CN114940985B - 兼具脱氧腺苷二磷酸激酶和乙酸激酶活性的蛋白及应用 - Google Patents
兼具脱氧腺苷二磷酸激酶和乙酸激酶活性的蛋白及应用 Download PDFInfo
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- CN114940985B CN114940985B CN202210416216.0A CN202210416216A CN114940985B CN 114940985 B CN114940985 B CN 114940985B CN 202210416216 A CN202210416216 A CN 202210416216A CN 114940985 B CN114940985 B CN 114940985B
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Abstract
本发明公开了兼具脱氧腺苷二磷酸激酶和乙酸激酶活性的蛋白及应用，涉及生物工程领域，兼具脱氧腺苷二磷酸激酶和乙酸激酶活性的蛋白的氨基酸序列如SEQ ID NO.1所示；兼具脱氧腺苷二磷酸激酶和乙酸激酶活性的蛋白基因的碱基序列如SEQ ID NO.2所示，蛋白的制备方法包括：重组菌株获取、重组菌株培养、诱导表达和提取蛋白。本发明的优点在于：提出一种兼具脱氧腺苷二磷酸激酶和乙酸激酶活性的蛋白，利用本发明提供的蛋白可实现一步二酶催化制备脱氧腺苷三磷酸，减少了催化用酶种类，简化了催化工艺过程，从更具有市场竞争力，并且所述生产脱氧腺苷三磷酸的方法适用于大规模工业化生产。
Description
技术领域
本发明涉及生物工程领域，具体是涉及兼具脱氧腺苷二磷酸激酶和乙酸激酶活性的蛋白及应用。
背景技术
脱氧腺苷三磷酸(dATP)分子量491，是由一个脱氧腺苷基团和3个磷酸基团组成，分子式是C10H16N5O12P3。它是生物体内DNA链的基本组成单元，是PCR(聚合酶链反应)所用的dNTP核苷酸小分子之一，广泛应用于基因合成、基因检测等领域。
dATP的合成主要有化学合成法、生物酶催化法。其中，传统的化学法制备dATP，使用多聚磷酸高温进行磷酸化，对磷酸化次数没有选择性，会产生3-5次磷酸化的dAP4-6副产物，产率仅有40％左右；而生物酶催化法可以避免化学法污染和收率低的问题，成为更具优势的合成方法。Pattama H和Somchai P以脱氧腺苷单磷酸(dAMP)为底物，分别添加脱氧腺苷单磷酸激酶、脱氧腺苷二磷酸激酶和丙酮酸激酶，成功合成dATP,但是酶种类多导致酶用量配比难以控制，且因为丙酮酸激酶对ADP和ATP磷酸化存在反应平衡，导致产率只有40-60％，不利于dATP的分离纯化，制备成本没用优势。专利号为CN101768617B的专利文件中公开了一种使用酵母细胞全细胞催化dAMP合成dATP，转化率达到93.6％，但是底物浓度仅有5mmol/L,没有规模化生产价值。
发明内容
为解决上述技术问题，提供一种兼具脱氧腺苷二磷酸激酶和乙酸激酶活性的蛋白及应用，本技术方案针对现有技术中所存在的添加酶种类多、反应转化率低的缺陷，本发明提供了一种脱氧腺苷二磷酸激酶和乙酸激酶双功能的蛋白(dACK)，并将所述蛋白与腺苷单磷酸激酶(dAMPK)配合，实现脱氧腺苷单磷酸(dAMP)高效转化为脱氧腺苷三磷酸(dATP)的制备方法。
为达到以上目的，本发明采用的技术方案为：
一种兼具脱氧腺苷二磷酸激酶和乙酸激酶活性的蛋白，所述兼具脱氧腺苷二磷酸激酶和乙酸激酶活性的蛋白的氨基酸序列如SEQ ID NO.1所示；
进一步的，提出一种兼具脱氧腺苷二磷酸激酶和乙酸激酶活性的蛋白的蛋白编码基因，用于编码如上述的兼具脱氧腺苷二磷酸激酶和乙酸激酶活性的蛋白。
优选的，所述兼具脱氧腺苷二磷酸激酶和乙酸激酶活性的蛋白的蛋白编码基因的碱基序列如SEQ ID NO.2所示；
再进一步的，提出一种蛋白编码基因重组载体，所述蛋白编码基因重组载体为包含上的兼具脱氧腺苷二磷酸激酶和乙酸激酶活性的蛋白编码基因的重组质粒pET-22b。
再进一步的，提出一种蛋白编码基因重组菌种，所述蛋白编码基因重组菌种为包含上述兼具脱氧腺苷二磷酸激酶和乙酸激酶活性的蛋白编码基因的重组表达基因工程菌E.coli BL21-pET-22b-dACK。
再进一步的，提出一种蛋白的制备方法，用于制备上述兼具脱氧腺苷二磷酸激酶和乙酸激酶活性的蛋白，其特征在于，包括如下步骤：
重组菌株获取，根据上述的蛋白编码基因针对大肠杆菌做密码子优化，将优化后的DNA序列合成并重组到表达载体pET-22b(含酶切位点BamH I、Hind III)上，重组质粒转化至E.coli BL21，获得重组表达基因工程菌E.coli BL21-pET-22b-dACK；
重组菌株培养，将上述含有所述蛋白编码基因的基因工程菌E.coli BL21-pET-22b-dACK接种到含有氨苄青霉素的5mL LB液体培养基的50mL的摇管中，在摇床上37℃恒温培养8h，转速200rpm；
诱导表达，将培养菌液按2％接种量接入含有100mL诱导培养基TB中的500mL摇瓶中，于200rpm，37℃培养2h，待OD600达到0.2左右时转16℃诱导24h离心收集菌体；
提取蛋白，超声破菌，离心取上清液为粗酶液dACK，置于4℃冰箱保存。
可选的，所述LB液体培养基由如下浓度的成分组成：
蛋白胨10g/L，氯化钠10g/L，酵母提取物5g/L；
所述诱导培养基TB由如下浓度的成分组成：
酵母粉25g/L，胰蛋白胨15g/L，氯化钠10g/L，葡萄糖2g/L，乳糖3g/L。
在进一步的，提出一种制备脱氧腺苷三磷酸的方法，包括如下步骤：
S1.向反应器中加入包含脱氧腺苷单磷酸、ATP和乙酰磷酸的溶液，调节所述溶液的pH至6.0；
S2.向所述溶液中加入上述兼具脱氧腺苷二磷酸激酶和乙酸激酶活性的蛋白和脱氧腺苷单磷酸激酶，得到反应体系并搅拌均匀；
S3.将所述反应体系在恒温水浴摇床中进行反应，摇床转速设置为50rpm，控制反应体系温度在40℃，保持反应体系的pH在6.0-7.0之间；
S4..获得含脱氧腺苷三磷酸的粗溶液；
S5.将所述粗溶液过滤，纯化，干燥，得到脱氧腺苷三磷酸。
精选的，所述包含脱氧腺苷单磷酸、ATP和乙酰磷酸的溶液中按摩尔浓度计由如下成分组成：
脱氧腺苷单磷酸300mmol/L、ATP 2mmol/L、MgCl2 150mmol/L、乙酰磷酸600mmol/L；
所述脱氧腺苷单磷酸激酶的浓度为50ml/L。
精选的，所述蛋白以酶溶液、酶冻干粉、含酶细胞、固定化酶或固定化酶的细胞其中一种或多种形式使用。
与现有技术相比，本发明的优点在于：
构建了含有兼具脱氧腺苷二磷酸激酶和乙酸激酶活性的蛋白编码基因的重组质粒，并在工程菌E.coli BL21中实现的兼具脱氧腺苷二磷酸激酶和乙酸激酶活性的蛋白的高效表达，与现有乙酸激酶或者脱氧腺苷二磷酸激酶相比，本发明的蛋白兼具脱氧腺苷二磷酸激酶和乙酸激酶活性，因此，利用本发明提供的蛋白实现一步二酶催化制备脱氧腺苷三磷酸，减少了催化用酶种类，简化了催化工艺过程，从更具有市场竞争力，并且所述生产脱氧腺苷三磷酸的方法适用于大规模工业化生产。
附图说明
图1为本发明提出的兼具脱氧腺苷二磷酸激酶和乙酸激酶活性的蛋白的电泳图；
图2为本发明实施例三中的反应过程；
图3-5分别与dAMP、dADP和dATP的标准品成分谱系图；
图6为本发明中实施例三种的dAMP反应液的成分谱系图。
具体实施方式
以下描述用于揭露本发明以使本领域技术人员能够实现本发明。以下描述中的优选实施例只作为举例，本领域技术人员可以想到其他显而易见的变型。
下述各实施例中，各英文简称含义为：
dACK：本发明制得的兼具脱氧腺苷二磷酸激酶和乙酸激酶活性的蛋白；
dAMP：脱氧腺苷单磷酸；
dAMPK：脱氧腺苷单磷酸激酶；
dATP：脱氧腺苷三磷酸；
dADP：脱氧腺苷二磷酸。
实施例一：
重组菌株的获得：
根据兼具脱氧腺苷二磷酸激酶和乙酸激酶活性的蛋白的蛋白编码基因针对大肠杆菌做密码子优化，将优化后的DNA序列合成并重组到表达载体pET-22b(含酶切位点BamHI、Hind III)上，重组质粒转化至E.coli BL21，获得重组表达基因工程菌E.coli BL21-pET-22b-dACK。
实施例二：
酶的制备：
将上述含有所述蛋白编码基因的基因工程菌E.coli BL21-pET-22b-dACK接种到含有氨苄青霉素的5mL LB液体培养基的50mL的摇管中，在摇床上37℃恒温培养8h，转速200rpm，之后将培养菌液按2％接种量接入含有100mL诱导培养基TB中的500mL摇瓶中，于200rpm，37℃培养2h，待OD 600达到0.2左右时转16℃诱导24h离心收集菌体，之后进行超声破菌，离心取上清液为粗酶液dACK，置于4℃冰箱保存，如图1所示，重组蛋白分子量为40kDa；
其中LB液体培养基由如下浓度的成分组成：蛋白胨10g/L，氯化钠10g/L，酵母提取物5g/L；
诱导培养基TB由如下浓度的成分组成：酵母粉25g/L，胰蛋白胨15g/L，氯化钠10g/L，葡萄糖2g/L，乳糖3g/L。
实施例三：
酶活鉴定：
为了验证dACK酶具有脱氧腺苷二磷酸激酶和乙酸激酶活性，设计如图2的反应过程，假设dACK具有两种酶活性，那么在以脱氧腺苷单磷酸dAMP、乙酰磷酸和少量ATP为底物的反应条件下，添加dACK和dAMPK两种酶，大部分的dAMP可以转化为dATP；假设dACK只具有乙酸激酶活性，那么以脱氧腺苷单磷酸dAMP、乙酰磷酸和少量ATP为底物的反应条件下，大部分的dAMP可以转化为dADP；假设dACK没有任何一种活性，那么以脱氧腺苷单磷酸dAMP、乙酰磷酸和少量ATP为底物的反应条件下，大部分的dAMP不会发生反应。实验条件：100ml纯化水中加入50mmol/L脱氧腺苷单磷酸、100mmol/L乙酰磷酸、2mmol/L ATP、30mmol/L氯化镁，用30％NaOH调整PH至6.0,加入dACK酶液10ml和dAMPK酶液5ml，40℃反应3小时，加入浓盐酸调整PH至1.5中止反应，反应液HPLC检测dATP生成量，结果如图6所示，参照图3-5，可得dATP反应液中，绝大多dAMP转化为dATP，证明本发明蛋白具有脱氧腺苷二磷酸激酶和乙酸激酶酶活。
实施例四：
dATP制备：
向反应器中加入含有300mmol/L dAMP、2mmol/L ATP、150mM MgCl2、600mmol/L乙酰磷酸的底物溶液，调节pH至6.0。然后加入催化酶，添加量分别为：100ml/L的离心上清dACK酶和50ml/LdAMPK酶，搅拌均匀后，在恒温水浴摇床中进行反应。摇床转速设置为50rpm，反应温度控制在40℃，pH保持在6.0-7.0。反应4小时后，得到含粗品dATP的溶液，含282mmol/L dATP，转化率达94％，经过滤，纯化，干燥，得到产物dATP；
本实施例中，dACK酶采用离心上清的形式添加，在其他实施例中，dACK酶还可采用酶冻干粉、含酶细胞、固定化酶或固定化酶的细胞其中一种或多种形式使用。
综上所述，本发明的优点在于：提出一种兼具脱氧腺苷二磷酸激酶和乙酸激酶活性的蛋白，利用本发明提供的蛋白可实现一步二酶催化制备脱氧腺苷三磷酸，减少了催化用酶种类，简化了催化工艺过程，从更具有市场竞争力，并且所述生产脱氧腺苷三磷酸的方法适用于大规模工业化生产。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是本发明的原理，在不脱离本发明精神和范围的前提下本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明的范围内。本发明要求的保护范围由所附的权利要求书及其等同物界定。
序列表
<110> 苏州酶泰生物科技有限公司
<120> 兼具脱氧腺苷二磷酸激酶和乙酸激酶活性的蛋白及其应用
<130> 2022.04.04
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Claims (4)
1.一种制备脱氧腺苷三磷酸的方法，其特征在于，包括如下步骤：
S1.向反应器中加入包含脱氧腺苷单磷酸、ATP和乙酰磷酸的溶液，调节所述溶液的pH至6.0；
S2.向所述溶液中加入兼具脱氧腺苷二磷酸激酶和乙酸激酶活性的蛋白和脱氧腺苷单磷酸激酶，得到反应体系并搅拌均匀；
S3.将所述反应体系在恒温水浴摇床中进行反应，摇床转速设置为50rpm，控制反应体系温度在40℃，保持反应体系的pH在6.0-7.0之间；
S4.获得含脱氧腺苷三磷酸的粗溶液；
S5.将所述粗溶液过滤，纯化，干燥，得到脱氧腺苷三磷酸；
其中，所述的兼具脱氧腺苷二磷酸激酶和乙酸激酶活性的蛋白的制备方法包括如下步骤：
重组菌株获取，根据兼具脱氧腺苷二磷酸激酶和乙酸激酶活性的蛋白的蛋白编码基因针对大肠杆菌做密码子优化，将优化后的DNA序列合成并重组到表达载体pET-22b(含酶切位点BamH I、HindIII)上，重组质粒转化至E.coli BL21，获得重组表达基因工程菌E.coliBL21-pET-22b-dACK；
重组菌株培养，将上述含有所述蛋白编码基因的基因工程菌E.coli BL21-pET-22b-dACK接种到含有氨苄青霉素的5mL LB液体培养基的50mL的摇管中，在摇床上37℃恒温培养8h，转速200rpm；
诱导表达，将培养菌液按2％接种量接入含有100mL诱导培养基TB中的500mL摇瓶中，于200rpm，37℃培养2h，待OD 600达到0.2左右时转16℃诱导24h离心收集菌体；
提取蛋白，超声破菌，离心取上清液为粗酶液dACK，置于4℃冰箱保存；
所述兼具脱氧腺苷二磷酸激酶和乙酸激酶活性的蛋白的氨基酸序列如SEQ ID NO.1所示；
所述兼具脱氧腺苷二磷酸激酶和乙酸激酶活性的蛋白的蛋白编码基因的碱基序列如SEQID NO.2所示。
2.根据权利要求1所述的一种制备脱氧腺苷三磷酸的方法，其特征在于，所述LB液体培养基由如下浓度的成分组成：
蛋白胨10g/L，氯化钠10g/L，酵母提取物5g/L；
所述诱导培养基TB由如下浓度的成分组成：
酵母粉25g/L，胰蛋白胨15g/L，氯化钠10g/L，葡萄糖2g/L，乳糖3g/L。
3.根据权利要求1所述的一种制备脱氧腺苷三磷酸的方法，其特征在于，所述包含脱氧腺苷单磷酸、ATP和乙酰磷酸的溶液中按摩尔浓度计由如下成分组成：
脱氧腺苷单磷酸300mmol/L、ATP2mmol/L、MgCl 2 150mmol/L、乙酰磷酸600mmol/L；
所述脱氧腺苷单磷酸激酶的浓度为50ml/L。
4.根据权利要求1所述的一种制备脱氧腺苷三磷酸的方法，其特征在于，所述蛋白以酶溶液、酶冻干粉、含酶细胞或固定化酶其中一种或多种形式使用。
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