CN115161303A - 一种磷脂酶突变体及其合成甘油磷脂的方法 - Google Patents
一种磷脂酶突变体及其合成甘油磷脂的方法 Download PDFInfo
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Abstract
本发明属于酶的基因工程技术领域,本发明属于酶的基因工程技术领域,具体涉及通过易错PCR技术及重叠PCR技术体外定向进化获得比酶活提高的磷脂酶D突变体,然后将高活力的磷脂酶D基因分别在枯草芽孢杆菌表达系统、解淀粉芽孢杆菌表达系统和地衣芽孢杆菌表达系统中进行表达,表达后,检测高活力磷脂酶D的比酶活较野生型磷脂酶D最高可提高500%。将高活力磷脂酶D在枯草芽孢杆菌表达系统、地衣芽孢杆菌表达系统、解淀粉芽孢杆菌表达系统内发酵,并且利用高活力磷脂酶D有效地制备磷脂酸、磷酯酰丝氨酸、磷脂酰乙醇胺,磷脂酰甘油和磷脂酰肌醇。
Description
本申请为发明专利申请201911149105.2的分案申请,201911149105.2的申请日为2019年11月21日,申请号为201911149105.2,发明名称为:一种新型磷脂酶D及其制备功能性磷脂的方法。
技术领域:
本发明属于酶的基因工程技术领域,具体涉及通过易错PCR技术及重叠PCR技术体外定向进化获得比酶活提高的磷脂酶D突变体,并提供由高活力磷脂酶D催化制备功能性磷脂的方法。
背景技术:
磷脂酶D(phospholipase D,PLD)来源广泛,主要分布在动物、植物及微生物中。PLD可催化发生两种反应:其一,水解磷脂生成磷脂酸和羟基化合物;其二,当有另一种含羟基的化合物存在时,可催化其结合到磷脂的碱基上,形成新的磷脂,此为PLD的转磷酯化反应或碱基交换反应。两种反应中尤为重要的是PLD的转磷酯化作用,它可以将自然界大量存在的磷脂酰胆碱(phosphatidylcholine,PC)催化合成为其他的稀有磷脂,如磷脂酸(phosphatidicacid,PA)、磷脂酰甘油(phosphatidylglycerol,PG)、磷脂酰丝氨酸(phosphatidylserine,PS)、磷脂酰乙醇胺(phosphati-dylethanolamine,PE)、磷脂酰肌醇(phosphatidylinositol,PI)等。因此,磷脂酶及其催化反应成为目前一个很活跃的研究领域。
PA是一种简单而又常见的磷脂,它可促进细胞有丝分裂、促进细胞内超氧化物的形成、引起肌肉的收缩、促进激素分泌、诱发血小板聚集等作用。PG是一种天然稀有磷脂。PG不但能降低肺表面张力,维持肺泡结构和功能的稳定,还可作为抗癌药物载体,对病灶进行靶向治疗。PS是磷脂中的一种,天然含量稀少,它能提高脑细胞的活力,改善大脑功能、修复大脑损伤。PE是一种常见的磷脂,其中高纯度PE在提高人体记忆力与增强大脑功能方面有很大的潜力,同时它具有维持及改善认知的功能。PI约占细胞总磷脂的5~10%,它在细胞中对于细胞形态、代谢调控、信号传导和细胞的各种生理功能起着非常重要的作用。
然而,这些稀有磷脂不能由人体自身完全合成。目前稀有磷脂的制备有溶剂提取法、化学合成法和酶转化法。采用溶剂提取法由于其安全性而受到质疑,化学合成法成本较高,工艺复杂,且纯度和收率有待提高,酶转化法具有反应条件温和、反应易控、高效简单的优势,因此越来越受到关注。
与动、植物来源的PLD相比,微生物PLD具有较好的转磷脂活性、较广泛的底物特异性和较强的底物耐受性。至今已报道的产PLD的微生物种类主要有链霉菌(Streptomyces)、大肠杆菌(Escherichia coli)、沙门式杆菌(Salmonella)以及假单胞菌(Pseudomonas)等。其中,链霉菌来源的PLD是目前报道最多的,且它们具有较好的水解和转磷脂活性。但由于链霉菌发酵周期长,培养条件复杂,因此用异源表达的方式生产高活力的PLD是十分必要的。
定向进化,又称为酶的非理性设计,即在不需明确酶蛋白的结构信息和催化机制的情况下,通过模拟自然进化中出现的随机突变、重组等进化机制,并在后期筛选中定向施压,可在短时间内获得具有某些特定优秀性能的突变体。本发明所使用的易错PCR是非理性设计中的经典方法,其原理是通过改变PCR反应条件来调整PCR反应中的突变频率,降低聚合酶固有的突变序列的倾向性,提高突变谱的多样性,使得错误碱基随机地以一定的频率掺入到扩增的基因中,从而得到随机突变的DNA群体,最后用合适的载体克隆突变基因。
定点突变,又称为理性设计,就是在已知的DNA序列中插入、缺失或取代一定长度的核苷酸序列,由于其迅速、高效的提高DNA所表达的目的蛋白的性状及表征,是基因研究工作中一种非常有用的手段。本发明所使用的重叠PCR技术是定点突变技术的一种,该技术可以简单、快速的将两个或多个基因片段通过末端互补、重叠延伸进行体外基因的拼接。重叠PCR技术可以获得依靠限制性内切酶消化难以得到的产物,并且方便快速,在进行大片段基因的定点突变、基因片段删除以及多个编码序列嵌合连接上具有独有的优势。
枯草芽孢杆菌属于革兰氏阳性菌。枯草芽孢杆菌表达系统具有以下优点:1、能够高效地分泌各种蛋白质;2、许多枯草芽孢杆菌在发酵工业上的使用已有相当长的历史,无致病性,不产生任何内毒素;3、芽孢杆菌属微生物遗传学背景研究的十分清楚,并且生长迅速,对营养物质无特殊要求等优点;4、密码子偏爱性不明显;5、发酵过程简单,枯草芽孢杆菌属于好氧菌,无需厌氧发酵设备,发酵结束后,简单的分离发酵液和细菌菌体,即可进入目的蛋白的分离、纯化回收阶段;6、具有抗逆性,可以生产多种耐热性酶制剂。枯草芽孢杆菌表达系统已成为现代分子生物学研究最重要的工具和模型,是表达外源基因较为理想的工具。
地衣芽孢杆菌属于革兰氏阳性菌。地衣芽孢杆菌表达系统具有以下优点:1、蛋白被直接分泌到胞外的培养基中,不会积聚,有利于蛋白的下游回收和纯化,降低整个生产链的操作成本;2、胞外蛋白分泌量大,生长温度较高适合用作工业生产宿主菌;3、作为单细胞生物在发酵过程中可以达到很高的细胞密度,且培养基相对比较简单,成本低、产出高,符合工业生产的要求。
解淀粉芽孢杆菌是一种革兰氏阳性菌。解淀粉芽孢杆菌表达系统具有以下优点:1、次级代谢产物丰富,能够产生抗菌蛋白、脂肽类和聚酮类等多种抗菌物质;2、解淀粉芽孢杆菌产生的蛋白具有较好的亲水性、稳定性和较高活性;3、解淀粉芽孢杆菌可以产生多种生理活性物质和氨基酸类物质;4、在解淀粉芽孢杆菌中,蛋白质及酶类抑菌物质合成所涉及的基因数量较少,有利于基因重组及表达;5、解淀粉芽孢杆菌具有较广的抑菌谱,它具无污染、不产生抗药性、有利于人体安全等优点,有较好的发展前景。
在本发明中,高活力磷脂酶D突变体基因在枯草芽孢杆菌表达系统、地衣芽孢杆菌和解淀粉芽孢杆菌表达系统中进行表达,分别得到枯草芽孢杆菌、地衣芽孢杆菌和解淀粉芽孢杆菌的高活力磷脂酶D游离表达重组菌株,重组菌株发酵后,经过相应的处理,可以得到高活力磷脂酶D,纯化后与底物反应,催化制备功能性磷脂。
发明内容:
本发明的目的在于提供一种新型磷脂酶D突变体及利用其制备功能性磷脂的方法。
为了实现上述目的,本发明提供的技术方案之一为:以申请人实验室保藏的一株抗生素链霉菌(Streptomyces antibioticus)基因组为模板,克隆出磷脂酶D野生型基因pld(如SEQ ID NO:1所示)后,通过酶切、连接等构建重组载体,通过易错PCR技术向基因中引入随机突变,建立突变文库来筛选可获得较高磷脂酶D产量的突变体。磷脂酶D突变体是基于SEQ ID No:2所示的磷脂酶D氨基酸序列,其中的第84、153、270、316、452位中的至少一个氨基酸被置换为以下氨基酸:第84位:D 84 I;第153位:N 153 I;第270位:G 270 F;第316位:P 316 W;第452位:A 452 F。之后由重叠PCR技术将单个突变点基因嵌合连接,得到组合突变体(对照表见表1)。
为了实现上述目的,本发明提供的技术方案之二为:将上述突变体基因重新构建重组载体,并在枯草芽孢杆菌、地衣芽孢杆菌和解淀粉芽孢杆菌中的高效表达,得到产高活力磷脂酶D的重组菌株,通过发酵、提取等技术获得高活力磷脂酶D。
用于表达所述的磷脂酶D突变体的宿主细胞分别为枯草芽孢杆菌、地衣芽孢杆菌和解淀粉芽孢杆菌,表达载体为pBSA43;
优选地,枯草芽孢杆菌为枯草芽孢杆菌WB600;
优选地,地衣芽孢杆菌为地衣芽孢杆菌TCCC11965;
优选地,解淀粉芽孢杆菌为解淀粉芽孢杆菌CGMCC No.11218;
为了实现上述目的,本发明提供的技术方案之三为:上述磷脂酶D突变体在制备磷脂中的应用,特别是在制备PA、PS、PE、PG和PI中的应用。
在本发明中采用如下定义:
1.氨基酸和DNA核酸序列的命名法
使用氨基酸残基的公认IUPAC命名法,用单字母代码形式。DNA核酸序列采用公认IUPAC命名法。
2.磷脂酶D突变体的标识
采用“原始氨基酸位置替换的氨基酸”来表示磷脂酶D突变体中突变的氨基酸。如D84 I,表示位置84的氨基酸由野生型磷脂酶D的D替换成I。位置的编号对应于SEQ ID NO:2中磷脂酶D的氨基酸序列编号。核苷酸的变化同样采用“原始核苷酸位置替换的核苷酸”来表示,位置编号对应SEQ ID NO:1中野生型磷脂酶D的核苷酸序列编号。
在本发明中,pld表示野生型磷脂酶D,氨基酸序列即原始序列(如SEQ ID NO:2所示)。用pldm加AxB的方式表示在pld基础上获得的各个突变体,A、B代表氨基酸,x分别为84、153、270、316、452,其中pldmD84I代表第84氨基酸由D替换成I的突变体,pldmN153I代表第153位氨基酸由N替换成I的突变体,pldmG270F代表第270位氨基酸由G替换成F的突变体,pldmP316W代表第316位氨基酸由P替换成W的突变体,pldmA452F代表第452位氨基酸由A替换成F的突变体;AxB也可以是AxmB/…/CxnD的形式,表示若干位点的组合突变体,如pldmD84I/N153I表示第84氨基酸由D替换成I,第153位氨基酸由N替换成I的突变体(其中,D:Asp;I:Ile;N:Asn;G:Gly;F:Phe;P:Pro;W:Trp;A:Ala)。各突变体的编码基因则以其氨基酸表示形式的斜体表示,如突变体pldmD84I的编码基因为pldmD84I。
本发明中,氨基酸的组合突变,包含如下:
pldmD84I/N153I、pldmD84I/G270F、pldmD84I/P316W、pldmD84I/A452F、pldmN153I/G270F、pldmN153I/P316W、pldmN153I/A452F、pldmG270F/P316W、pldmG270F/A452F、pldmP316W/A452F、pldmD84I/N153I/G270F、pldmD84I/N153I/P316W、pldmD84I/N153I/A452F、pldmD84I/G270F/P316W、pldmD84I/G270F/A452F、pldmD84I/P316W/A452F、pldmN153I/G270F/P316W、pldmN153I/G270F/A452F、pldmG270F/P316W/A452F、pldmN153I/P316W/A452F、pldmD84I/N153I/P316W/A452F、pldmD84I/N153I/G270F/P316W、pldmD84I/N153I/G270F/A452F、pldmD84I/G270F/P316W/A452F、pldmN153I/G270F/P316W/A452F、pldmD84I/N153I/G270F/P316W/A452F;
表1:突变位点对照表
本发明的实验步骤具体如下:
1、一种获得高活力磷脂酶D突变体编码基因的过程包括如下步骤:
以来自抗生素链霉菌(Streptomyces antibioticus)的野生型磷脂酶D基因(SEQID NO.1所示)与载体pET22b连接,构建重组质粒pET22b-pld,利用易错PCR技术向磷脂酶D基因中引入随机突变,构建重组质粒pET22b-pldmAxB,建立突变文库筛选获得较高磷脂酶D产量的突变体编码基因pldmD84I、pldmN153I、pldmG270F、pldmP316W、pldmA452F。通过重叠PCR将多个突变点基因嵌合连接,得到高活力磷脂酶D突变体编码基因pldmD84I/N153I、pldmD84I/G270F、pldmD84I/P316W、pldmD84I/A452F、pldmN153I/G270F、pldmN153I/P316W、pldmN153I/A452F、pldmG270F/P316W、pldmG270F/A452F、pldmP316W/A452F、pldmD84I/N153I/G270F、pldmD84I/N153I/P316W、pldmD84I/N153I/A452F、pldmD84I/G270F/P316W、pldmD84I/G270F/A452F、pldmD84I/P316W/A452F、pldmN153I/G270F/P316W、pldmN153I/G270F/A452F、pldmG270F/P316W/A452F、pldmN153I/P316W/A452F、pldmD84I/N153I/P316W/A452F、pldmD84I/N153I/G270F/P316W、pldmD84I/N153I/G270F/A452F、pldmD84I/G270F/P316W/A452F、pldmN153I/G270F/P316W/A452F、pldmD84I/N153I/G270F/P316W/A452F。
2、一株含有上述高活力磷脂酶D基因的枯草芽孢杆菌重组菌株及以此制备高活力磷脂酶D的过程包括如下步骤:
(1)将上述含有高活力磷脂酶D突变体编码基因的pET22b-pldmAxB进行酶切,将得到的高活力磷脂酶D突变体编码基因与载体大肠杆菌-枯草芽孢杆菌穿梭质粒pBSA43通过连接得到了新的重组载体;
(2)将重组载体转化入枯草芽孢杆菌WB600中,得到重组菌株,之后将重组菌株发酵,得到高活力磷脂酶D。
(3)之后进行发酵,制备高活力磷脂酶D。
3、一株含有上述高活力磷脂酶D基因的地衣芽孢杆菌重组菌株及以此制备高活力磷脂酶D的过程包括如下步骤:
(1)将上述含有高活力磷脂酶D突变体编码基因的pET22b-pldmAxB进行酶切,将得到的高活力磷脂酶D突变体编码基因与表达载体大肠杆菌-地衣芽孢杆菌穿梭质粒pBSA43通过连接得到了新的重组载体;
(2)将重组载体转化入地衣芽孢杆菌TCCC11965中,得到的重组菌株经过遗传霉素筛选和磷脂酶D的酶活测定,得到高活力磷脂酶D的高产菌株;
(3)之后进行发酵,制备高活力磷脂酶D。
4、一株含有上述高活力磷脂酶D基因的解淀粉芽孢杆菌重组菌株及以此制备高活力磷脂酶D的过程包括如下步骤:
(1)将上述含有高活力磷脂酶D突变体编码基因的pET22b-pldmAxB进行酶切,将得到的高活力磷脂酶D突变体编码基因与表达载体大肠杆菌-解淀粉芽孢杆菌穿梭质粒pBSA43通过连接得到了新的重组载体;
(2)将重组载体转化入解淀粉芽孢杆菌CGMCC No.11218中,得到的重组菌株经过遗传霉素筛选和磷脂酶D的酶活测定,得到高活力磷脂酶D的高产菌株;
(3)之后进行发酵,制备高活力磷脂酶D。
有益效果:
1、本发明利用易错PCR技术向基因中引入随机突变,通过建立突变文库筛选出5个活力较高的磷脂酶D突变体及基因,利用重叠PCR技术对磷脂酶D突变基因进行突变点组合(pldmD84I/N153I、pldmD84I/G270F、pldmD84I/P316W、pldmD84I/A452F、pldmN153I/G270F、pldmN153I/P316W、pldmN153I/A452F、pldmG270F/P316W、pldmG270F/A452F、pldmP316W/A452F、pldmD84I/N153I/G270F、pldmD84I/N153I/P316W、pldmD84I/N153I/A452F、pldmD84I/G270F/P316W、pldmD84I/G270F/A452F、pldmD84I/P316W/A452F、pldmN153I/G270F/P316W、pldmN153I/G270F/A452F、pldmG270F/P316W/A452F、pldmN153I/P316W/A452F、pldmD84I/N153I/P316W/A452F、pldmD84I/N153I/G270F/P316W、pldmD84I/N153I/G270F/A452F、pldmD84I/G270F/P316W/A452F、pldmN153I/G270F/P316W/A452F、pldmD84I/N153I/G270F/P316W/A452F),并进一步表达得到高活力磷脂酶D。
2、本发明分别使用了枯草芽孢杆菌表达系统、地衣芽孢杆菌表达系统,解淀粉芽孢杆菌表达系统,高活力磷脂酶D在各表达系统内发酵酶活最高值分别为319.1U/mL、952.2U/mL、1304.5U/mL,较野生型分别提高480%、425%、500%。
3、本发明采用高活力磷脂酶D生产PA、PS、PE、PG和PI的转化率分别达到93%、79.3%、53.9%、60.1%、32.1%。
附图说明:
图1为本发明野生型磷脂酶D基因的PCR扩增电泳图
其中:M为DNA Marker,1、2分别为磷脂酶D基因;
图2为本发明枯草芽孢杆菌中重组质粒pBSA43-pldmD84I酶切验证图
其中:M为DNA Marker,1为重组质粒pBSA43-pldmD84I经EcoR I和Not I双酶切图;
图3为本发明地衣芽孢杆菌中重组质粒pBSA43-pldmD84I酶切验证图
其中:M为DNA Marker,1为重组质粒pBSA43-pldmD84I经EcoR I和Not I双酶切图;
图4为本发明解淀粉芽孢杆菌中重组质粒pBSA43-pldmD84I酶切验证图
其中:M为DNA Marker,1为重组质粒pBSA43-pldmD84I经EcoR I和Not I双酶切图;
图5实施例7纯化样品SDS-PAGE;
图6实施例8纯化样品SDS-PAGE;
图7实施例9纯化样品SDS-PAGE。
具体实施方式:
下面结合实施例对本发明的技术内容做进一步说明,但本发明不只限于这些实施例,不能以下述实施例来限定本发明的保护范围。
本发明所使用的地衣芽胞杆菌为TCCC11965,公开于:Development andapplication of a CRISPR/Cas9 system for Bacillus licheniformis genome editing[J].International Journal of Biological Macromolecules,2019,122:329-337,目前保存于天津科技大学微生物菌种保藏管理中心,公众可从该中心获取菌种。
实施例1:野生型磷脂酶D基因的获得
1.野生型磷脂酶D基因来自一株抗生素链霉菌(Streptomyces antibioticus),提取其基因组DNA。
其中抗生素链霉菌基因组DNA的提取步骤如下:
(1)从培养菌体的平板上挑取一环菌接种于50mL适当培养基中,26℃,150r/min培养2-3d。
(2)之后取1mL培养液于1.5mL EP管中,8000r/min离心20min,倒上清,用200μL溶液I或灭菌水重悬。
(3)加20-50μL的50mg/mL溶菌酶在37℃下消化0.5-1h。
(4)加入100μL的2%SDS溶液,充分反应至菌悬液呈现粘稠状。
(5)加入等体积的Tris平衡酚:氯仿=1:1,混合均匀,12000r/min离心5min,将上清转移到另一EP管中。
(6)反复抽提两次,直至无蛋白层出现,最后再用等体积氯仿抽提一次。
(7)加入等体积的异丙醇沉淀DNA,12000r/min离心5min,弃去上清,用500μL 75%乙醇洗涤2次,每次吹打后12000r/min离心5min。
(8)将EP管倒置于滤纸或置于55℃金属浴中,晾干至无酒精味后用TE缓冲液或灭菌水溶解,-20℃保存。
2.由磷脂酶D基因设计野生型磷脂酶D基因的扩增引物,序列如下:
上游P1(SEQ ID NO:5):CCGGAATTCGGCGGACACACCGCC
下游P2(SEQ ID NO:6):AAGGAAAAAAGCGGCCGCGCCCGCCTGGCG
PCR扩增的反应体系为50μL,其组成为:
2×buffer | 25μL |
dNTPs(2.5mmol/L each) | 2μL |
上游引物P1(20μmol/L) | 5μL |
下游引物P2(20μmol/L) | 5μL |
DNA模版 | 2μL |
Pyrobest酶 | 0.5μL |
ddH<sub>2</sub>O | 10.5μL |
总体积 | 50μL |
扩增程序的设置为:扩增条件为:95℃预变性10min;94℃变性30s,53℃退火45s,72℃延伸1min45s,反应30个循环;72℃延伸10min。将PCR产物进行琼脂糖凝胶电泳,可以看到野生型磷脂酶D基因的条带,共1527bp(见图1),再由小量DNA回收试剂盒回收PCR产物,得到了野生型磷脂酶D基因,即pld(SEQ ID NO.1所示)。
纯化后的pld与pET22b表达载体进行连接,随后将重组质粒化转入大肠杆菌DH5α中,通过EcoR I和Not I双酶切,成功验证野生型磷脂酶D基因已克隆至pET22b载体上。
实施例2:高活力磷脂酶D基因的筛选
1.基于易错PCR技术进行随机突变,构建新型磷脂酶D,设计引物如下:
上游P1(SEQ ID NO:5):CCGGAATTCGGCGGACACACCGCC
下游P2(SEQ ID NO:6):AAGGAAAAAAGCGGCCGCGCCCGCCTGGCG
在易错PCR反应体系中,以P1和P2作为上下游引物,以pET22b-pld即野生型磷脂酶D基因与pET22b载体连接的重组载体为模板,进行易错PCR。
其扩增的反应条件为:
扩增条件为:95℃预变性10min;98℃变性10s,53℃退火30s,72℃延伸1min45s反应30个循环;72℃延伸10min。
2.将磷脂酶D易错PCR产物克隆入表达载体pET22b中,转化大肠杆菌BL21(DE3),接种于每孔含200μL LB液体培养基(含30μg/mL的Kan)的96孔细胞培养板中,于37℃条件下200r/min摇床培养,当OD600达到0.6,向每个孔中加入IPTG(终浓度1mmol/L),在16℃下诱导16h,4℃下4000r/min离心15min后收集上清获得粗酶液,随后进行酶活力检测。
3.筛选高活力磷脂酶D基因
(1)磷脂酶D酶活测定原理
采用酶联比色法进行活性检测:磷脂酶D催化水解L-α-卵磷脂生成胆碱,胆碱在胆碱氧化酶的作用下生成过氧化氢,过氧化氢在过氧化酶的作用下与4-氨基氨替比林和苯酚生成醌亚胺显色物质,在500nm波长下有吸光值。
酶活定义:pH=8.0、T=37℃时,磷脂酶D1min内催化水解L-α-卵磷脂释放1.0μmol的胆碱所需要的酶量。
(2)高活力磷脂酶D酶活筛选方法
卵磷脂乳液:0.345g卵磷脂,2mL乙醚,3mL7.5%TritonX-100,20mLH2O,充分混匀。
反应终止液:1M Tris-HCl,0.5M EDTA,pH8.0。
磷脂酶D的筛选步骤:
向96孔板中加入115μL卵磷脂乳液,10μL 100mM Tris-HCl,5μL CaCl2,10μL粗酶液(如为酶粉,则采用PBS溶解后制成酶液;如为发酵液,则离心后取上清做酶液),37℃水浴反应10min,随后加入20μL反应终止液,煮沸5min,冷至室温。随后加入含有2U含胆碱氧化酶、4U过氧化酶、2mg 4-安替比林、1mg苯酚、20mg Triton X-100的180μL 10mM Tris-HCl,37℃反应20min,随后在500nm处测吸光值。
空白样品以水取代反应中的酶液,以此调零。
(3)磷脂酶D酶活测定
经测定,筛选出5个比野生型活力高的突变体,测序(北京华大生物工程公司)结果表明,5个磷脂酶D变体编码基因分别为:pldmD84I、pldmN153I、pldmG270F、pldmP316W和pldmA452F,酶活分别比pld编码的磷脂酶D提高了33%、29%、46%、38%、27%。
实施例3:在单个氨基酸突变的基础上获得多个氨基酸突变的磷脂酶D变体,以重叠PCR技术在pldmD84I突变体的基础上进行N153I、G270F、P316W、A452F突变为例,最终基因序列如SEQ ID NO:3所示,最终氨基酸序列如SEQ ID NO:4所示。
具体策略是:先在单个突变的基础上实现双突变,随后进行第三、第四和第五个氨基酸的突变。
首先在D84I基础上实现N153I的突变,步骤与实施例2一致,设计重叠引物,如下:
上游P1(SEQ ID NO.5):CCGGAATTCGGCGGACACACCGCC
下游P2(SEQ ID NO.6):AAGGAAAAAAGCGGCCGCGCCCGCCTGGCG
重叠引物P5(SEQ ID NO.7):CGGCAAGGTCACGCTCATCGTCGCCTC
重叠引物P6(SEQ ID NO.8):GAGGCGACGATGAGCGTGACCTTGCCG
重叠引物P5与P6包含了对153位氨基酸残基的突变。
将重组质粒pET22b-pldmD84I,即编码突变体pldmD84I的基因与pET22b载体连接的重组载体,为模板进行PCR扩增;
PCR1,反应体系为50μL,其组成为:
10×PCR buffer | 5μL |
dNTPs | 5μL |
上游引物P1 | 2μL |
下游引物P6 | 2μL |
pET22b-pldmD84I | 2μL |
Pyrobest酶 | 0.5μL |
ddH<sub>2</sub>O | 10.5μL |
总体积 | 50μL |
PCR2,反应体系为50μL,其组成为:
PCR1和PCR2扩增程序的设置为:95℃预变性10min;94℃变性30s,53℃退火45s,72℃延伸45s反应30个循环;72℃延伸10min。
PCR3,反应体系为46μL,其组成为:
10×PCR buffer | 5μL |
dNTPs | 5μL |
PCR1产物 | 2μL |
PCR2产物 | 2μL |
Pyrobest酶 | 0.5μL |
ddH<sub>2</sub>O | 31.5μL |
总体积 | 46μL |
PCR3扩增程序的设置为:95℃预变性10min;94℃变性30s,60℃退火45s,72℃延伸1min45 s反应30个循环;72℃延伸10min。
PCR4,反应体系为50μL,其组成为:
10×PCR buffer | 5μL |
dNTPs | 5μL |
上游引物P1 | 2μL |
下游引物P2 | 2μL |
PCR3产物 | 2μL |
Pyrobest酶 | 0.5μL |
ddH2O | 31.5μL |
总体积 | 50μL |
PCR4扩增程序的设置为:95℃预变性10min;94℃变性30s,53℃退火45s,72℃延伸1min45s反应30个循环;72℃延伸10min。
最终得到的PCR产物进行测序(北京华大生物工程公司),结果表明,此时扩增得到D84I和N153I双突变的磷脂酶D基因片段pldmD84I/N153I,氨基酸和碱基突变位点如表1所示。
继续进行其他各个突变,步骤与实施例2步骤一致,所有突变体引物序列见下表3,在pldmD84I/N153I的基础上按上述步骤,仅按表3更换引物,依次进行G270F,P316W,A452F的点突变及其组合突变,并送至测序公司测序,最终得到26株具有高活力磷脂酶D的菌株,分别为:BL21/pET22b-pldmD84I/N153I、BL21/pET22b-pldmD84I/G270F、BL21/pET22b-pldmD84I/P316W、BL21/pET22b-pldmD84I/A452F、BL21/pET22b-pldmN153I/G270F、BL21/pET22b-pldmN153I/P316W、BL21/pET22b-pldmN153I/A452F、BL21/pET22b-pldmG270F/P316W、BL21/pET22b-pldmG270F/A452F、BL21/pET22b-pldmP316W/A452F、BL21/pET22b-pldmD84I/N153I/G270F、BL21/pET22b-pldmD84I/N153I/P316W、BL21/pET22b-pldmD84I/N153I/A452F、BL21/pET22b-pldmD84I/G270F/P316W、BL21/pET22b-pldmD84I/G270F/A452F、BL21/pET22b-pldmD84I/P316W/A452F、BL21/pET22b-pldmN153I/G270F/P316W、BL21/pET22b-pldmN153I/G270F/A452F、BL21/pET22b-pldmG270F/P316W/A452F、BL21/pET22b-pldmN153I/P316W/A452F、BL21/pET22b-pldmD84I/N153I/P316W/A452F、BL21/pET22b-pldmD84I/N153I/G270F/P316W、BL21/pET22b-pldmD84I/N153I/G270F/A452F、BL21/pET22b-pldmD84I/G270F/P316W/A452F、BL21/pET22b-pldmN153I/G270F/P316W/A452F、BL21/pET22b-pldmD84I/N153I/G270F/P316W/A452F。
表3重叠PCR引物
突变位点 | F端引物 | R端引物 |
N153I | P5:SEQ ID NO.7 | P6:SEQ ID NO.8 |
G270F | P7:SEQ ID NO.9 | P8:SEQ ID NO.10 |
P316W | P9:SEQ ID NO.11 | P10:SEQ ID NO.12 |
A452F | P11:SEQ ID NO.13 | P12:SEQ ID NO.14 |
实施例4:枯草芽孢杆菌高活力磷脂酶D重组菌的构建
1.表达载体pBSA43的构建
以大肠杆菌-枯草芽孢杆菌穿梭克隆载体pBE2为骨架,克隆入一个强的芽孢杆菌组成型启动子P43和能够使重组蛋白直接分泌到培养基中的果聚糖蔗糖酶信号序列sacB获得了表达载体pBSA43。它带有Ampr和Kmr基因,可以在大肠杆菌中利用氨苄青霉素抗性作为筛选标记,同时又可以在枯草芽孢杆菌、地衣芽孢杆菌中利用卡那霉素抗性作为筛选标记。
2.构建高活力磷脂酶D表达载体pBSA43-pldmAxB
将上述获得的高活力磷脂酶D基因及野生型磷脂酶D基因分别与枯草芽孢杆菌表达载体pBSA43都经EcoR I和NotI双酶切,随后进行连接,构建得到重组质粒pBSA43-pldmAxB,转化至大肠杆菌DH5α感受态细胞,挑选阳性转化子,提取质粒进行酶切验证并测序,确定构建成功,即获得重组菌株pBSA43-pldmAxB。
3.表达载体pBSA43-pldmAxB转化枯草芽孢杆菌WB600
向预冷的1mm电转杯中加入60μL感受态细胞中和1μL(50ng/μL)pBSA43-pldmx,混匀并冰浴5min,设置参数(25μF,200Ω,4.5-5.0ms),电击一次,随后立即加入1mL复苏培养基(LB+0.5mol/L山梨醇+0.5mol/L甘露醇),混匀后吸取至1.5mLEP管中,37℃摇床震荡培养3h,离心后留取200μL复苏物涂布在具有抗性的LB平板上,37℃培养24h,挑取转化子,提质粒、酶切验证(pBSA43-pldmD84I酶切验证如图2所示,其他突变体基因重组质粒酶切验证同图2),得到枯草芽孢杆菌重组菌株WB600/pBSA43-pldmAxB。
实施例5:地衣芽孢杆菌高活力磷脂酶D重组菌的构建
1.表达载体pBSA43的构建
以大肠杆菌-地衣芽孢杆菌穿梭克隆载体pBE2为骨架,克隆入一个强的芽孢杆菌组成型启动子P43和能够使重组蛋白直接分泌到培养基中的果聚糖蔗糖酶信号序列sacB获得了表达载体pBSA43。它带有Ampr和Kmr基因,可以在大肠杆菌中利用氨苄青霉素抗性作为筛选标记,同时又可以在枯草芽孢杆菌、地衣芽孢杆菌中利用卡那霉素抗性作为筛选标记。
2.构建高活力磷脂酶D表达载体pBSA43-pldmAxB
将上述获得的高活力磷脂酶D基因及野生型磷脂酶D基因分别与地衣芽孢杆菌表达载体pBSA43都经EcoR I和Not I双酶切,随后进行连接,构建得到重组质粒pBSA43-pldmAxB,转化至大肠杆菌DH5α感受态细胞,挑选阳性转化子,提取质粒进行酶切验证并测序,确定构建成功,即获得重组菌株pBSA43-pldmAxB。
3.表达载体pBSA43-pldmAxB转化地衣芽孢杆菌TCCC11965
向预冷的1mm电转杯中加入60μL感受态细胞中和1μL(50ng/μL)pBSA43-pldmAxB,混匀并冰浴5min,设置参数(25μF,200Ω,4.5-5.0ms),电击一次,随后立即加入1mL复苏培养基(LB+0.5mol/L山梨醇+0.5mol/L甘露醇),混匀后吸取至1.5mLEP管中,37℃摇床震荡培养3h,离心后留取200μL复苏物涂布在具有抗性的LB平板上,37℃培养24h,挑取转化子,提质粒、酶切验证(pBSA43-pldmD84I酶切验证如图3所示,其他突变体基因重组质粒酶切验证同图3),得到地衣芽孢杆菌重组菌株TCCC11965/pBSA43-pldmAxB。
实施例6:解淀粉芽孢杆菌高活力磷脂酶D重组菌的构建
1.表达载体pBSA43的构建
以大肠杆菌-解淀粉芽孢杆菌穿梭克隆载体pBE2为骨架,克隆入一个强的芽孢杆菌组成型启动子P43和能够使重组蛋白直接分泌到培养基中的果聚糖蔗糖酶信号序列sacB获得了表达载体pBSA43。它带有Ampr和Kmr基因,可以在大肠杆菌中利用氨苄青霉素抗性作为筛选标记,同时又可以在枯草芽孢杆菌、地衣芽孢杆菌中利用卡那霉素抗性作为筛选标记。
2.构建高活力磷脂酶D表达载体pBSA43-pldmAxB
将上述获得的高活力磷脂酶D基因及野生型磷脂酶D基因分别与解淀粉芽孢杆菌表达载体pBSA43都经EcoR I和Not I双酶切,随后进行连接,构建得到重组质粒pBSA43-pldmAxB,转化至大肠杆菌DH5α感受态细胞,挑选阳性转化子,提取质粒进行酶切验证并测序,确定构建成功,即获得重组菌株pBSA43-pldmAxB。
3.表达载体pBSA43-pldmAxB转化解淀粉芽孢杆菌CGMCC No.11218
向预冷的1mm电转杯中加入60μL感受态细胞中和1μL(50ng/μL)pBSA43-pldmAxB,混匀并冰浴5min,设置参数(25μF,200Ω,4.5-5.0ms),电击一次,随后立即加入1mL复苏培养基(LB+0.5mol/L山梨醇+0.5mol/L甘露醇),混匀后吸取至1.5mLEP管中,37℃摇床震荡培养3h,离心后留取200μL复苏物涂布在具有抗性的LB平板上,37℃培养24h,挑取转化子,提质粒、酶切验证(pBSA43-pldmD84I酶切验证如图4所示,其他突变体基因重组质粒酶切验证同图2),得到解淀粉芽孢杆菌重组菌株CGMCCNo.11218/pBSA43-pldmAxB。
实施例7:高活力磷脂酶D在枯草芽孢杆菌重组菌中的表达及制备
①将枯草芽孢杆菌重组菌株WB600/pBSA43-pldmAxB接种于含卡纳霉素(50μg/mL)LB液体培养基中,37℃,220r/min培养过夜;
②按1%接种量转接于50mL的LB培养基中,37℃,220r/min培养48h,4000r/min离心15min后收集上清获得粗酶液;
③将粗酶液先以25%饱和度的硫酸铵盐析除去杂蛋白,再把饱和度加大到65%,沉淀目的蛋白。溶解后,透析除盐,再将透析脱盐后得到的活性组分用0.02mol/LTris-HCl(pH7.0)缓冲液溶解,上样后用同样的缓冲液先洗脱未吸附的蛋白,再用含0~1mol/LNaCl的0.02mol/LTris-HCl(pH7.0)缓冲液梯度洗脱,收集目的蛋白。离子交换得到的活性组分先用含0.15mol/L NaCl的0.02mol/L Tris-HCl(pH7.0)缓冲液平衡,上样后用相同的缓冲液以0.5mL/min的速度洗脱,获得纯化的酶液。取纯化后酶液进行SDS-PAGE分析,结果如图5所示。将脱色完全的凝胶放入凝胶成像系统中拍照,并使用图像分析软件对目的蛋白进行纯度分析,分析结果显示蛋白纯度为98%。
④纯化后酶液经冷冻干燥制得高活力磷脂酶D纯酶酶粉。
⑤采用实施例2中的方法对酶粉进行酶活测定,经计算,得到野生型、pldmD84I、pldmN153I、pldmG270F、pldmP316W、pldmA452F、pldmD84I/N153I、pldmD84I/G270F、pldmD84I/P316W、pldmD84I/A452F、pldmN153I/G270F、pldmN153I/P316W、pldmN153I/A452F、pldmG270F/P316W、pldmG270F/A452F、pldmP316W/A452F、pldmD84I/N153I/G270F、pldmD84I/N153I/P316W、pldmD84I/N153I/A452F、pldmD84I/G270F/P316W、pldmD84I/G270F/A452F、pldmD84I/P316W/A452F、pldmN153I/G270F/P316W、pldmN153I/G270F/A452F、pldmG270F/P316W/A452F、pldmN153I/P316W/A452F、pldmD84I/N153I/P316W/A452F、pldmD84I/N153I/G270F/P316W、pldmD84I/N153I/G270F/A452F、pldmD84I/G270F/P316W/A452F、pldmN153I/G270F/P316W/A452F、pldmD84I/N153I/G270F/P316W/A452F的酶的比活分别为:17.9U/mg、25.1U/mg、21.7U/mg、28.3U/mg、24.6U/mg、20.4U/mg、36.7U/mg、42.8U/mg、38.2U/mg、31.4U/mg、39.9U/mg、37.9U/mg、30.4U/mg、45.5U/mg、39.7U/mg、36.1U/mg、62.6U/mg、58.8U/mg、46.8U/mg、68.3U/mg、61.2U/mg、53.7U/mg、66.7U/mg、59.7U/mg、48.5U/mg、62.6U/mg、85.3U/mg、75.1U/mg、72.4U/mg、81.5U/mg、80.3U/mg、103.9U/mg。
实施例8:高活力磷脂酶D在地衣芽孢杆菌重组菌中的表达及制备
①将地衣芽孢杆菌重组菌株TCCC11965/pBSA43-pldmAxB接种于含卡纳霉素(50μg/mL)LB液体培养基中,37℃,220r/min培养过夜;
②按1%接种量转接于50mL的LB培养基中,37℃,220r/min培养48h,4000r/min离心15min后收集上清获得粗酶液;
③随后采用实施例7的方法,使用分级盐析法沉淀酶蛋白,收集蛋白质沉淀,溶解后,透析除盐,再经离子交换层析、凝胶层析后,获得纯化的酶液。取纯化后酶液进行SDS-PAGE分析,结果如图6所示。将脱色完全的凝胶放入凝胶成像系统中拍照,并使用图像分析软件对目的蛋白进行纯度分析,分析结果显示蛋白纯度为96%。
④纯化后酶液经冷冻干燥制得高活力磷脂酶D纯酶酶粉。
⑤采用实施例2中的方法对酶粉进行酶活测定,经计算,得到野生型、pldmD84I、pldmN153I、pldmG270F、pldmP316W、pldmA452F、pldmD84I/N153I、pldmD84I/G270F、pldmD84I/P316W、pldmD84I/A452F、pldmN153I/G270F、pldmN153I/P316W、pldmN153I/A452F、pldmG270F/P316W、pldmG270F/A452F、pldmP316W/A452F、pldmD84I/N153I/G270F、pldmD84I/N153I/P316W、pldmD84I/N153I/A452F、pldmD84I/G270F/P316W、pldmD84I/G270F/A452F、pldmD84I/P316W/A452F、pldmN153I/G270F/P316W、pldmN153I/G270F/A452F、pldmG270F/P316W/A452F、pldmN153I/P316W/A452F、pldmD84I/N153I/P316W/A452F、pldmD84I/N153I/G270F/P316W、pldmD84I/N153I/G270F/A452F、pldmD84I/G270F/P316W/A452F、pldmN153I/G270F/P316W/A452F、pldmD84I/N153I/G270F/P316W/A452F的酶的比活分别为:18.1U/mg、25.2U/mg、21.5U/mg、28.6U/mg、24.4U/mg、20.3U/mg、36.9U/mg、42.7U/mg、38.8U/mg、31.8U/mg、39.5U/mg、37.0U/mg、30.2U/mg、45.5U/mg、39.6U/mg、36.6U/mg、62.1U/mg、58.0U/mg、46.6U/mg、68.1U/mg、60.9U/mg、53.8U/mg、66.5U/mg、60.2U/mg、48.3U/mg、62.7U/mg、85.8U/mg、75.3U/mg、72.1U/mg、81.7U/mg、80.4U/mg、104.2U/mg。
实施例9:高活力磷脂酶D在解淀粉芽孢杆菌重组菌中的表达及制备
①将解淀粉芽孢杆菌重组菌株CGMCC No.11218/pBSA43-pldmAxB接种于含卡纳霉素(50μg/mL)LB液体培养基中,37℃,220r/min培养过夜;
②按1%接种量转接于50mL的LB培养基中,37℃,220r/min培养48h,4000r/min离心15min后收集上清获得粗酶液;
③随后采用实施例7的方法,使用分级盐析法沉淀酶蛋白,收集蛋白质沉淀,溶解后,透析除盐,再经离子交换层析、凝胶层析后,获得纯化的酶液。取纯化后酶液进行SDS-PAGE分析,结果如图7所示。将脱色完全的凝胶放入凝胶成像系统中拍照,并使用图像分析软件对目的蛋白进行纯度分析,分析结果显示蛋白纯度为98%。
④纯化后酶液经冷冻干燥制得高活力磷脂酶D纯酶酶粉。
⑤采用实施例2中的方法对酶粉进行酶活测定,经计算,得到野生型、pldmD84I、pldmN153I、pldmG270F、pldmP316W、pldmA452F、pldmD84I/N153I、pldmD84I/G270F、pldmD84I/P316W、pldmD84I/A452F、pldmN153I/G270F、pldmN153I/P316W、pldmN153I/A452F、pldmG270F/P316W、pldmG270F/A452F、pldmP316W/A452F、pldmD84I/N153I/G270F、pldmD84I/N153I/P316W、pldmD84I/N153I/A452F、pldmD84I/G270F/P316W、pldmD84I/G270F/A452F、pldmD84I/P316W/A452F、pldmN153I/G270F/P316W、pldmN153I/G270F/A452F、pldmG270F/P316W/A452F、pldmN153I/P316W/A452F、pldmD84I/N153I/P316W/A452F、pldmD84I/N153I/G270F/P316W、pldmD84I/N153I/G270F/A452F、pldmD84I/G270F/P316W/A452F、pldmN153I/G270F/P316W/A452F、pldmD84I/N153I/G270F/P316W/A452F的酶的比活分别为:17.7U/mg、24.9U/mg、21.9U/mg、28.5U/mg、24.3U/mg、20.7U/mg、36.4U/mg、42.4U/mg、38.0U/mg、31.3U/mg、39.6U/mg、37.7U/mg、30.8U/mg、45.2U/mg、40.1U/mg、36.5U/mg、62.9U/mg、58.7U/mg、46.4U/mg、68.9U/mg、61.4U/mg、53.3U/mg、66.6U/mg、59.8U/mg、48.6U/mg、62.5U/mg、85.1U/mg、75.9U/mg、72.2U/mg、81.7U/mg、80.3U/mg、103.8U/mg。
实施例10:发酵液中磷脂酶D活力测定
磷脂酶D发酵液酶活测定,测定实施例7-9发酵得到的高活力磷脂酶D的发酵液酶活列表如下:
实施例11:以高活力磷脂酶D制备磷脂酸
底物为1g大豆卵磷脂(PC含量90%),溶于10mL pH7.0的磷酸缓冲液中,按每毫升的反应体系加入50U高活力磷脂酶D,其中高活力磷脂酶D为本发明实施例7-9制备(可由任意突变体发酵得到,催化时酶粉添加量达到50U/mL即可)。反应温度40℃,在磁力搅拌器搅拌作用下反应12h,随后通过30mL氯仿/甲醇(2:1)萃取获得磷脂酸,其制备磷脂酸的转化率为93%,PA转化率(摩尔%)=PA量/初始PC量×100%。
实施例12:以高活力磷脂酶D制备PS、PE、PG和PI
取1g大豆卵磷脂(PC含量90%)分别与2.5g丝氨酸、1mL乙醇胺、5mL甘油、5mL肌醇混合,溶于5mL pH5.5的乙酸-乙酸钠缓冲液中,最后混合,至使总体积为10mL,按每毫升的反应体系加入100U高活力磷脂酶D,其中高活力磷脂酶D为本发明实施例7-9制备(可由任意突变体发酵得到,催化时酶粉添加量达到100U/mL即可)。反应温度40℃,在磁力搅拌器搅拌作用下反应12h,随后通过30mL氯仿/甲醇(2:1)萃取获得PS、PE、PG和PI,其制备PS、PE、PG和PI的转化率分别为79.3%、53.9%、60.1%、32.1%。转化率(摩尔%)=产物量/初始PC量×100%。
SEQUENCE LISTING
<110> 天津科技大学
<120> 一种磷脂酶突变体及其合成甘油磷脂的方法
<130> 1
<160> 14
<170> PatentIn version 3.5
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gcggacacac cgcccacccc ccatctggac gccatcgagc ggtcgctgcg cgacacctcc 60
cccggcctcg aaggctcggt gtggcagcgc acggacggca accgcctgga cgccccggac 120
ggcgaccccg ccggctggct gctgcagacc cccggctgct ggggcgacgc cggctgcaag 180
gaccgcgccg gcacccggcg gctgctcgac aagatgaccc gcaacatcgc cgacgcccgg 240
cacaccgtgg acatctcctc gctggccccc ttccccaacg gcgggttcga ggacgcggtc 300
gtcgacggcc tcaaggcggt cgtcgcggcg gggcactccc cgcgggtgcg catcctggtc 360
ggcgccgccc cgatctacca cctcaacgtg gtgccgtccc gctaccgcga cgagctgatc 420
ggcaagctcg gcgcggcggc cggcaaggtc acgctcaacg tcgcctcgat gaccacgtcc 480
aagacgtcgc tctcctggaa ccactccaag ctcctcgtgg tcgacgggaa gacggccatc 540
acgggcggga tcaacggctg gaaggacgac tacctcgaca ccgcccaccc ggtgtcggac 600
gtggacatgg cgctcagcgg cccggccgcc gcctcggcgg ggaagtacct cgacaccctc 660
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ggcgcctcct gcatgccgtc gatggagcag gacgaggcgg gatccgcccc cgccgagccc 780
accggtgacg tccccgtcat cgcggtcggc ggcctcggcg tgggcatcaa ggagtccgac 840
ccctcctcgg gataccaccc ggacctgccg acggccccgg acaccaagtg caccgtgggg 900
ctgcacgaca acaccaacgc cgaccgcgac tacgacacgg tcaaccccga ggagaacgcg 960
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Ala Asp Thr Pro Pro Thr Pro His Leu Asp Ala Ile Glu Arg Ser Leu
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Gly Asn Arg Leu Asp Ala Pro Asp Gly Asp Pro Ala Gly Trp Leu Leu
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Gln Thr Pro Gly Cys Trp Gly Asp Ala Gly Cys Lys Asp Arg Ala Gly
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Thr Arg Arg Leu Leu Asp Lys Met Thr Arg Asn Ile Ala Asp Ala Arg
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His Thr Val Asp Ile Ser Ser Leu Ala Pro Phe Pro Asn Gly Gly Phe
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Glu Asp Ala Val Val Asp Gly Leu Lys Ala Val Val Ala Ala Gly His
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Ser Pro Arg Val Arg Ile Leu Val Gly Ala Ala Pro Ile Tyr His Leu
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Asn Val Val Pro Ser Arg Tyr Arg Asp Glu Leu Ile Gly Lys Leu Gly
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Ala Ala Ala Gly Lys Val Thr Leu Asn Val Ala Ser Met Thr Thr Ser
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Lys Thr Ser Leu Ser Trp Asn His Ser Lys Leu Leu Val Val Asp Gly
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Lys Thr Ala Ile Thr Gly Gly Ile Asn Gly Trp Lys Asp Asp Tyr Leu
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Asp Thr Ala His Pro Val Ser Asp Val Asp Met Ala Leu Ser Gly Pro
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Ala Ala Ala Ser Ala Gly Lys Tyr Leu Asp Thr Leu Trp Asp Trp Thr
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Cys Arg Asn Ala Ser Asp Pro Ala Lys Val Trp Leu Ala Thr Ser Asn
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Gly Ala Ser Cys Met Pro Ser Met Glu Gln Asp Glu Ala Gly Ser Ala
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Pro Ala Glu Pro Thr Gly Asp Val Pro Val Ile Ala Val Gly Gly Leu
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Gly Val Gly Ile Lys Glu Ser Asp Pro Ser Ser Gly Tyr His Pro Asp
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Leu Pro Thr Ala Pro Asp Thr Lys Cys Thr Val Gly Leu His Asp Asn
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Thr Asn Ala Asp Arg Asp Tyr Asp Thr Val Asn Pro Glu Glu Asn Ala
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Leu Arg Ser Leu Ile Ala Ser Ala Arg Ser His Val Glu Ile Ser Gln
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Gln Asp Leu Asn Ala Thr Cys Pro Pro Leu Pro Arg Tyr Asp Ile Arg
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Thr Tyr Asp Thr Leu Ala Gly Lys Leu Ala Ala Gly Val Lys Val Arg
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Ile Val Val Ser Asp Pro Ala Asn Arg Gly Ala Val Gly Ser Gly Gly
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Tyr Ser Gln Ile Lys Ser Leu Asp Glu Ile Ser Asp Thr Leu Arg Thr
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Arg Leu Val Ala Leu Thr Gly Asp Asn Glu Lys Ala Ser Arg Ala Leu
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Cys Gly Asn Leu Gln Leu Ala Ser Phe Arg Ser Ser Asp Ala Ala Lys
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ggcgaccccg ccggctggct gctgcagacc cccggctgct ggggcgacgc cggctgcaag 180
gaccgcgccg gcacccggcg gctgctcgac aagatgaccc gcaacatcgc cgacgcccgg 240
cacaccgtga tcatctcctc gctggccccc ttccccaacg gcgggttcga ggacgcggtc 300
gtcgacggcc tcaaggcggt cgtcgcggcg gggcactccc cgcgggtgcg catcctggtc 360
ggcgccgccc cgatctacca cctcaacgtg gtgccgtccc gctaccgcga cgagctgatc 420
ggcaagctcg gcgcggcggc cggcaaggtc acgctcatcg tcgcctcgat gaccacgtcc 480
aagacgtcgc tctcctggaa ccactccaag ctcctcgtgg tcgacgggaa gacggccatc 540
acgggcggga tcaacggctg gaaggacgac tacctcgaca ccgcccaccc ggtgtcggac 600
gtggacatgg cgctcagcgg cccggccgcc gcctcggcgg ggaagtacct cgacaccctc 660
tgggactgga cctgccgcaa cgcgtccgac ccggccaagg tgtggctcgc cacgtcgaac 720
ggcgcctcct gcatgccgtc gatggagcag gacgaggcgg gatccgcccc cgccgagccc 780
accggtgacg tccccgtcat cgcggtcttc ggcctcggcg tgggcatcaa ggagtccgac 840
ccctcctcgg gataccaccc ggacctgccg acggccccgg acaccaagtg caccgtgggg 900
ctgcacgaca acaccaacgc cgaccgcgac tacgacacgg tcaactggga ggagaacgcg 960
ctgcgttcgc tcatcgccag cgcgcgcagc cacgtcgaga tctcccagca ggacctcaac 1020
gccacctgcc cgccgttgcc gcgctacgac atccggacct acgacaccct cgcgggcaag 1080
ctggccgccg gggtcaaggt ccgcatcgtc gtcagcgatc ccgccaaccg cggcgccgtc 1140
ggcagcgggg gctactccca gatcaagtcc ctggacgaga tcagcgacac cctccgcacg 1200
cgtctcgtcg ccctgaccgg cgacaacgag aaggcgtcgc gggccctgtg cggcaacctg 1260
cagctcgcct cgttccgcag ctcggacgcc gcgaagtggg ccgacggcaa gccgtacgcg 1320
ctgcaccaca agctggtgtc ggtggacgac tcgttcttct acatcggctc caagaacctc 1380
tacccggcct ggctgcagga cttcggctac atcgtcgaga gccccgccgc ggcccagcag 1440
ctcaagaccg agctgctcga cccggagtgg aagtactccc agcaggcggc ggccaccccg 1500
gccggctgcc cggctcgcca ggcgggc 1527
<210> 4
<211> 509
<212> PRT
<213> 人工序列
<400> 4
Ala Asp Thr Pro Pro Thr Pro His Leu Asp Ala Ile Glu Arg Ser Leu
1 5 10 15
Arg Asp Thr Ser Pro Gly Leu Glu Gly Ser Val Trp Gln Arg Thr Asp
20 25 30
Gly Asn Arg Leu Asp Ala Pro Asp Gly Asp Pro Ala Gly Trp Leu Leu
35 40 45
Gln Thr Pro Gly Cys Trp Gly Asp Ala Gly Cys Lys Asp Arg Ala Gly
50 55 60
Thr Arg Arg Leu Leu Asp Lys Met Thr Arg Asn Ile Ala Asp Ala Arg
65 70 75 80
His Thr Val Ile Ile Ser Ser Leu Ala Pro Phe Pro Asn Gly Gly Phe
85 90 95
Glu Asp Ala Val Val Asp Gly Leu Lys Ala Val Val Ala Ala Gly His
100 105 110
Ser Pro Arg Val Arg Ile Leu Val Gly Ala Ala Pro Ile Tyr His Leu
115 120 125
Asn Val Val Pro Ser Arg Tyr Arg Asp Glu Leu Ile Gly Lys Leu Gly
130 135 140
Ala Ala Ala Gly Lys Val Thr Leu Ile Val Ala Ser Met Thr Thr Ser
145 150 155 160
Lys Thr Ser Leu Ser Trp Asn His Ser Lys Leu Leu Val Val Asp Gly
165 170 175
Lys Thr Ala Ile Thr Gly Gly Ile Asn Gly Trp Lys Asp Asp Tyr Leu
180 185 190
Asp Thr Ala His Pro Val Ser Asp Val Asp Met Ala Leu Ser Gly Pro
195 200 205
Ala Ala Ala Ser Ala Gly Lys Tyr Leu Asp Thr Leu Trp Asp Trp Thr
210 215 220
Cys Arg Asn Ala Ser Asp Pro Ala Lys Val Trp Leu Ala Thr Ser Asn
225 230 235 240
Gly Ala Ser Cys Met Pro Ser Met Glu Gln Asp Glu Ala Gly Ser Ala
245 250 255
Pro Ala Glu Pro Thr Gly Asp Val Pro Val Ile Ala Val Phe Gly Leu
260 265 270
Gly Val Gly Ile Lys Glu Ser Asp Pro Ser Ser Gly Tyr His Pro Asp
275 280 285
Leu Pro Thr Ala Pro Asp Thr Lys Cys Thr Val Gly Leu His Asp Asn
290 295 300
Thr Asn Ala Asp Arg Asp Tyr Asp Thr Val Asn Trp Glu Glu Asn Ala
305 310 315 320
Leu Arg Ser Leu Ile Ala Ser Ala Arg Ser His Val Glu Ile Ser Gln
325 330 335
Gln Asp Leu Asn Ala Thr Cys Pro Pro Leu Pro Arg Tyr Asp Ile Arg
340 345 350
Thr Tyr Asp Thr Leu Ala Gly Lys Leu Ala Ala Gly Val Lys Val Arg
355 360 365
Ile Val Val Ser Asp Pro Ala Asn Arg Gly Ala Val Gly Ser Gly Gly
370 375 380
Tyr Ser Gln Ile Lys Ser Leu Asp Glu Ile Ser Asp Thr Leu Arg Thr
385 390 395 400
Arg Leu Val Ala Leu Thr Gly Asp Asn Glu Lys Ala Ser Arg Ala Leu
405 410 415
Cys Gly Asn Leu Gln Leu Ala Ser Phe Arg Ser Ser Asp Ala Ala Lys
420 425 430
Trp Ala Asp Gly Lys Pro Tyr Ala Leu His His Lys Leu Val Ser Val
435 440 445
Asp Asp Ser Phe Phe Tyr Ile Gly Ser Lys Asn Leu Tyr Pro Ala Trp
450 455 460
Leu Gln Asp Phe Gly Tyr Ile Val Glu Ser Pro Ala Ala Ala Gln Gln
465 470 475 480
Leu Lys Thr Glu Leu Leu Asp Pro Glu Trp Lys Tyr Ser Gln Gln Ala
485 490 495
Ala Ala Thr Pro Ala Gly Cys Pro Ala Arg Gln Ala Gly
500 505
<210> 5
<211> 24
<212> DNA
<213> 人工序列
<400> 5
ccggaattcg gcggacacac cgcc 24
<210> 6
<211> 30
<212> DNA
<213> 人工序列
<400> 6
aaggaaaaaa gcggccgcgc ccgcctggcg 30
<210> 7
<211> 27
<212> DNA
<213> 人工序列
<400> 7
cggcaaggtc acgctcatcg tcgcctc 27
<210> 8
<211> 27
<212> DNA
<213> 人工序列
<400> 8
gaggcgacga tgagcgtgac cttgccg 27
<210> 9
<211> 25
<212> DNA
<213> 人工序列
<400> 9
tccccgtcat cgcggtcttc ggcct 25
<210> 10
<211> 25
<212> DNA
<213> 人工序列
<400> 10
aggccgaaga ccgcgatgac gggga 25
<210> 11
<211> 32
<212> DNA
<213> 人工序列
<400> 11
tacgacacgg tcaactggga ggagaacgcg ct 32
<210> 12
<211> 32
<212> DNA
<213> 人工序列
<400> 12
agcgcgttct cctcccagtt gaccgtgtcg ta 32
<210> 13
<211> 39
<212> DNA
<213> 人工序列
<400> 13
ggtggacgac tcgttcttct acatcggctc caagaacct 39
<210> 14
<211> 39
<212> DNA
<213> 人工序列
<400> 14
aggttcttgg agccgatgta gaagaacgag tcgtccacc 39
Claims (6)
1.一种磷脂酶D突变体,其特征在于,基于SEQ ID No.2所示的磷脂酶D氨基酸序列,发生如下突变获得:A452F。
2.权利要求1所述磷脂酶D突变体的编码基因。
3.权利要求1所述磷脂酶D突变体或权利要求2所述的基因的用途,其特征在于,用于磷脂酸、磷脂酰丝氨酸、磷脂酰乙醇胺、磷脂酰甘油和磷脂酰肌醇的制备。
4.一种包含权利要求2所述的基因的重组载体或重组菌。
5.如权利要求4所述的重组载体或重组菌,其特征在于,表达载体为pBSA43,宿主细胞为枯草芽孢杆菌WB600;表达载体为pBSA43,宿主细胞为解淀粉芽孢杆菌CGMCC No.11218;表达载体为pBSA43,宿主细胞为地衣芽孢杆菌TCCC11965。
6.权利要求1所述磷脂酶D突变体的制备方法,其特征在于,步骤如下:
(1)将权利要求2所述的基因进行酶切,与表达载体连接得到新的重组载体;
(2)将重组载体转化入宿主细胞中,得到重组菌株,之后将重组菌株发酵,得到高活力磷脂酶D。
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