CN111051505B - 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 PDFInfo
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- CN111051505B CN111051505B CN201880037979.6A CN201880037979A CN111051505B CN 111051505 B CN111051505 B CN 111051505B CN 201880037979 A CN201880037979 A CN 201880037979A CN 111051505 B CN111051505 B CN 111051505B
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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
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 IDNO：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′-GGTAAGCTTTCAGAGCGAGCAGACGCCCC-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：
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
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 CaCl2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 CaCl2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
序列表
<110> 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
<120> 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
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1623
<212> DNA
<213> 肉桂链霉菌(Streptomyces cinnamoneus)
<400> 1
atgctccgcc accggctccg ccgtttacac cgtctgaccc gcagtgcggc ggtctcggcc 60
gtcgtcctgg ccgccctgcc cgcggctccg gccttcgcga gcagcccctc gcccgccccg 120
cacctggacg ccgtggagaa ggcgctgcgc gaggtctcac cggggctgga gggtgacgtc 180
tggcagcgca ccgacggcaa caagctggac gcctccgccg cggacccctc cgactggctg 240
ctgcagaccc ccggttgctg gggcgacgcc gcgtgcaagg agcgtcccgg caccgagcgc 300
ctgctcgcca aggtgacgga gaacatctcc aaggccaggc gcacggtgga catctccacg 360
ctcgcgccct tcccgaacgg tgcgttccag gacgcgatag ccgccggcct caaggcgtcg 420
gtcgcgtccg gcaacaagcc gaaggtccgc gtcctggtcg gcgccgcgcc ggtctaccac 480
atgaacgtac tgccctcgaa gtaccgggac gacctcaagg cccggctcgg caaggccgcc 540
gacgacatca cgctgaacgt cgcgtcgatg acgacgtcga agaccagctt ctcctggaac 600
cactccaagc tcctcgtcgt ggacggcgag tcggccgtca ccggtggcat caacagctgg 660
aaggacgact acgtcgacac ccagcacccg gtgaccgacg tggacctggc gctgaccggc 720
cccgccgcga gctccgccgg ccgctacctg gacacgctct ggacgtggac gtgccagaac 780
aagagcaaca tcgccagtgt gtggttcgcg gcctcgggcg gcgactgcat ggccacgatg 840
gagaaggacg cgaaccccag gcccgccggg cccacgggca acgtccccgt gatcgccgtg 900
ggcggcctcg gcgtcggcat caaggactcc gaccccgcct ggacgttccg cccgcagctg 960
ccctccgccc cggacaccaa gtgcgtcgtc ggcctgcccg acaagaccaa cgccgaccgt 1020
gactacgaca cggtcaaccc cgaggagagc gccctgcggg ccctggtggc cagcgccgac 1080
cgccagatcg tcatctccca gcaggacctg aacgccacct gcccgcccat cgcccgctac 1140
gacgtccgcc tctacgacat cctcgccgcc aagatggcgg ccggggtgaa ggtgcgcatc 1200
gtcgtcagcg accccgccaa ccgcggcgcg gtcggcagcg gcggctactc gcaaatcaag 1260
tccctggccg agatcagcga cacgctccgc aaccgtctcg ccctgctcaa gggcggcgac 1320
cagcagaagg ccaaggcggc catgtgctcc accctccagc tggggacctt ccgcagctcc 1380
gcgagcgcca cgtgggccga cgggcacccc tacgccctgc accacaagct ggtggcggtc 1440
gacagctccg ccttcaacat cggctccaag aacctctacc cctcgtggct gcaggacttc 1500
ggctacatcg tggagagccc ggaggccgcc aagcagcttg aggccaagct cctcgacccc 1560
gagtggaagt tctcgcagga gaccgcgacg gtcgaccacg cgcggggcgt ctgctcgctc 1620
tga 1623
<210> 2
<211> 540
<212> PRT
<213> 肉桂链霉菌(Streptomyces cinnamoneus)
<400> 2
Met Leu Arg His Arg Leu Arg Arg Leu His Arg Leu Thr Arg Ser Ala
1 5 10 15
Ala Val Ser Ala Val Val Leu Ala Ala Leu Pro Ala Ala Pro Ala Phe
20 25 30
Ala Ser Ser Pro Ser Pro Ala Pro His Leu Asp Ala Val Glu Lys Ala
35 40 45
Leu Arg Glu Val Ser Pro Gly Leu Glu Gly Asp Val Trp Gln Arg Thr
50 55 60
Asp Gly Asn Lys Leu Asp Ala Ser Ala Ala Asp Pro Ser Asp Trp Leu
65 70 75 80
Leu Gln Thr Pro Gly Cys Trp Gly Asp Ala Ala Cys Lys Glu Arg Pro
85 90 95
Gly Thr Glu Arg Leu Leu Ala Lys Val Thr Glu Asn Ile Ser Lys Ala
100 105 110
Arg Arg Thr Val Asp Ile Ser Thr Leu Ala Pro Phe Pro Asn Gly Ala
115 120 125
Phe Gln Asp Ala Ile Ala Ala Gly Leu Lys Ala Ser Val Ala Ser Gly
130 135 140
Asn Lys Pro Lys Val Arg Val Leu Val Gly Ala Ala Pro Val Tyr His
145 150 155 160
Met Asn Val Leu Pro Ser Lys Tyr Arg Asp Asp Leu Lys Ala Arg Leu
165 170 175
Gly Lys Ala Ala Asp Asp Ile Thr Leu Asn Val Ala Ser Met Thr Thr
180 185 190
Ser Lys Thr Ser Phe Ser Trp Asn His Ser Lys Leu Leu Val Val Asp
195 200 205
Gly Glu Ser Ala Val Thr Gly Gly Ile Asn Ser Trp Lys Asp Asp Tyr
210 215 220
Val Asp Thr Gln His Pro Val Thr Asp Val Asp Leu Ala Leu Thr Gly
225 230 235 240
Pro Ala Ala Ser Ser Ala Gly Arg Tyr Leu Asp Thr Leu Trp Thr Trp
245 250 255
Thr Cys Gln Asn Lys Ser Asn Ile Ala Ser Val Trp Phe Ala Ala Ser
260 265 270
Gly Gly Asp Cys Met Ala Thr Met Glu Lys Asp Ala Asn Pro Arg Pro
275 280 285
Ala Gly Pro Thr Gly Asn Val Pro Val Ile Ala Val Gly Gly Leu Gly
290 295 300
Val Gly Ile Lys Asp Ser Asp Pro Ala Trp Thr Phe Arg Pro Gln Leu
305 310 315 320
Pro Ser Ala Pro Asp Thr Lys Cys Val Val Gly Leu Pro Asp Lys Thr
325 330 335
Asn Ala Asp Arg Asp Tyr Asp Thr Val Asn Pro Glu Glu Ser Ala Leu
340 345 350
Arg Ala Leu Val Ala Ser Ala Asp Arg Gln Ile Val Ile Ser Gln Gln
355 360 365
Asp Leu Asn Ala Thr Cys Pro Pro Ile Ala Arg Tyr Asp Val Arg Leu
370 375 380
Tyr Asp Ile Leu Ala Ala Lys Met Ala Ala Gly Val Lys Val Arg Ile
385 390 395 400
Val Val Ser Asp Pro Ala Asn Arg Gly Ala Val Gly Ser Gly Gly Tyr
405 410 415
Ser Gln Ile Lys Ser Leu Ala Glu Ile Ser Asp Thr Leu Arg Asn Arg
420 425 430
Leu Ala Leu Leu Lys Gly Gly Asp Gln Gln Lys Ala Lys Ala Ala Met
435 440 445
Cys Ser Thr Leu Gln Leu Gly Thr Phe Arg Ser Ser Ala Ser Ala Thr
450 455 460
Trp Ala Asp Gly His Pro Tyr Ala Leu His His Lys Leu Val Ala Val
465 470 475 480
Asp Ser Ser Ala Phe Asn Ile Gly Ser Lys Asn Leu Tyr Pro Ser Trp
485 490 495
Leu Gln Asp Phe Gly Tyr Ile Val Glu Ser Pro Glu Ala Ala Lys Gln
500 505 510
Leu Glu Ala Lys Leu Leu Asp Pro Glu Trp Lys Phe Ser Gln Glu Thr
515 520 525
Ala Thr Val Asp His Ala Arg Gly Val Cys Ser Leu
530 535 540
Claims (7)
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位或第93位和第215位有突变，所述突变体的突变为K93A或G215S/K93A。
2.一种基因序列，其特征在于：所述基因序列编码权利要求1所述的磷脂酶D突变体。
3.一种生物材料，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2所述的基因序列。
4.权利要求1所述的磷脂酶D突变体在制备磷脂酰丝氨酸中的应用。
5.根据权利要求4所述的磷脂酶D突变体在制备磷脂酰丝氨酸中的应用，其特征在于：权利要求1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。
6.一种制备磷脂酰丝氨酸的方法，其特征在于：用权利要求1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。
7.根据权利要求6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Tris-HCl缓冲液存在的条件下，用权利要求1所述磷脂酶D突变体催化制备磷脂酰丝氨酸。
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