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CN114525266B - 一种来源于南极细菌的磷脂酶d突变体及其应用 - Google Patents

一种来源于南极细菌的磷脂酶d突变体及其应用 Download PDF

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CN114525266B
CN114525266B CN202210160732.1A CN202210160732A CN114525266B CN 114525266 B CN114525266 B CN 114525266B CN 202210160732 A CN202210160732 A CN 202210160732A CN 114525266 B CN114525266 B CN 114525266B
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王方华
毛雪静
王永华
李力浪
崔瑞国
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Abstract

本发明公开了一种来源于南极细菌的磷脂酶D突变体及其应用，其氨基酸序列为SEQ ID NO:4或SEQ ID NO:6或SEQ ID NO:8。本发明所构建的突变体S148C‑T206C和D225C‑A328C相比于野生型酶蛋白，在保持其最适反应温度条件不变的情况下，将其在最适反应温度条件下的半衰期提高到原来的1.4和2.0倍。S148C‑T206C和D225C‑A328C叠加突变后，其在最适反应温度条件下的半衰期提高到原来的3.2倍，且酶活力提高到原来的1.4倍。本发明获得的MsPLD突变体可适用于磷脂改性，生产磷脂酸及各类天然稀有磷脂和非天然磷脂化合物，应用于生物、食品、医药、日化等领域。

Description

一种来源于南极细菌的磷脂酶D突变体及其应用
技术领域
本发明属于酶的基因工程技术领域，具体地说涉及利用分子生物学技术获得一种催化稳定性提高的磷脂酶D突变体及其大肠杆菌重组表达制备方法和应用。
背景技术
酶蛋白半衰期是评价酶性能和应用潜力的重要指标，筛选和改造获得温度稳定性较高的酶蛋白成为研究者所关注的焦点。通常，分子改造提高酶蛋白稳定性主要通过随机突变和基于结构进行理性设计两种方法来实现。随机突变因涉及到突变体建库，给后续筛选带来较大的工作量，且筛选效率较低。在酶蛋白结构中理性设计引入二硫键是被公认的可以提高酶蛋白稳定性的方法，但该方法需要了解酶蛋白结构信息，且对于二硫键引入位点的选择上需要综合考虑引入位点之间的空间距离、角度等因素。基于结构的理性设计手段已经被成功用于多种酶蛋白的改造以提高其热稳定性研究中，例如：ɑ-淀粉酶、纤维素酶、几丁质酶、脂肪酶、内切葡聚糖酶等。相比于随机突变，虽然大大节省了突变体构建筛选的时间和成本，但仍无法保证所预测设计的位点与预期结果相符。
磷脂酶D(PLD)作为一种重要的工具酶，被用于磷脂改性，在食品、医药和日化行业中具有极大的应用价值。在应用过程中，酶蛋白催化活力和催化反应稳定性是影响用酶成本的关键因素，开发高活力且高催化稳定性的磷脂酶D具有重要意义。现已报道磷脂酶D的酶活力都普遍较低，且以往对于该类酶的研究主要集中在酶蛋白底物选择性改造上，目前尚无基于二硫键的分子改造技术提升该类酶催化活性和稳定性的报道。
发明内容
本发明在前期对来源于南极细菌的PLD酶学性质及蛋白结构解析的基础上，尝试基于结构的酶蛋白二硫键引入策略来提升该酶在最适反应温度条件下的催化稳定性，以延长酶反应条件下的半衰期。通过借助分子动力学模拟和二硫键预测手段，在酶蛋白结构中理性设计引入了18个二硫键。在此基础上，开展实验验证，最终在筛选效果较好的两个突变体基础上，通过进一步叠加突变，进一步提升酶蛋白的温度稳定性，为该酶的下游应用奠定基础。
本发明的技术方案如下：
一种来源于南极细菌的磷脂酶D突变体，是在氨基酸序列为SEQ ID NO:1的亲本序列基础上突变亲本序列中第148和206、225和328相应位点突变为半胱氨酸，从而形成二硫键而获得，其氨基酸序列为SEQID NO:4或SEQID NO:6或SEQID NO:8。
一种编码上述磷脂酶D突变体的基因，其核苷酸序列为SEQID NO:3或SEQID NO:5或SEQID NO:7。
一种含上述基因的重组基因工程菌。
所述重组基因工程菌的制备方法：将所述磷脂酶D突变体的基因克隆到表达载体pET-21a、pET28a或pET32a上，转化大肠杆菌感受态细胞，获得重组基因工程菌。
所述磷脂酶D突变体可用于催化合成PA、PS等天然稀有磷脂和非天然磷脂化合物。
与现有技术相比，本发明具有如下有益效果：
本发明所构建的突变体S148C-T206C和D225C-A328C相比于野生型酶蛋白，在保持其最适反应温度条件不变的情况下，将其在最适反应温度条件下的半衰期提高到原来的1.4和2.0倍。S148C-T206C和D225C-A328C叠加突变后，其在最适反应温度条件下的半衰期提高到原来的3.2倍，且酶活力提高到原来的1.4倍。本发明获得的MsPLD突变体可适用于磷脂改性，生产磷脂酸及各类天然稀有磷脂和非天然磷脂化合物，应用于生物、食品、医药、日化、农业、工业等领域。
附图说明
图1为野生型MsPLD表达及纯化效果SDS-PAGE电泳图。1：破碎后总菌蛋白；2：破碎总菌蛋白离心后上清液；3：破碎总菌蛋白离心后沉淀；4:破碎上清液过Ni2+亲和层析柱后，200mM咪唑浓度洗脱蛋白。
图2为MsPLD野生型及构建突变体蛋白经脱盐柱纯化后SDS-PAGE检测结果图；1:MsPLD；2:N60C-R283C；3:S62C-V111C；4:E74C-A155C；5:K101C-S162C；6:N145C-T205C；7:S147C-T205C；8:D224C-A327C；9:S449C-V551C；10:S486C-I549C；11:S147C-T205C/D224C-A327C。
具体实施方式
下面结合具体实施例对本发明作进一步具体详细描述，但本发明的实施方式不限于此，对于未特别注明的工艺参数，可参照常规技术进行。
实施例1：野生型MsPLD及突变体重组表达载体及表达菌株的构建
(1)参照南极细菌(Moritella sp.JT01)脂酶D完整蛋白序列(GenBank:KXO13223.1基础上去除N端28个氨基酸获得(命名为MsPLD)，在编码该蛋白序列的基因上游引入NdeⅠ酶切位点及编码6个His蛋白标签，下游引入Xho I，其重组表达完整氨基酸序列如SEQ IDNO:1所示。按照大肠杆菌表达密码子偏好性对编码该蛋白序列进行密码子优化，优化后的基因序列如SEQ ID NO:2所示；
(2)将(1)所合成的MsPLD基因用限制性内切酶NdeⅠ和XhoI分别对纯化的基因片段和质粒pET21a进行双酶切消化，连接，转化至大肠杆菌E.coliTop10感受态细胞，涂布于LB(含100μg/mL氨苄青霉素)平板。挑取阳性克隆通过菌落PCR及基因测序。采用质粒提取试剂盒提取获得野生型MsPLD的pET 21a-MsPLD重组质粒。
(3)采用重叠PCR方法构建本发明所述突变体，反应条件如下：
反应条件1：
Figure BDA0003513914550000031
其中突变体构建所用上游引物和下游引物序列为：
表1.构建突变体所用的引物列表
Figure BDA0003513914550000032
Figure BDA0003513914550000041
Figure BDA0003513914550000051
注：下划线序列为突变位点序列
PCR扩增条件：98℃，3min；98℃，15s；60℃，25s；72℃，130s；30个循环；72℃，2min。扩增产物使用DpnⅠ酶切消化模板质粒，消化体系如下：
Figure BDA0003513914550000061
将DpnⅠ酶切消化体系置于37℃条件下2h。将消化产物转化至E.coliTop10感受态细胞。涂布于LB(含100μg/mL氨苄青霉素)平板。挑取阳性克隆通过NdeⅠ和XhoI双酶切鉴定及基因测序，获得pET21a-MsPLD-突变体质粒。
(4)分别将(2)、(3)所得的重组质粒转化到大肠杆菌感受态细胞，挑选阳性克隆并测序验证，即获得重组pET21a-MsPLD野生型及各突变体的大肠杆菌重组表达菌株。
实施例2：野生型MsPLD及其突变体重组表达菌株发酵及重组蛋白纯化(1)将实施例1中测序正确的单克隆划线至含有100μg/mL氨苄青霉素的LB平板上，37℃培养12～16h，待菌落生长至合适大小时挑取单克隆于5mL含有100μg/mL氨苄青霉素的LB培养基(NaCl10g/L，蛋白胨10g/L，酵母提取物5g/L，pH7.2～7.4)中，在37℃、220r/min条件下培养至OD600为0.8～0.9之间，按2％的接种量接种至含有50mL LB培养基的500mL锥形瓶中，在37℃、220r/min条件下培养至OD600为0.8～0.9之间作为种子液；(2)将(1)中所述种子液按5％的接种量接种到500mL LB培养基中，于37℃，220r/min摇瓶培养至OD600为0.8～0.9，降温至20℃，再加入终浓度为0.2mM的IPTG，于16℃，200r/min条件下诱导培养20h；
(3)将(2)中所得发酵液离心(8000r/min,20min)，收集菌体沉淀，用Tris-HCl缓冲液(pH 8.0)重悬并超声破碎细胞，将细胞破碎液离心(10000r/min,30min)，取上清，即为制备得到的磷脂酶MsPLD或突变体粗酶液；
(4)将(3)中所得磷脂酶粗酶液用镍柱亲和层析柱纯化，流速4mL/min，最后用含10-500mM咪唑的Tris-HCl缓冲液(pH 8.0)梯度洗脱，目标蛋白在200mM咪唑浓度处被洗脱下来。将洗脱下来的目的蛋白过G-25脱盐柱后洗脱得到目的蛋白(见附图1，2)。
实施例3：磷脂酶D MsPLD野生型及其突变体最适反应温度条件下半衰期及催化动力学参数的测定
采用酶联比色法测定MsPLD及其突变体在35℃条件下的酶活力。反应体系(100μL)包含0.1M Tris-HCl(pH8.0)，5mM soyPC，15mM SDS，15mM TritonX-100和10μL纯化后的酶溶液，于各温度下反应5min，加热终止反应，待溶液冷却后，加入含10U/mL胆碱氧化酶、1U/mL过氧化物酶、5mM 4-氨基安替林和7mM苯酚的显色液，30℃孵育30min，最后添加1％TrionX-100终止显色反应。于500nm处测定吸光值。测定在35℃孵育不同时间(0～60min)下的MsPLD的残余酶活，实验重复三次，实验结果用相对酶活力表示，测定的最大酶活力定为100％。结果表明MsPLD的热稳定性较差，根据其失活曲线，能计算出其在35℃下的半衰期为117min，突变体S148C-T206C的半衰期为168min，突变体D225C-A328C的半衰期为245min，突变体S148C-T206C/D225C-A328C的半衰期为369min，分别为野生型的1.4、2.0、3.2倍，酶突变体的温度稳定性大幅提高。
以大豆磷脂酰胆碱为底物，测定野生型及酶突变体的反应动力学参数，S148C-T206C和D225C-A328C叠加突变体的催化效率提高到原来的1.4倍。
表2.设计突变体重组表达情况及最适反应温度条件下半衰期和催化效率测定结果
Figure BDA0003513914550000071
注：表达情况栏中：“+”代表能够重组表达，“-”代表无法实现重组表达；t1/2栏中，“-”代表表达蛋白无活性。催化效率栏中，“-”代表未测定。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
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<110> 华南理工大学
<120> 一种来源于南极细菌的磷脂酶D突变体及其应用
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Asn Ser Glu Phe Val Tyr Asn Tyr Leu Leu Asn Val Leu His Lys Ala
450 455 460
Pro Tyr Tyr Leu Glu Arg Asn Tyr Ala Lys Thr Phe Leu Asp Lys Asn
465 470 475 480
Leu His Ile Asn Phe Ile Ser Ile Asn Gly Arg Glu Thr Asn Asn Met
485 490 495
Ser His Asn Lys Leu Trp Ile Val Asp Asp Lys Val Phe Tyr Val Gly
500 505 510
Ser His Asn Ile Tyr Pro Ser Ser Leu Gln Gln Phe Gly Val Ile Val
515 520 525
Asp Asp Lys Asp Ala Thr Ala Gln Leu Glu Lys Gln Leu Trp Thr Pro
530 535 540
Met Trp Lys Asn Ser Ile His Val Pro Ile Asn Asn Ser
545 550 555
<210> 2
<211> 1674
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgcaccacc accaccacca tagcaccaac gaactggatg tgaacgatat ctatgatcat 60
ctgaacgaaa aatactctca gttcaacgat gttaccttca gcaaaccgtc taccaactac 120
ctgaaaccgg gctggattct ggatacccac ttcaccttcg gcacttccag cgaattttac 180
aacaaatcct tcgacgcgct gagcttcaac cacgttgact ctgaattcaa catgtctacc 240
tgtaacgacg atagcgaatg cggcggcgtt agcacctgca cagcaccggc gtacaccaaa 300
aacaaagatg gtgatgctaa aaaactgtgc accgttccgg ctgataaaat tctggatgcg 360
atctacgata acatcgtttc tgcgaaacgc agcgttgaca tcgtgaccct gcagccgatg 420
gatatcagcc acctgaacct gagtttttct agcggtgctt tcaccgcgac cattaaaaac 480
gcgctgagcc agctggcgaa aaacacccag tactctgatc accatattac cgttcgtctg 540
ctgcagggca gcttcacccc gatgctgggc tacgacgcag aaagcgaaga agaagaaatc 600
cgccagctgt ctctgaccca gaccaactac ctgagcgaaa tcgcgtccgt tctgccggaa 660
gttaacaacc tggatattac cgttggtagc gtgcgttctt gcaacaaact gatctctaac 720
tgcggcaaca acaacagcca gaaagatgtt ctgctgaacg ttgcttggaa ccatggtaaa 780
attatcaacg ttgacaacca gagcgttatt accggcggcc acaacctgtg gggcgcggat 840
tatctgcagc gtaacccggt gaacgatctg tctattaaca tcctgggtcc gatcgcgagc 900
accgccacca aatacggtaa caccctgtgg aactacgtgt gcaacaacac cggtaccatc 960
accaacacct ttgtgaccta cgctaacggt cagtacacct acgattgccc ggcgcacatc 1020
tccagcacct acgtggcacc gaccgatgcg aaaaacggcc tggccgtgaa agtgatgagc 1080
atttctaaac tgaacaacgg cgtgctggat aaagatgcgg atcagagcga agttgcgcgc 1140
gtttatgcgt tcaaaaacgc gaccaaatct attaaaatca gccagcaggc tctgttcttc 1200
aaaggtgctt tcggtaaagt tctgcacccg ctgaaaacca tcgatggcac cgttatggaa 1260
gcactggcgt ctgcaatcta caaaggcgtg accgtggata ttgtgacctc ttccctggat 1320
ggtggtatct atagctctgg ttacaacagc gaattcgttt ataactacct gctgaacgtt 1380
ctgcacaaag ctccgtacta cctggaacgt aactacgcta aaaccttcct tgataagaac 1440
ctgcacatca acttcatctc catcaacggt cgcgaaacca acaacatgtc ccataacaaa 1500
ctgtggattg tggatgataa agttttctac gttggcagcc ataatatcta tccgtctagc 1560
ctgcagcagt tcggcgttat cgttgatgat aaagatgcta ccgcacagct ggaaaaacag 1620
ctgtggaccc cgatgtggaa aaactccatc cacgttccga tcaacaacag ctaa 1674
<210> 3
<211> 1674
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgcaccacc accaccacca tagcaccaac gaactggatg tgaacgatat ctatgatcat 60
ctgaacgaaa aatactctca gttcaacgat gttaccttca gcaaaccgtc taccaactac 120
ctgaaaccgg gctggattct ggatacccac ttcaccttcg gcacttccag cgaattttac 180
aacaaatcct tcgacgcgct gagcttcaac cacgttgact ctgaattcaa catgtctacc 240
tgtaacgacg atagcgaatg cggcggcgtt agcacctgca cagcaccggc gtacaccaaa 300
aacaaagatg gtgatgctaa aaaactgtgc accgttccgg ctgataaaat tctggatgcg 360
atctacgata acatcgtttc tgcgaaacgc agcgttgaca tcgtgaccct gcagccgatg 420
gatatcagcc acctgaacct gtgcttttct agcggtgctt tcaccgcgac cattaaaaac 480
gcgctgagcc agctggcgaa aaacacccag tactctgatc accatattac cgttcgtctg 540
ctgcagggca gcttcacccc gatgctgggc tacgacgcag aaagcgaaga agaagaaatc 600
cgccagctgt ctctgtgcca gaccaactac ctgagcgaaa tcgcgtccgt tctgccggaa 660
gttaacaacc tggatattac cgttggtagc gtgcgttctt gcaacaaact gatctctaac 720
tgcggcaaca acaacagcca gaaagatgtt ctgctgaacg ttgcttggaa ccatggtaaa 780
attatcaacg ttgacaacca gagcgttatt accggcggcc acaacctgtg gggcgcggat 840
tatctgcagc gtaacccggt gaacgatctg tctattaaca tcctgggtcc gatcgcgagc 900
accgccacca aatacggtaa caccctgtgg aactacgtgt gcaacaacac cggtaccatc 960
accaacacct ttgtgaccta cgctaacggt cagtacacct acgattgccc ggcgcacatc 1020
tccagcacct acgtggcacc gaccgatgcg aaaaacggcc tggccgtgaa agtgatgagc 1080
atttctaaac tgaacaacgg cgtgctggat aaagatgcgg atcagagcga agttgcgcgc 1140
gtttatgcgt tcaaaaacgc gaccaaatct attaaaatca gccagcaggc tctgttcttc 1200
aaaggtgctt tcggtaaagt tctgcacccg ctgaaaacca tcgatggcac cgttatggaa 1260
gcactggcgt ctgcaatcta caaaggcgtg accgtggata ttgtgacctc ttccctggat 1320
ggtggtatct atagctctgg ttacaacagc gaattcgttt ataactacct gctgaacgtt 1380
ctgcacaaag ctccgtacta cctggaacgt aactacgcta aaaccttcct tgataagaac 1440
ctgcacatca acttcatctc catcaacggt cgcgaaacca acaacatgtc ccataacaaa 1500
ctgtggattg tggatgataa agttttctac gttggcagcc ataatatcta tccgtctagc 1560
ctgcagcagt tcggcgttat cgttgatgat aaagatgcta ccgcacagct ggaaaaacag 1620
ctgtggaccc cgatgtggaa aaactccatc cacgttccga tcaacaacag ctaa 1674
<210> 4
<211> 557
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met His His His His His His Ser Thr Asn Glu Leu Asp Val Asn Asp
1 5 10 15
Ile Tyr Asp His Leu Asn Glu Lys Tyr Ser Gln Phe Asn Asp Val Thr
20 25 30
Phe Ser Lys Pro Ser Thr Asn Tyr Leu Lys Pro Gly Trp Ile Leu Asp
35 40 45
Thr His Phe Thr Phe Gly Thr Ser Ser Glu Phe Tyr Asn Lys Ser Phe
50 55 60
Asp Ala Leu Ser Phe Asn His Val Asp Ser Glu Phe Asn Met Ser Thr
65 70 75 80
Cys Asn Asp Asp Ser Glu Cys Gly Gly Val Ser Thr Cys Thr Ala Pro
85 90 95
Ala Tyr Thr Lys Asn Lys Asp Gly Asp Ala Lys Lys Leu Cys Thr Val
100 105 110
Pro Ala Asp Lys Ile Leu Asp Ala Ile Tyr Asp Asn Ile Val Ser Ala
115 120 125
Lys Arg Ser Val Asp Ile Val Thr Leu Gln Pro Met Asp Ile Ser His
130 135 140
Leu Asn Leu Cys Phe Ser Ser Gly Ala Phe Thr Ala Thr Ile Lys Asn
145 150 155 160
Ala Leu Ser Gln Leu Ala Lys Asn Thr Gln Tyr Ser Asp His His Ile
165 170 175
Thr Val Arg Leu Leu Gln Gly Ser Phe Thr Pro Met Leu Gly Tyr Asp
180 185 190
Ala Glu Ser Glu Glu Glu Glu Ile Arg Gln Leu Ser Leu Cys Gln Thr
195 200 205
Asn Tyr Leu Ser Glu Ile Ala Ser Val Leu Pro Glu Val Asn Asn Leu
210 215 220
Asp Ile Thr Val Gly Ser Val Arg Ser Cys Asn Lys Leu Ile Ser Asn
225 230 235 240
Cys Gly Asn Asn Asn Ser Gln Lys Asp Val Leu Leu Asn Val Ala Trp
245 250 255
Asn His Gly Lys Ile Ile Asn Val Asp Asn Gln Ser Val Ile Thr Gly
260 265 270
Gly His Asn Leu Trp Gly Ala Asp Tyr Leu Gln Arg Asn Pro Val Asn
275 280 285
Asp Leu Ser Ile Asn Ile Leu Gly Pro Ile Ala Ser Thr Ala Thr Lys
290 295 300
Tyr Gly Asn Thr Leu Trp Asn Tyr Val Cys Asn Asn Thr Gly Thr Ile
305 310 315 320
Thr Asn Thr Phe Val Thr Tyr Ala Asn Gly Gln Tyr Thr Tyr Asp Cys
325 330 335
Pro Ala His Ile Ser Ser Thr Tyr Val Ala Pro Thr Asp Ala Lys Asn
340 345 350
Gly Leu Ala Val Lys Val Met Ser Ile Ser Lys Leu Asn Asn Gly Val
355 360 365
Leu Asp Lys Asp Ala Asp Gln Ser Glu Val Ala Arg Val Tyr Ala Phe
370 375 380
Lys Asn Ala Thr Lys Ser Ile Lys Ile Ser Gln Gln Ala Leu Phe Phe
385 390 395 400
Lys Gly Ala Phe Gly Lys Val Leu His Pro Leu Lys Thr Ile Asp Gly
405 410 415
Thr Val Met Glu Ala Leu Ala Ser Ala Ile Tyr Lys Gly Val Thr Val
420 425 430
Asp Ile Val Thr Ser Ser Leu Asp Gly Gly Ile Tyr Ser Ser Gly Tyr
435 440 445
Asn Ser Glu Phe Val Tyr Asn Tyr Leu Leu Asn Val Leu His Lys Ala
450 455 460
Pro Tyr Tyr Leu Glu Arg Asn Tyr Ala Lys Thr Phe Leu Asp Lys Asn
465 470 475 480
Leu His Ile Asn Phe Ile Ser Ile Asn Gly Arg Glu Thr Asn Asn Met
485 490 495
Ser His Asn Lys Leu Trp Ile Val Asp Asp Lys Val Phe Tyr Val Gly
500 505 510
Ser His Asn Ile Tyr Pro Ser Ser Leu Gln Gln Phe Gly Val Ile Val
515 520 525
Asp Asp Lys Asp Ala Thr Ala Gln Leu Glu Lys Gln Leu Trp Thr Pro
530 535 540
Met Trp Lys Asn Ser Ile His Val Pro Ile Asn Asn Ser
545 550 555
<210> 5
<211> 1674
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atgcaccacc accaccacca tagcaccaac gaactggatg tgaacgatat ctatgatcat 60
ctgaacgaaa aatactctca gttcaacgat gttaccttca gcaaaccgtc taccaactac 120
ctgaaaccgg gctggattct ggatacccac ttcaccttcg gcacttccag cgaattttac 180
aacaaatcct tcgacgcgct gagcttcaac cacgttgact ctgaattcaa catgtctacc 240
tgtaacgacg atagcgaatg cggcggcgtt agcacctgca cagcaccggc gtacaccaaa 300
aacaaagatg gtgatgctaa aaaactgtgc accgttccgg ctgataaaat tctggatgcg 360
atctacgata acatcgtttc tgcgaaacgc agcgttgaca tcgtgaccct gcagccgatg 420
gatatcagcc acctgaacct gagtttttct agcggtgctt tcaccgcgac cattaaaaac 480
gcgctgagcc agctggcgaa aaacacccag tactctgatc accatattac cgttcgtctg 540
ctgcagggca gcttcacccc gatgctgggc tacgacgcag aaagcgaaga agaagaaatc 600
cgccagctgt ctctgaccca gaccaactac ctgagcgaaa tcgcgtccgt tctgccggaa 660
gttaacaacc tgtgcattac cgttggtagc gtgcgttctt gcaacaaact gatctctaac 720
tgcggcaaca acaacagcca gaaagatgtt ctgctgaacg ttgcttggaa ccatggtaaa 780
attatcaacg ttgacaacca gagcgttatt accggcggcc acaacctgtg gggcgcggat 840
tatctgcagc gtaacccggt gaacgatctg tctattaaca tcctgggtcc gatcgcgagc 900
accgccacca aatacggtaa caccctgtgg aactacgtgt gcaacaacac cggtaccatc 960
accaacacct ttgtgaccta ctgcaacggt cagtacacct acgattgccc ggcgcacatc 1020
tccagcacct acgtggcacc gaccgatgcg aaaaacggcc tggccgtgaa agtgatgagc 1080
atttctaaac tgaacaacgg cgtgctggat aaagatgcgg atcagagcga agttgcgcgc 1140
gtttatgcgt tcaaaaacgc gaccaaatct attaaaatca gccagcaggc tctgttcttc 1200
aaaggtgctt tcggtaaagt tctgcacccg ctgaaaacca tcgatggcac cgttatggaa 1260
gcactggcgt ctgcaatcta caaaggcgtg accgtggata ttgtgacctc ttccctggat 1320
ggtggtatct atagctctgg ttacaacagc gaattcgttt ataactacct gctgaacgtt 1380
ctgcacaaag ctccgtacta cctggaacgt aactacgcta aaaccttcct tgataagaac 1440
ctgcacatca acttcatctc catcaacggt cgcgaaacca acaacatgtc ccataacaaa 1500
ctgtggattg tggatgataa agttttctac gttggcagcc ataatatcta tccgtctagc 1560
ctgcagcagt tcggcgttat cgttgatgat aaagatgcta ccgcacagct ggaaaaacag 1620
ctgtggaccc cgatgtggaa aaactccatc cacgttccga tcaacaacag ctaa 1674
<210> 6
<211> 557
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Met His His His His His His Ser Thr Asn Glu Leu Asp Val Asn Asp
1 5 10 15
Ile Tyr Asp His Leu Asn Glu Lys Tyr Ser Gln Phe Asn Asp Val Thr
20 25 30
Phe Ser Lys Pro Ser Thr Asn Tyr Leu Lys Pro Gly Trp Ile Leu Asp
35 40 45
Thr His Phe Thr Phe Gly Thr Ser Ser Glu Phe Tyr Asn Lys Ser Phe
50 55 60
Asp Ala Leu Ser Phe Asn His Val Asp Ser Glu Phe Asn Met Ser Thr
65 70 75 80
Cys Asn Asp Asp Ser Glu Cys Gly Gly Val Ser Thr Cys Thr Ala Pro
85 90 95
Ala Tyr Thr Lys Asn Lys Asp Gly Asp Ala Lys Lys Leu Cys Thr Val
100 105 110
Pro Ala Asp Lys Ile Leu Asp Ala Ile Tyr Asp Asn Ile Val Ser Ala
115 120 125
Lys Arg Ser Val Asp Ile Val Thr Leu Gln Pro Met Asp Ile Ser His
130 135 140
Leu Asn Leu Ser Phe Ser Ser Gly Ala Phe Thr Ala Thr Ile Lys Asn
145 150 155 160
Ala Leu Ser Gln Leu Ala Lys Asn Thr Gln Tyr Ser Asp His His Ile
165 170 175
Thr Val Arg Leu Leu Gln Gly Ser Phe Thr Pro Met Leu Gly Tyr Asp
180 185 190
Ala Glu Ser Glu Glu Glu Glu Ile Arg Gln Leu Ser Leu Thr Gln Thr
195 200 205
Asn Tyr Leu Ser Glu Ile Ala Ser Val Leu Pro Glu Val Asn Asn Leu
210 215 220
Cys Ile Thr Val Gly Ser Val Arg Ser Cys Asn Lys Leu Ile Ser Asn
225 230 235 240
Cys Gly Asn Asn Asn Ser Gln Lys Asp Val Leu Leu Asn Val Ala Trp
245 250 255
Asn His Gly Lys Ile Ile Asn Val Asp Asn Gln Ser Val Ile Thr Gly
260 265 270
Gly His Asn Leu Trp Gly Ala Asp Tyr Leu Gln Arg Asn Pro Val Asn
275 280 285
Asp Leu Ser Ile Asn Ile Leu Gly Pro Ile Ala Ser Thr Ala Thr Lys
290 295 300
Tyr Gly Asn Thr Leu Trp Asn Tyr Val Cys Asn Asn Thr Gly Thr Ile
305 310 315 320
Thr Asn Thr Phe Val Thr Tyr Cys Asn Gly Gln Tyr Thr Tyr Asp Cys
325 330 335
Pro Ala His Ile Ser Ser Thr Tyr Val Ala Pro Thr Asp Ala Lys Asn
340 345 350
Gly Leu Ala Val Lys Val Met Ser Ile Ser Lys Leu Asn Asn Gly Val
355 360 365
Leu Asp Lys Asp Ala Asp Gln Ser Glu Val Ala Arg Val Tyr Ala Phe
370 375 380
Lys Asn Ala Thr Lys Ser Ile Lys Ile Ser Gln Gln Ala Leu Phe Phe
385 390 395 400
Lys Gly Ala Phe Gly Lys Val Leu His Pro Leu Lys Thr Ile Asp Gly
405 410 415
Thr Val Met Glu Ala Leu Ala Ser Ala Ile Tyr Lys Gly Val Thr Val
420 425 430
Asp Ile Val Thr Ser Ser Leu Asp Gly Gly Ile Tyr Ser Ser Gly Tyr
435 440 445
Asn Ser Glu Phe Val Tyr Asn Tyr Leu Leu Asn Val Leu His Lys Ala
450 455 460
Pro Tyr Tyr Leu Glu Arg Asn Tyr Ala Lys Thr Phe Leu Asp Lys Asn
465 470 475 480
Leu His Ile Asn Phe Ile Ser Ile Asn Gly Arg Glu Thr Asn Asn Met
485 490 495
Ser His Asn Lys Leu Trp Ile Val Asp Asp Lys Val Phe Tyr Val Gly
500 505 510
Ser His Asn Ile Tyr Pro Ser Ser Leu Gln Gln Phe Gly Val Ile Val
515 520 525
Asp Asp Lys Asp Ala Thr Ala Gln Leu Glu Lys Gln Leu Trp Thr Pro
530 535 540
Met Trp Lys Asn Ser Ile His Val Pro Ile Asn Asn Ser
545 550 555
<210> 7
<211> 1674
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atgcaccacc accaccacca tagcaccaac gaactggatg tgaacgatat ctatgatcat 60
ctgaacgaaa aatactctca gttcaacgat gttaccttca gcaaaccgtc taccaactac 120
ctgaaaccgg gctggattct ggatacccac ttcaccttcg gcacttccag cgaattttac 180
aacaaatcct tcgacgcgct gagcttcaac cacgttgact ctgaattcaa catgtctacc 240
tgtaacgacg atagcgaatg cggcggcgtt agcacctgca cagcaccggc gtacaccaaa 300
aacaaagatg gtgatgctaa aaaactgtgc accgttccgg ctgataaaat tctggatgcg 360
atctacgata acatcgtttc tgcgaaacgc agcgttgaca tcgtgaccct gcagccgatg 420
gatatcagcc acctgaacct gtgcttttct agcggtgctt tcaccgcgac cattaaaaac 480
gcgctgagcc agctggcgaa aaacacccag tactctgatc accatattac cgttcgtctg 540
ctgcagggca gcttcacccc gatgctgggc tacgacgcag aaagcgaaga agaagaaatc 600
cgccagctgt ctctgtgcca gaccaactac ctgagcgaaa tcgcgtccgt tctgccggaa 660
gttaacaacc tgtgcattac cgttggtagc gtgcgttctt gcaacaaact gatctctaac 720
tgcggcaaca acaacagcca gaaagatgtt ctgctgaacg ttgcttggaa ccatggtaaa 780
attatcaacg ttgacaacca gagcgttatt accggcggcc acaacctgtg gggcgcggat 840
tatctgcagc gtaacccggt gaacgatctg tctattaaca tcctgggtcc gatcgcgagc 900
accgccacca aatacggtaa caccctgtgg aactacgtgt gcaacaacac cggtaccatc 960
accaacacct ttgtgaccta ctgcaacggt cagtacacct acgattgccc ggcgcacatc 1020
tccagcacct acgtggcacc gaccgatgcg aaaaacggcc tggccgtgaa agtgatgagc 1080
atttctaaac tgaacaacgg cgtgctggat aaagatgcgg atcagagcga agttgcgcgc 1140
gtttatgcgt tcaaaaacgc gaccaaatct attaaaatca gccagcaggc tctgttcttc 1200
aaaggtgctt tcggtaaagt tctgcacccg ctgaaaacca tcgatggcac cgttatggaa 1260
gcactggcgt ctgcaatcta caaaggcgtg accgtggata ttgtgacctc ttccctggat 1320
ggtggtatct atagctctgg ttacaacagc gaattcgttt ataactacct gctgaacgtt 1380
ctgcacaaag ctccgtacta cctggaacgt aactacgcta aaaccttcct tgataagaac 1440
ctgcacatca acttcatctc catcaacggt cgcgaaacca acaacatgtc ccataacaaa 1500
ctgtggattg tggatgataa agttttctac gttggcagcc ataatatcta tccgtctagc 1560
ctgcagcagt tcggcgttat cgttgatgat aaagatgcta ccgcacagct ggaaaaacag 1620
ctgtggaccc cgatgtggaa aaactccatc cacgttccga tcaacaacag ctaa 1674
<210> 8
<211> 557
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Met His His His His His His Ser Thr Asn Glu Leu Asp Val Asn Asp
1 5 10 15
Ile Tyr Asp His Leu Asn Glu Lys Tyr Ser Gln Phe Asn Asp Val Thr
20 25 30
Phe Ser Lys Pro Ser Thr Asn Tyr Leu Lys Pro Gly Trp Ile Leu Asp
35 40 45
Thr His Phe Thr Phe Gly Thr Ser Ser Glu Phe Tyr Asn Lys Ser Phe
50 55 60
Asp Ala Leu Ser Phe Asn His Val Asp Ser Glu Phe Asn Met Ser Thr
65 70 75 80
Cys Asn Asp Asp Ser Glu Cys Gly Gly Val Ser Thr Cys Thr Ala Pro
85 90 95
Ala Tyr Thr Lys Asn Lys Asp Gly Asp Ala Lys Lys Leu Cys Thr Val
100 105 110
Pro Ala Asp Lys Ile Leu Asp Ala Ile Tyr Asp Asn Ile Val Ser Ala
115 120 125
Lys Arg Ser Val Asp Ile Val Thr Leu Gln Pro Met Asp Ile Ser His
130 135 140
Leu Asn Leu Cys Phe Ser Ser Gly Ala Phe Thr Ala Thr Ile Lys Asn
145 150 155 160
Ala Leu Ser Gln Leu Ala Lys Asn Thr Gln Tyr Ser Asp His His Ile
165 170 175
Thr Val Arg Leu Leu Gln Gly Ser Phe Thr Pro Met Leu Gly Tyr Asp
180 185 190
Ala Glu Ser Glu Glu Glu Glu Ile Arg Gln Leu Ser Leu Cys Gln Thr
195 200 205
Asn Tyr Leu Ser Glu Ile Ala Ser Val Leu Pro Glu Val Asn Asn Leu
210 215 220
Cys Ile Thr Val Gly Ser Val Arg Ser Cys Asn Lys Leu Ile Ser Asn
225 230 235 240
Cys Gly Asn Asn Asn Ser Gln Lys Asp Val Leu Leu Asn Val Ala Trp
245 250 255
Asn His Gly Lys Ile Ile Asn Val Asp Asn Gln Ser Val Ile Thr Gly
260 265 270
Gly His Asn Leu Trp Gly Ala Asp Tyr Leu Gln Arg Asn Pro Val Asn
275 280 285
Asp Leu Ser Ile Asn Ile Leu Gly Pro Ile Ala Ser Thr Ala Thr Lys
290 295 300
Tyr Gly Asn Thr Leu Trp Asn Tyr Val Cys Asn Asn Thr Gly Thr Ile
305 310 315 320
Thr Asn Thr Phe Val Thr Tyr Cys Asn Gly Gln Tyr Thr Tyr Asp Cys
325 330 335
Pro Ala His Ile Ser Ser Thr Tyr Val Ala Pro Thr Asp Ala Lys Asn
340 345 350
Gly Leu Ala Val Lys Val Met Ser Ile Ser Lys Leu Asn Asn Gly Val
355 360 365
Leu Asp Lys Asp Ala Asp Gln Ser Glu Val Ala Arg Val Tyr Ala Phe
370 375 380
Lys Asn Ala Thr Lys Ser Ile Lys Ile Ser Gln Gln Ala Leu Phe Phe
385 390 395 400
Lys Gly Ala Phe Gly Lys Val Leu His Pro Leu Lys Thr Ile Asp Gly
405 410 415
Thr Val Met Glu Ala Leu Ala Ser Ala Ile Tyr Lys Gly Val Thr Val
420 425 430
Asp Ile Val Thr Ser Ser Leu Asp Gly Gly Ile Tyr Ser Ser Gly Tyr
435 440 445
Asn Ser Glu Phe Val Tyr Asn Tyr Leu Leu Asn Val Leu His Lys Ala
450 455 460
Pro Tyr Tyr Leu Glu Arg Asn Tyr Ala Lys Thr Phe Leu Asp Lys Asn
465 470 475 480
Leu His Ile Asn Phe Ile Ser Ile Asn Gly Arg Glu Thr Asn Asn Met
485 490 495
Ser His Asn Lys Leu Trp Ile Val Asp Asp Lys Val Phe Tyr Val Gly
500 505 510
Ser His Asn Ile Tyr Pro Ser Ser Leu Gln Gln Phe Gly Val Ile Val
515 520 525
Asp Asp Lys Asp Ala Thr Ala Gln Leu Glu Lys Gln Leu Trp Thr Pro
530 535 540
Met Trp Lys Asn Ser Ile His Val Pro Ile Asn Asn Ser
545 550 555

Claims (6)

1.一种来源于南极细菌的磷脂酶D突变体，其特征在于，其氨基酸序列为SEQ ID NO:4或SEQ ID NO:6或SEQ ID NO:8。
2.一种编码权利要求1所述磷脂酶D突变体的基因。
3.根据权利要求2所述的基因，其特征在于，其核苷酸序列为SEQ ID NO:3或SEQ IDNO:5或SEQ ID NO:7。
4.一种含权利要求2或3所述基因的重组基因工程菌。
5.权利要求4所述重组基因工程菌的制备方法，其特征在于，将权利要求3或4所述基因克隆到表达载体pET-21a、pET28a或pET32a上，转化大肠杆菌感受态细胞，获得重组基因工程菌。
6.权利要求1所述磷脂酶D突变体的应用，其特征在于，所述磷脂酶D突变体用于催化合成天然稀有磷脂和非天然磷脂化合物。
CN202210160732.1A 2022-02-22 2022-02-22 一种来源于南极细菌的磷脂酶d突变体及其应用 Active CN114525266B (zh)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996040939A2 (en) * 1995-06-07 1996-12-19 Cadus Pharmaceutical Corporation Expression of functional vertebrate phospholipases in yeast
CN1235636A (zh) * 1996-10-31 1999-11-17 诺沃挪第克公司 新型磷脂酶,及其生产和应用
JP2001136984A (ja) * 1999-09-03 2001-05-22 Ajinomoto Co Inc 変異型ヌクレオシド−５’−リン酸生産酵素
CN108118041A (zh) * 2017-12-29 2018-06-05 华南理工大学 一种磷脂酶d突变体、重组基因工程菌及其制备方法和应用
CN112899256A (zh) * 2021-01-29 2021-06-04 华南理工大学 一种来源于南极细菌的耐低温磷脂酶d及其制备方法和应用
CN113801862A (zh) * 2021-08-20 2021-12-17 华南理工大学 一种海洋链霉菌磷脂酶d突变体及其重组表达菌株的制备方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996040939A2 (en) * 1995-06-07 1996-12-19 Cadus Pharmaceutical Corporation Expression of functional vertebrate phospholipases in yeast
CN1235636A (zh) * 1996-10-31 1999-11-17 诺沃挪第克公司 新型磷脂酶,及其生产和应用
JP2001136984A (ja) * 1999-09-03 2001-05-22 Ajinomoto Co Inc 変異型ヌクレオシド−５’−リン酸生産酵素
CN108118041A (zh) * 2017-12-29 2018-06-05 华南理工大学 一种磷脂酶d突变体、重组基因工程菌及其制备方法和应用
CN112899256A (zh) * 2021-01-29 2021-06-04 华南理工大学 一种来源于南极细菌的耐低温磷脂酶d及其制备方法和应用
CN113801862A (zh) * 2021-08-20 2021-12-17 华南理工大学 一种海洋链霉菌磷脂酶d突变体及其重组表达菌株的制备方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Purification, biochemical characterization, and cloning of phospholipase D from Streptomyces racemochromogenes strain 10-3;Yozo Nakazawa等;《Protein J》;第29卷(第8期);第598-608页 *

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