，与如SEQ ID NO：2所示的野生型磷脂酶D的氨基酸序列相比，本发明的磷脂酶D突变体具有K93A、V105M、G215S、G216S、Q228A和N486Y中的至少一个突变。即，本发明的磷脂酶D突变体的突变为K93A、V105M、G215S、G216S、Q228A和N486Y中的任意一个，或者为K93A、V105M、G215S、G216S、Q228A和N486Y中的任意两个或者两个以上的组合。
更优选地，与如SEQ ID NO：2所示的野生型磷脂酶D的氨基酸序列相比，本发明的磷脂酶D突变体的突变为K93A、V105M、Q228A、N486Y、G215S/K93A或G216S/Q228A。
本发明还提供了一种基因序列，该基因序列编码本发明提供的上述磷脂酶D突变体。
本发明还提供了一种生物材料，包括重组载体、重组细胞或者重组微生物，该生物材料含有上述本发明提供的基因序列。即，本发明提供的生物材料是含有上述本发明提供的基因序列的重组载体、含有上述本发明提 供的基因序列的重组细胞或者含有上述本发明提供的基因序列的重组微生物。
本发明还提供了上述本发明的磷脂酶D突变体的用途，该用途为将本发明的磷脂酶D应用在制备磷脂以及磷脂衍生物中。
具体地，上述本发明的应用是指将上述本发明的磷脂酶D突变体用于催化磷脂类物质和亲核供体发生转酯反应。
优选地，上述磷脂衍生物为磷脂酰丝氨酸、磷脂酰肌醇、磷脂酰甘油、磷脂酰葡萄糖或磷脂酰乙醇胺。
本发明还提供了一种制备磷脂酰丝氨酸的方法，包括用上述本发明的磷脂酶D突变体催化卵磷脂和丝氨酸转化成磷脂酰丝氨酸。
优选地，本发明的制备磷脂酰丝氨酸的方法包括：将卵磷脂溶解于乙醚中，然后加入丝氨酸的水溶液，在氯化钙和缓冲液存在的条件下，用上述本发明的磷脂酶D突变体催化制备磷脂酰丝氨酸。
有益效果：
1、与野生型磷脂酶D相比，本发明提供的磷脂酶D突变体在酶活力得到显著提高的同时，大大降低了其水解活力与磷脂酰转移活力的比值，使得酶活更偏向于磷脂酰转移活性。
2、与野生型磷脂酶D相比，将本发明提供的磷脂酶D突变体应用于磷脂酰丝氨酸的酶转化法制备时，可使磷脂酰丝氨酸的产率由71.5％提高至81.0％～94.5％，降低了生产成本。
具体实施方式
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。若无特别说明，本发明实施例中所使用的原料及试剂皆为市售商品，实施例中未注明具体条件者，按常规条件或制造商建议的条件进行。
1、野生型磷脂酶D质粒的制备
设计引物对5′-GGACATATGCTCCGCCACCGGCTCCG-3′和5′-GGT AAGCTTTCAGAGCGAGCAGACGCCCC-3′，利用PCR扩增技术对 如SEQ ID NO：1所示的来源于肉桂链霉菌(streptomyces cinnamoneus)的野生型磷脂酶D(其氨基酸序列如SEQ ID NO：2所示)的核苷酸序列进行扩增，然后将扩增产物插入表达载体PL97中的NdeI和HindIII位点，得到野生型磷脂酶D质粒。
2、磷脂酶D突变体质粒的制备
通过反向PCR技术分别对野生型磷脂酶D的第93位、第105位、第215位、第216位、第228位和第486位进行点突变，利用表1中各突变位点对应的上下游引物，以第1部分制备得到的野生型磷脂酶D质粒为模板，分别进行反向PCR扩增，PCR产物中加入1uL的Dpn I酶切处理1h后转化大肠杆菌DH5α，挑取转化子测序验证正确后保存。
PCR扩增反应体系和PCR扩增反应程序如下：
PCR扩增反应体系：
Figure PCTCN2018125222-appb-000001
PCR扩增反应程序：
95℃，30s；95℃10s，60℃30s，72℃4min，25个循环；72℃延伸5min。
表1
Figure PCTCN2018125222-appb-000002
Figure PCTCN2018125222-appb-000003
3、磷脂酶D酶液的制备
将第1部分制备得到的野生型磷脂酶D质粒和第2部分制备得到的磷脂酶D突变体质粒分别转入链霉菌中，30℃发酵3天后离心收集发酵液的上清液，即得野生型磷脂酶D酶液和磷脂酶D突变体酶液。与如SEQ ID NO：2所示的野生型磷脂酶D的氨基酸序列相比，本部分制备得到的磷脂酶D突变体的突变分别为K93A、V105M、Q228A、N486Y、G215S/K93A或G216S/Q228A。
4、酶活力测定
参照如下(1)和(2)所示的方法分别测定第3部分制备得到的磷脂酶D酶液的水解活力和磷脂酰转移活力，然后计算每种酶液的磷脂酰转移活力与水解活力的比值，测定及计算结果如表2所示。
(1)水解活性测定
反应液：0.5％磷脂酰胆碱PC，0.1％Triton X-100，40mM Tris-HCl(pH7.4),10uL酶液。37℃反应10分钟，加入50uL的终止液(50mM EDTA和100mM Tris-HCl，pH7.4)，冷却至室温，然后加入500uL显色反应液(20mM pH7.6的磷酸钾缓冲液中含有21mM苯酚，0.59mM 4-氨基安替比林，170ug胆碱氧化酶和1ug辣根过氧化物酶)，37℃显色5分钟，505nm读取吸光值。通过胆碱的释放量来定义磷脂酶D的水解活力，1个单位酶活力定义为每分钟释放1umol胆碱所需要的酶量。
(2)磷脂酰转移活性测定
将卵磷脂溶于乙醚，配置成5mM的溶液，取1mL加入0.5mL 2M的丝氨酸水溶液，加入1mL 0.1M的Tris-HCl缓冲液(pH7.4),0.1mL 15mM CaCl 2溶液和10uL磷脂酶D酶液。37℃，300rpm震荡反应10min，加入50uL的终止液(50mM EDTA和100mM Tris-HCl，pH7.4)，冷却至室温，通过HPLC检测磷脂酰丝氨酸的含量。1个单位酶活力定义为每分钟产生1umol磷脂酰丝氨酸所需要的酶量。
表2
类型 水解活力U/mL 磷脂酰转移活力U/mL 转移活力/水解活力
野生型 50.0 155.1 3.1
K93A 43.8 332.9 7.6
V105M 55.3 259.9 4.7
Q228A 38.5 242.5 6.3
G215S+K93A 26.7 309.7 11.6
G216S+Q228 62.5 593.8 9.5
N486Y 47.1 244.9 5.2
5、磷脂酰丝氨酸产率测定
将卵磷脂溶于乙醚，配置成5mM的溶液，取1mL加入0.5mL 1M的丝氨酸水溶液，再加入1mL 0.1M的Tris-HCl缓冲液(pH7.4)，0.1mL 15mM CaCl 2溶液和20uL第3部分制备得到的磷脂酶D酶液。37℃、600rpm震荡反应2小时后通过HPLC检测磷脂酰丝氨酸(PS)的含量，然后计算PS产率(PS产率＝PS含量/卵磷脂转化成PS的理论最大量)。各磷脂酶D酶液对应的PS含量以及PS产率的数据如表3所示。
表3
类型 PS含量 PS产率
野生型 2.68mg 71.5％
K93A 3.30mg 88.1％
V105M 3.04mg 81.0％
Q228A 3.22mg 85.8％
G215S+K93A 3.54mg 94.5％
G216S+Q228 3.48mg 92.9％
N486Y 3.13mg 83.4％

Claims (10)

  1. 一种磷脂酶D突变体，其特征在于：与如SEQ ID NO：2所示的野生型磷脂酶D的氨基酸序列相比，所述突变体在第93位、第105位、第215位、第216位、第228位和第486位的至少一个位点处有突变。
  2. 根据权利要求1所述的磷脂酶D突变体，其特征在于：与如SEQ ID NO：2所示的野生型磷脂酶D的氨基酸序列相比，所述突变体具有K93A、V105M、G215S、G216S、Q228A和N486Y中的至少一个突变。
  3. 根据权利要求1所述的磷脂酶D突变体，其特征在于：与如SEQ ID NO：2所示的野生型磷脂酶D的氨基酸序列相比，所述突变体的突变为K93A、V105M、Q228A、N486Y、G215S/K93A或G216S/Q228A。
  4. 一种基因序列，其特征在于：所述基因序列编码权利要求1-3任一项所述的磷脂酶D突变体。
  5. 一种生物材料，包括重组载体、重组细胞或者重组微生物，其特征在于：所述生物材料含有权利要求4所述的基因序列。
  6. 权利要求1至3任一项所述的磷脂酶D突变体在制备磷脂以及磷脂衍生物中的应用。
  7. 根据权利要求6所述的磷脂酶D突变体在制备磷脂以及磷脂衍生物中的应用，其特征在于：所述磷脂酶D突变体催化磷脂类物质和亲核供体发生转酯反应。
  8. 根据权利要求6或7所述的磷脂酶D突变体在制备磷脂以及磷脂衍生物中的应用，其特征在于：所述磷脂衍生物为磷脂酰丝氨酸、磷脂酰肌醇、磷脂酰甘油、磷脂酰葡萄糖或磷脂酰乙醇胺。
  9. 一种制备磷脂酰丝氨酸的方法，其特征在于：用权利要求1-3任一项所述的磷脂酶D突变体催化卵磷脂和丝氨酸转化成磷脂酰丝氨酸。
  10. 根据权利要求9所述的制备磷脂酰丝氨酸的方法，其特征在于所述方法包括：将卵磷脂溶解于乙醚中，然后加入丝氨酸的水溶液，在氯化钙和缓冲液存在的条件下，用所述磷脂酶D突变体催化制备磷脂酰丝氨酸。
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