CN114480340A - 嗜盐胆碱激酶突变体及其应用 - Google Patents

嗜盐胆碱激酶突变体及其应用 Download PDF

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CN114480340A
CN114480340A CN202210067132.0A CN202210067132A CN114480340A CN 114480340 A CN114480340 A CN 114480340A CN 202210067132 A CN202210067132 A CN 202210067132A CN 114480340 A CN114480340 A CN 114480340A
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应汉杰
瞿明金
温庆仕
郑诚
王骏之
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Nanjing Tech University
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Abstract

本发明公开一种嗜盐胆碱激酶突变体，所述突变体由序列为SEQ ID NO:1的胆碱激酶突变而来。本发明的嗜盐胆碱激酶突变体M1‑M3相较于野生型的嗜盐胆碱激酶具有更高的催化活性和耐盐性，可以更好的应用于胞苷二磷酸胆碱的制备上。

Description

嗜盐胆碱激酶突变体及其应用
技术领域
本发明涉及基因工程领域，具体涉及一种嗜盐胆碱激酶突变体及其应用。
背景技术
在胞苷二磷酸胆碱的生产过程中，随着反应进行盐浓度逐渐升高，当盐浓度超过1200mM时，胆碱激酶CKI(NP_013234.1)活性降低了75％～80％(刘毅，王杰等.ATP供体模块驱动的高效多酶催化CDP胆碱生产[J]，2017)。为了适应反应的盐环境，我们需要寻找一种高效耐盐的胆碱激酶。
发明内容
发明目的：为解决现有技术中存在的技术问题，本发明基于酿酒酵母S288C的CKI基因，构建一种改造的嗜盐胆碱激酶，这种酶可以应用于高盐环境下胞苷二磷酸胆碱的生成。
为实现上述目的，本发明在序列为SEQ ID NO:1的胆碱激酶的基础上，进行定点突变，所述突变为如下突变中的至少一种：
(1)将第46位氨基酸异亮氨酸突变成氨基酸谷氨酸；
(2)将第59位氨基酸亮氨酸突变成氨基酸天冬氨酸；
(3)将第77位氨基酸丙氨酸突变成氨基酸天冬氨酸；
(4)将第344位氨基酸天冬酰胺突变成氨基酸谷氨酸；
(5)将第346位氨基酸脯氨酸突变成氨基酸谷氨酸；
(6)将第347位氨基酸丝氨酸突变成氨基酸天冬氨酸；
(7)将第509位氨基酸赖氨酸突变造成氨基酸天冬氨酸；
(8)将第558位氨基酸异亮氨酸突变成氨基酸谷氨酸。
在一种优选地实施方式中，本发明提供了一种嗜盐胆碱激酶突变体M1，所述突变体由序列为SEQ ID NO:1的胆碱激酶突变而来，所述突变为上述突变中的(1)、(3)和(5)，即：I46E，A77D和P346E。所述嗜盐胆碱激酶突变体M1的氨基酸序列为SEQ ID NO:2。
在另一种优选地实施方式中，本发明提供了一种嗜盐胆碱激酶突变体M2，所述突变体由序列为SEQ ID NO:1的胆碱激酶突变而来，所述突变包括(1)、(3)、(4)、(5)和(7)，即：I46E，A77D，N344E，P346E，K509D。所述嗜盐胆碱激酶突变体M2的氨基酸序列为SEQ IDNO:3。
在又一种优选地实施方式中，本发明提供了一种嗜盐胆碱激酶突变体M3，所述突变体由序列为SEQ ID NO:1的胆碱激酶突变而来，所述突变包括(1)-(8)，即：I46E、L59D、A77D、N344E、P346E、S347D、K509D和I558E。所述嗜盐胆碱激酶突变体M3的氨基酸序列为SEQID NO:4。
编码上述嗜盐胆碱激酶突变体的核苷酸序列也在本发明的保护范围之内。
具体地，编码SEQ ID NO.2的核苷酸序列如SEQ ID NO.5所示；编码SEQ ID NO.3的核苷酸序列如SEQ ID NO.6所示；编码SEQ ID NO.4的核苷酸序列如SEQ ID NO.7所示。
进一步地，本发明提出了包含上述编码嗜盐胆碱激酶突变体的核苷酸序列的表达载体，优选地，所述表达载体为pET-28a。
更进一步地，本发明提出了一种宿主细胞，所述宿主细胞包含上述表达载体。优选地，所述宿主细胞为大肠杆菌BL21(DE3)。
本发明进一步提出了上述嗜盐胆碱激酶突变体M1-M3在制备胞苷二磷酸胆碱上上的应用。
作为本发明的另一方面，本发明提出了一种提高嗜盐胆碱激酶的催化活性和耐盐性的方法，即通过对序列为SEQ ID NO:1的胆碱激酶进行上述突变。
有益效果：与现有技术相比，本发明的嗜盐胆碱激酶突变体M1-M3相较于野生型的嗜盐胆碱激酶具有更高的催化活性和耐盐性，可以更好的应用于胞苷二磷酸胆碱的制备上。
具体实施方式
下面结合具体实施例对本发明做进一步详细说明，实施例将有助于理解本发明，但是本发明的保护范围不限于下述的实施例。
实施例1嗜盐胆碱激酶突变体M1-M3的改造。
野生胆碱激酶WT由酿酒酵母S288C基因组经体外PCR技术扩增而来，其序列为SEQID NO:1所示。
将野生胆碱激酶WT进行改造，改造位点及改造方法如下：
将原酶的序列的第46位氨基酸I改造成氨基酸E；将第59位氨基酸L改造成氨基酸D；将第77位氨基酸A改造成氨基酸D；将第344位氨基酸N改造成氨基酸E；将第346位氨基酸P改造成氨基酸E；将第347位氨基酸S改造成氨基酸D；将第509位氨基酸K改造成氨基酸D；将第558位氨基酸I改造成氨基酸E。
由此获得三条氨基酸序列如下的嗜盐胆碱激酶：
胆碱激酶突变体M1的序列如SEQ ID NO:2所示，其进行如下突变：I46E，A77D和P346E。
胆碱激酶突变体M2的序列如SEQ ID NO:3所示，其进行如下突变：I46E，A77D，N344E，P346E，K509D。
胆碱激酶突变体M3的序列如SEQ ID NO:4所示，其进行如下突变：I46E、L59D、A77D、N344E、P346E、S347D、K509D和I558E。
按照上述胆碱激酶的氨基酸序列人工合成对应的碱基序列，其中，编码SEQ IDNO.2的核苷酸序列如SEQ ID NO.5所示；编码SEQ ID NO.3的核苷酸序列如SEQ ID NO.6所示；编码SEQ ID NO.4的核苷酸序列如SEQ ID NO.7所示，扩增构建表达载体，表达蛋白并制备含有胆碱激酶的菌体通透液。
具体地，按照上述磷酸胞苷胆碱酰转移酶突变体的氨基酸序列人工合成对应的碱基序列，并插入pET-28a表达载体，转化至大肠杆菌BL21(DE3)细胞中。含有突变体表达载体的大肠杆菌在TB培养基中培养至OD600＝0.6-0.8时，加入终浓度为0.2mM的IPTG以诱导蛋白表达。培养24h后收集菌体，用于催化反应。胆碱激酶的菌体通透液由向含有20g/L突变体菌体的水溶液中添加10g/L二甲苯，在30℃下以200rpm搅拌反应混合物获得。
实施例2嗜盐胆碱激酶突变体的效果检测。
将本专利中制备得到的嗜盐胆碱激酶M1-M3，以及合成的胆碱激酶WT的菌体通透液稀释到同一OD值(0.5)，制备标准反应混合物(50ml)：20g/LCMP.2Na，10g/L氯化胆碱，40g/L乙酰磷酸，8g/LMgCl2.6H2O，1g/LATP，10g/L二甲苯，20g/L胞苷酸激酶，20g/L核苷二磷酸激酶，20g/L磷酸胆碱胞苷基转移酶，20g/L乙酸激酶，20g/L上述菌体通透液，在32℃下以750rpm搅拌反应混合物1h并通过添加5mol/L碱溶液将反应pH维持在7.5，评估浓度范围为300至2000mM的四种醋酸盐溶液(醋酸钾、醋酸锂、醋酸钠、醋酸铵)对活性的影响。(催化活性定义为1分钟内，每g含酶湿菌体催化生成的CDPC的μmol量)
表1 WT和突变体的功能参数(M1–M3)
Figure BDA0003480683110000041
表2针对WT和M1-M3的CKI活性，醋酸盐的IC50值(mmol)汇总
Figure BDA0003480683110000042
实验结果如表1、表2所示，结果表明，在没有盐存在的情况下，所有三个突变体的活性与WT相似。然而，当醋酸盐浓度较高时，WT的活性明显受到抑制，三个突变体在所有条件下均表现出比WT更高的活性。温度升高时，M2和M3的活性降低程度高于野生型，表明他们性对不稳定，但是随着温度升高，他们在高盐浓度下仍保持着比WT更高的催化活性。由此可见，经过改造后的嗜盐胆碱激酶的催化活性更高，耐盐性也更好。
本发明提供了嗜盐胆碱激酶突变体的改造思路及方法，具体实现该技术方案的方法和途径很多，以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。本实施例中未明确的各组成部分均可用现有技术加以实现。
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Arg Gln Arg Ser Ser Gln Arg Leu Ile Arg Thr Glu Ser Ile Glu Ser
35 40 45
Asp Val Ser Asn Ile Thr Asp Asp Asp Asp Leu Arg Ala Val Asn Glu
50 55 60
Gly Val Ala Gly Val Gln Leu Asp Val Asp Glu Thr Ala Asn Lys Gly
65 70 75 80
Pro Arg Arg Ala Ser Ala Thr Asp Val Thr Asp Ser Leu Gly Ser Thr
85 90 95
Ser Ser Glu Tyr Ile Glu Ile Pro Phe Val Lys Glu Thr Leu Asp Ala
100 105 110
Ser Leu Pro Ser Asp Tyr Leu Lys Gln Asp Ile Leu Asn Leu Ile Gln
115 120 125
Ser Leu Lys Ile Ser Lys Trp Tyr Asn Asn Lys Lys Ile Gln Pro Val
130 135 140
Ala Gln Asp Met Asn Leu Val Lys Ile Ser Gly Ala Met Thr Asn Ala
145 150 155 160
Ile Phe Lys Val Glu Tyr Pro Lys Leu Pro Ser Leu Leu Leu Arg Ile
165 170 175
Tyr Gly Pro Asn Ile Asp Asn Ile Ile Asp Arg Glu Tyr Glu Leu Gln
180 185 190
Ile Leu Ala Arg Leu Ser Leu Lys Asn Ile Gly Pro Ser Leu Tyr Gly
195 200 205
Cys Phe Val Asn Gly Arg Phe Glu Gln Phe Leu Glu Asn Ser Lys Thr
210 215 220
Leu Thr Lys Asp Asp Ile Arg Asn Trp Lys Asn Ser Gln Arg Ile Ala
225 230 235 240
Arg Arg Met Lys Glu Leu His Val Gly Val Pro Leu Leu Ser Ser Glu
245 250 255
Arg Lys Asn Gly Ser Ala Cys Trp Gln Lys Ile Asn Gln Trp Leu Arg
260 265 270
Thr Ile Glu Lys Val Asp Gln Trp Val Gly Asp Pro Lys Asn Ile Glu
275 280 285
Asn Ser Leu Leu Cys Glu Asn Trp Ser Lys Phe Met Asp Ile Val Asp
290 295 300
Arg Tyr His Lys Trp Leu Ile Ser Gln Glu Gln Gly Ile Glu Gln Val
305 310 315 320
Asn Lys Asn Leu Ile Phe Cys His Asn Asp Ala Gln Tyr Gly Asn Leu
325 330 335
Glu Phe Glu Ala Pro Val Met Asn Thr Pro Ser Leu Tyr Thr Ala Pro
340 345 350
Ser Ser Thr Ser Leu Thr Ser Gln Ser Ser Ser Leu Phe Pro Ser Ser
355 360 365
Ser Asn Val Ile Val Asp Asp Ile Ile Asn Pro Pro Lys Gln Glu Gln
370 375 380
Ser Gln Asp Ser Lys Leu Val Val Ile Asp Phe Glu Tyr Ala Gly Ala
385 390 395 400
Asn Pro Ala Ala Tyr Asp Leu Ala Asn His Leu Ser Glu Trp Met Tyr
405 410 415
Asp Tyr Asn Asn Ala Lys Ala Pro His Gln Cys His Ala Asp Arg Tyr
420 425 430
Pro Asp Lys Glu Gln Val Leu Asn Phe Leu Tyr Ser Tyr Val Ser His
435 440 445
Leu Arg Gly Gly Ala Lys Glu Pro Ile Asp Glu Glu Val Gln Arg Leu
450 455 460
Tyr Lys Ser Ile Ile Gln Trp Arg Pro Thr Val Gln Leu Phe Trp Ser
465 470 475 480
Leu Trp Ala Ile Leu Gln Ser Gly Lys Leu Glu Lys Lys Glu Ala Ser
485 490 495
Thr Ala Asp Thr Arg Glu Glu Ile Gly Pro Asn Gly Lys Lys Tyr Ile
500 505 510
Ile Lys Thr Glu Pro Glu Ser Pro Glu Glu Asp Phe Val Glu Asn Asp
515 520 525
Asp Glu Pro Glu Ala Gly Val Ser Ile Asp Thr Phe Asp Tyr Met Ala
530 535 540
Tyr Gly Arg Asp Lys Ile Ala Val Phe Trp Gly Asp Leu Ile Gly Leu
545 550 555 560
Gly Ile Ile Thr Glu Glu Glu Cys Lys Asn Phe Ser Ser Phe Lys Phe
565 570 575
Leu Asp Thr Ser Tyr Leu
580
<210> 4
<211> 582
<212> PRT
<213> 胆碱激酶突变体M3氨基酸序列(Artificial Sequence)
<400> 4
Met Val Gln Glu Ser Arg Pro Gly Ser Val Arg Ser Tyr Ser Val Gly
1 5 10 15
Tyr Gln Ala Arg Ser Arg Ser Ser Ser Gln Arg Arg His Ser Leu Thr
20 25 30
Arg Gln Arg Ser Ser Gln Arg Leu Ile Arg Thr Glu Ser Ile Glu Ser
35 40 45
Asp Val Ser Asn Ile Thr Asp Asp Asp Asp Leu Arg Ala Val Asn Glu
50 55 60
Gly Val Ala Gly Val Gln Leu Asp Val Asp Glu Thr Ala Asn Lys Gly
65 70 75 80
Pro Arg Arg Ala Ser Ala Thr Asp Val Thr Asp Ser Leu Gly Ser Thr
85 90 95
Ser Ser Glu Tyr Ile Glu Ile Pro Phe Val Lys Glu Thr Leu Asp Ala
100 105 110
Ser Leu Pro Ser Asp Tyr Leu Lys Gln Asp Ile Leu Asn Leu Ile Gln
115 120 125
Ser Leu Lys Ile Ser Lys Trp Tyr Asn Asn Lys Lys Ile Gln Pro Val
130 135 140
Ala Gln Asp Met Asn Leu Val Lys Ile Ser Gly Ala Met Thr Asn Ala
145 150 155 160
Ile Phe Lys Val Glu Tyr Pro Lys Leu Pro Ser Leu Leu Leu Arg Ile
165 170 175
Tyr Gly Pro Asn Ile Asp Asn Ile Ile Asp Arg Glu Tyr Glu Leu Gln
180 185 190
Ile Leu Ala Arg Leu Ser Leu Lys Asn Ile Gly Pro Ser Leu Tyr Gly
195 200 205
Cys Phe Val Asn Gly Arg Phe Glu Gln Phe Leu Glu Asn Ser Lys Thr
210 215 220
Leu Thr Lys Asp Asp Ile Arg Asn Trp Lys Asn Ser Gln Arg Ile Ala
225 230 235 240
Arg Arg Met Lys Glu Leu His Val Gly Val Pro Leu Leu Ser Ser Glu
245 250 255
Arg Lys Asn Gly Ser Ala Cys Trp Gln Lys Ile Asn Gln Trp Leu Arg
260 265 270
Thr Ile Glu Lys Val Asp Gln Trp Val Gly Asp Pro Lys Asn Ile Glu
275 280 285
Asn Ser Leu Leu Cys Glu Asn Trp Ser Lys Phe Met Asp Ile Val Asp
290 295 300
Arg Tyr His Lys Trp Leu Ile Ser Gln Glu Gln Gly Ile Glu Gln Val
305 310 315 320
Asn Lys Asn Leu Ile Phe Cys His Asn Asp Ala Gln Tyr Gly Asn Leu
325 330 335
Glu Phe Glu Asp Pro Val Met Asn Thr Pro Ser Leu Tyr Thr Ala Pro
340 345 350
Ser Ser Thr Ser Leu Thr Ser Gln Ser Ser Ser Leu Phe Pro Ser Ser
355 360 365
Ser Asn Val Ile Val Asp Asp Ile Ile Asn Pro Pro Lys Gln Glu Gln
370 375 380
Ser Gln Asp Ser Lys Leu Val Val Ile Asp Phe Glu Tyr Ala Gly Ala
385 390 395 400
Asn Pro Ala Ala Tyr Asp Leu Ala Asn His Leu Ser Glu Trp Met Tyr
405 410 415
Asp Tyr Asn Asn Ala Lys Ala Pro His Gln Cys His Ala Asp Arg Tyr
420 425 430
Pro Asp Lys Glu Gln Val Leu Asn Phe Leu Tyr Ser Tyr Val Ser His
435 440 445
Leu Arg Gly Gly Ala Lys Glu Pro Ile Asp Glu Glu Val Gln Arg Leu
450 455 460
Tyr Lys Ser Ile Ile Gln Trp Arg Pro Thr Val Gln Leu Phe Trp Ser
465 470 475 480
Leu Trp Ala Ile Leu Gln Ser Gly Lys Leu Glu Lys Lys Glu Ala Ser
485 490 495
Thr Ala Asp Thr Arg Glu Glu Ile Gly Pro Asn Gly Lys Lys Tyr Ile
500 505 510
Ile Lys Thr Glu Pro Glu Ser Pro Glu Glu Asp Phe Val Glu Asn Asp
515 520 525
Asp Glu Pro Glu Ala Gly Val Ser Ile Asp Thr Phe Asp Tyr Met Ala
530 535 540
Tyr Gly Glu Asp Lys Ile Ala Val Phe Trp Gly Asp Leu Ile Gly Leu
545 550 555 560
Gly Ile Ile Thr Glu Glu Glu Cys Lys Asn Phe Ser Ser Phe Lys Phe
565 570 575
Leu Asp Thr Ser Tyr Leu
580
<210> 5
<211> 1746
<212> DNA
<213> 胆碱激酶突变体M1核苷酸序列(Artificial Sequence)
<400> 5
atggtgcagg aaagccgccc gggcagcgtg cgcagctata gcgtgggcta tcaggcgcgc 60
agccgcagca gcagccagcg ccgccatagc ctgacccgcc agcgcagcag ccagcgcctg 120
attcgcaccg aaagcattga aagcgatgtg agcaacatta ccgatgatga tgatctgcgc 180
gcggtgaacg aaggcgtggc gggcgtgcag ctggatgtgg atgaaaccgc gaacaaaggc 240
ccgcgccgcg cgagcgcgac cgatgtgacc gatagcctgg gcagcaccag cagcgaatat 300
attgaaattc cgtttgtgaa agaaaccctg gatgcgagcc tgccgagcga ttatctgaaa 360
caggatattc tgaacctgat tcagagcctg aaaattagca aatggtataa caacaaaaaa 420
attcagccgg tggcgcagga tatgaacctg gtgaaaatta gcggcgcgat gaccaacgcg 480
atttttaaag tggaatatcc gaaactgccg agcctgctgc tgcgcattta tggcccgaac 540
attgataaca ttattgatcg cgaatatgaa ctgcagattc tggcgcgcct gagcctgaaa 600
aacattggcc cgagcctgta tggctgcttt gtgaacggcc gctttgaaca gtttctggaa 660
aacagcaaaa ccctgaccaa agatgatatt cgcaactgga aaaacagcca gcgcattgcg 720
cgccgcatga aagaactgca tgtgggcgtg ccgctgctga gcagcgaacg caaaaacggc 780
agcgcgtgct ggcagaaaat taaccagtgg ctgcgcacca ttgaaaaagt ggatcagtgg 840
gtgggcgatc cgaaaaacat tgaaaacagc ctgctgtgcg aaaactggag caaatttatg 900
gatattgtgg atcgctatca taaatggctg attagccagg aacagggcat tgaacaggtg 960
aacaaaaacc tgattttttg ccataacgat gcgcagtatg gcaacctgct gtttgaagcg 1020
ccggtgatga acaccccgag cctgtatacc gcgccgagca gcaccagcct gaccagccag 1080
agcagcagcc tgtttccgag cagcagcaac gtgattgtgg atgatattat taacccgccg 1140
aaacaggaac agagccagga tagcaaactg gtggtgattg attttgaata tgcgggcgcg 1200
aacccggcgg cgtatgatct ggcgaaccat ctgagcgaat ggatgtatga ttataacaac 1260
gcgaaagcgc cgcatcagtg ccatgcggat cgctatccgg ataaagaaca ggtgctgaac 1320
tttctgtata gctatgtgag ccatctgcgc ggcggcgcga aagaaccgat tgatgaagaa 1380
gtgcagcgcc tgtataaaag cattattcag tggcgcccga ccgtgcagct gttttggagc 1440
ctgtgggcga ttctgcagag cggcaaactg gaaaaaaaag aagcgagcac cgcgattacc 1500
cgcgaagaaa ttggcccgaa cggcaaaaaa tatattatta aaaccgaacc ggaaagcccg 1560
gaagaagatt ttgtggaaaa cgatgatgaa ccggaagcgg gcgtgagcat tgataccttt 1620
gattatatgg cgtatggccg cgataaaatt gcggtgtttt ggggcgatct gattggcctg 1680
ggcattatta ccgaagaaga atgcaaaaac tttagcagct ttaaatttct ggataccagc 1740
tatctg 1746
<210> 6
<211> 1746
<212> DNA
<213> 胆碱激酶突变体M2核苷酸序列(Artificial Sequence)
<400> 6
atggtgcagg aaagccgccc gggcagcgtg cgcagctata gcgtgggcta tcaggcgcgc 60
agccgcagca gcagccagcg ccgccatagc ctgacccgcc agcgcagcag ccagcgcctg 120
attcgcaccg aaagcattga aagcgatgtg agcaacatta ccgatgatga tgatctgcgc 180
gcggtgaacg aaggcgtggc gggcgtgcag ctggatgtgg atgaaaccgc gaacaaaggc 240
ccgcgccgcg cgagcgcgac cgatgtgacc gatagcctgg gcagcaccag cagcgaatat 300
attgaaattc cgtttgtgaa agaaaccctg gatgcgagcc tgccgagcga ttatctgaaa 360
caggatattc tgaacctgat tcagagcctg aaaattagca aatggtataa caacaaaaaa 420
attcagccgg tggcgcagga tatgaacctg gtgaaaatta gcggcgcgat gaccaacgcg 480
atttttaaag tggaatatcc gaaactgccg agcctgctgc tgcgcattta tggcccgaac 540
attgataaca ttattgatcg cgaatatgaa ctgcagattc tggcgcgcct gagcctgaaa 600
aacattggcc cgagcctgta tggctgcttt gtgaacggcc gctttgaaca gtttctggaa 660
aacagcaaaa ccctgaccaa agatgatatt cgcaactgga aaaacagcca gcgcattgcg 720
cgccgcatga aagaactgca tgtgggcgtg ccgctgctga gcagcgaacg caaaaacggc 780
agcgcgtgct ggcagaaaat taaccagtgg ctgcgcacca ttgaaaaagt ggatcagtgg 840
gtgggcgatc cgaaaaacat tgaaaacagc ctgctgtgcg aaaactggag caaatttatg 900
gatattgtgg atcgctatca taaatggctg attagccagg aacagggcat tgaacaggtg 960
aacaaaaacc tgattttttg ccataacgat gcgcagtatg gcaacctgga atttgaagcg 1020
ccggtgatga acaccccgag cctgtatacc gcgccgagca gcaccagcct gaccagccag 1080
agcagcagcc tgtttccgag cagcagcaac gtgattgtgg atgatattat taacccgccg 1140
aaacaggaac agagccagga tagcaaactg gtggtgattg attttgaata tgcgggcgcg 1200
aacccggcgg cgtatgatct ggcgaaccat ctgagcgaat ggatgtatga ttataacaac 1260
gcgaaagcgc cgcatcagtg ccatgcggat cgctatccgg ataaagaaca ggtgctgaac 1320
tttctgtata gctatgtgag ccatctgcgc ggcggcgcga aagaaccgat tgatgaagaa 1380
gtgcagcgcc tgtataaaag cattattcag tggcgcccga ccgtgcagct gttttggagc 1440
ctgtgggcga ttctgcagag cggcaaactg gaaaaaaaag aagcgagcac cgcggatacc 1500
cgcgaagaaa ttggcccgaa cggcaaaaaa tatattatta aaaccgaacc ggaaagcccg 1560
gaagaagatt ttgtggaaaa cgatgatgaa ccggaagcgg gcgtgagcat tgataccttt 1620
gattatatgg cgtatggccg cgataaaatt gcggtgtttt ggggcgatct gattggcctg 1680
ggcattatta ccgaagaaga atgcaaaaac tttagcagct ttaaatttct ggataccagc 1740
tatctg 1746
<210> 7
<211> 1746
<212> DNA
<213> 胆碱激酶突变体M3核苷酸序列(Artificial Sequence)
<400> 7
atggtgcagg aaagccgccc gggcagcgtg cgcagctata gcgtgggcta tcaggcgcgc 60
agccgcagca gcagccagcg ccgccatagc ctgacccgcc agcgcagcag ccagcgcctg 120
attcgcaccg aaagcattga aagcgatgtg agcaacatta ccgatgatga tgatctgcgc 180
gcggtgaacg aaggcgtggc gggcgtgcag ctggatgtgg atgaaaccgc gaacaaaggc 240
ccgcgccgcg cgagcgcgac cgatgtgacc gatagcctgg gcagcaccag cagcgaatat 300
attgaaattc cgtttgtgaa agaaaccctg gatgcgagcc tgccgagcga ttatctgaaa 360
caggatattc tgaacctgat tcagagcctg aaaattagca aatggtataa caacaaaaaa 420
attcagccgg tggcgcagga tatgaacctg gtgaaaatta gcggcgcgat gaccaacgcg 480
atttttaaag tggaatatcc gaaactgccg agcctgctgc tgcgcattta tggcccgaac 540
attgataaca ttattgatcg cgaatatgaa ctgcagattc tggcgcgcct gagcctgaaa 600
aacattggcc cgagcctgta tggctgcttt gtgaacggcc gctttgaaca gtttctggaa 660
aacagcaaaa ccctgaccaa agatgatatt cgcaactgga aaaacagcca gcgcattgcg 720
cgccgcatga aagaactgca tgtgggcgtg ccgctgctga gcagcgaacg caaaaacggc 780
agcgcgtgct ggcagaaaat taaccagtgg ctgcgcacca ttgaaaaagt ggatcagtgg 840
gtgggcgatc cgaaaaacat tgaaaacagc ctgctgtgcg aaaactggag caaatttatg 900
gatattgtgg atcgctatca taaatggctg attagccagg aacagggcat tgaacaggtg 960
aacaaaaacc tgattttttg ccataacgat gcgcagtatg gcaacctgga atttgaagat 1020
ccggtgatga acaccccgag cctgtatacc gcgccgagca gcaccagcct gaccagccag 1080
agcagcagcc tgtttccgag cagcagcaac gtgattgtgg atgatattat taacccgccg 1140
aaacaggaac agagccagga tagcaaactg gtggtgattg attttgaata tgcgggcgcg 1200
aacccggcgg cgtatgatct ggcgaaccat ctgagcgaat ggatgtatga ttataacaac 1260
gcgaaagcgc cgcatcagtg ccatgcggat cgctatccgg ataaagaaca ggtgctgaac 1320
tttctgtata gctatgtgag ccatctgcgc ggcggcgcga aagaaccgat tgatgaagaa 1380
gtgcagcgcc tgtataaaag cattattcag tggcgcccga ccgtgcagct gttttggagc 1440
ctgtgggcga ttctgcagag cggcaaactg gaaaaaaaag aagcgagcac cgcggatacc 1500
cgcgaagaaa ttggcccgaa cggcaaaaaa tatattatta aaaccgaacc ggaaagcccg 1560
gaagaagatt ttgtggaaaa cgatgatgaa ccggaagcgg gcgtgagcat tgataccttt 1620
gattatatgg cgtatggcga agataaaatt gcggtgtttt ggggcgatct gattggcctg 1680
ggcattatta ccgaagaaga atgcaaaaac tttagcagct ttaaatttct ggataccagc 1740
tatctg 1746

Claims (8)

1.一种嗜盐胆碱激酶突变体，其特征在于，所述突变体由序列为SEQ ID NO:1的胆碱激酶突变而来，其中，所述突变为如下突变中的至少一种：
（1）将第46位异亮氨酸突变成谷氨酸；
（2）将第59位亮氨酸突变成天冬氨酸；
（3）将第77位丙氨酸突变成天冬氨酸；
（4）将第344位天冬酰胺突变成谷氨酸；
（5）将第346位脯氨酸突变成谷氨酸；
（6）将第347位丝氨酸突变成天冬氨酸；
（7）将第509位赖氨酸突变造成天冬氨酸；
（8）将第558位异亮氨酸突变成谷氨酸。
2.根据权利要求1所述的嗜盐胆碱激酶突变体，其特征在于，所述突变包括（1）-（8），得到的嗜盐胆碱激酶突变体命名为M3，所述嗜盐胆碱激酶突变体M3的氨基酸序列为SEQ IDNO:4。
3.根据权利要求1所述的嗜盐胆碱激酶突变体，其特征在于，所述突变包括（1）、（3）、（4）、（5）和（7），得到的嗜盐胆碱激酶突变体命名为M2，所述嗜盐胆碱激酶突变体M2的氨基酸序列为SEQ ID NO:3。
4.根据权利要求1所述的嗜盐胆碱激酶突变体，其特征在于，所述突变包括（1）、（3）和（5），得到的嗜盐胆碱激酶突变体命名为M1，所述嗜盐胆碱激酶突变体M1的氨基酸序列为SEQ ID NO:2。
5.编码权利要求1-4任一项所述的嗜盐胆碱激酶突变体的核苷酸序列。
6.一种表达载体，其特征在于，所述表达载体包含权利要求5所述的核苷酸序列中的任意一种。
7.一种宿主细胞，其特征在于，所述宿主细胞包含权利要求6所述的表达载体。
8.权利要求1-4任一项所述的嗜盐胆碱激酶突变体在制备胞苷二磷酸胆碱上的应用。
CN202210067132.0A 2022-01-20 2022-01-20 嗜盐胆碱激酶突变体及其应用 Pending CN114480340A (zh)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116240193A (zh) * 2023-05-06 2023-06-09 北京量维生物科技研究院有限公司 胆碱激酶突变体及其在胞磷胆碱生产中的应用

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CN104774799A (zh) * 2015-04-17 2015-07-15 南京工业大学 一株表达胆碱激酶及磷酰胆碱胞苷转移酶的基因工程菌及其构建方法与应用
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116240193A (zh) * 2023-05-06 2023-06-09 北京量维生物科技研究院有限公司 胆碱激酶突变体及其在胞磷胆碱生产中的应用

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