CN112522231B - 一种酰基转移酶及其编码基因和应用 - Google Patents
一种酰基转移酶及其编码基因和应用 Download PDFInfo
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- CN112522231B CN112522231B CN202011474193.6A CN202011474193A CN112522231B CN 112522231 B CN112522231 B CN 112522231B CN 202011474193 A CN202011474193 A CN 202011474193A CN 112522231 B CN112522231 B CN 112522231B
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Abstract
本发明公开了一种酰基转移酶及其编码基因和应用。所述的酰基转移酶Tri18，其氨基酸序列如SEQ ID NO.2所示。本发明首次从广藿香内生真菌露湿漆斑菌Myrothecium roridum A553中发掘了在D型单端孢霉烯大环结构形成过程中发挥重要作用的新型酰基转移酶Tri18。本发明所得的新型酰基转移酶Tri18能和乙酰CoA发生相互作用，发挥酰基转移的作用。本发明为解析单端孢霉烯的生物合成途径并促进酰基转移酶在生物催化中的应用奠定分子生物学基础，从而为解析露湿漆斑菌中单端孢霉烯的生物合成机制奠定了分子生物学基础，也为促进酰基转移酶在生物转化中的应用提供了物质基础。
Description
技术领域：
本发明属于基因工程领域，具体涉及一种新型植物内生真菌露湿漆斑菌来源的新型酰基转移酶及其编码基因和应用。
背景技术：
露湿漆斑菌是一种对大豆等经济作物具有显著致病性的病原菌，对我国大豆的产量造成了不可估量的损失。据研究报道，单端孢霉烯毒素是露湿漆斑菌的重要致病因子。本发明人前期从药用植物广藿香内生真菌中分离得到了露湿漆斑菌A553，从中分离获得了大量单端孢霉烯类化合物，对肿瘤细胞具有显著的细胞毒活性。单端孢霉烯毒素与靶向药物的结合在抗肿瘤药物开发方面有巨大的应用前景。
发明内容：
本发明的第一个目的是提供一种植物内生真菌露湿漆斑菌来源的新型酰基转移酶Tri18。
本发明的新型乙酰转移酶Tri18，是通过以下方法制备的：
以广藿香内生真菌露湿漆斑菌Myrothecium roridumA553基因组为模板，根据基因簇Tricluster中的Tri8基因序列(如SEQ ID NO.1所示)设计引物，通过分段式扩增融合PCR获得酰基转移酶Tri18的编码基因-基因Tri18。回收目的片段，采用ClonExpress UltraOne Step Cloning Kit(诺唯赞生物科技有限公司，南京)同源重组试剂盒将目的基因插入用限制性内切酶NdeI、HindⅢ双酶切的表达载体pET30a，用含50μg/mL Kana的LB平板筛选，挑取克隆扩大培养，采用菌液PCR扩增Tri18基因筛选阳性克隆并测序验证。Ni亲和层析柱对酰基转移酶Tri18进行纯化，用含不同浓度咪唑的洗脱液进行洗脱，从而获得纯的酰基转移酶Tri18。以乙酰CoA，丁醇为底物，加入酰基转移酶Tri18，并加入DTNB进行显色，通过检测412nm处的吸光值测定酰基转移酶Tri18的活力，其Vmax为307.7±10.91U/mg，Km为376.5±30.07μM，Kcat为1538.5S-1，Kcat/Km为245.18μM/min。酰基转移酶Tri18在250μM乙酰辅酶A，pH＝8.0，室温反应条件下的酶活大概是115U/mg。
本发明的酰基转移酶Tri18，其氨基酸序列如SEQ ID NO.2所示。
本发明的第二个目的是提供编码上述酰基转移酶Tri18的编码基因。
所述的编码基因，其核苷酸序列如SEQ ID NO.1所示。
本发明的第三个目的是提供上述酰基转移酶Tri18在催化酰基转移形成酯或酰胺中的应用。
酰基转移酶在活性次级代谢产物骨架的生物合成及其后修饰过程中发挥重要功能，本发明首次从广藿香内生真菌露湿漆斑菌Myrothecium roridum A553中发掘了在D型单端孢霉烯大环结构形成过程中发挥重要作用的新型酰基转移酶Tri18。本发明所得的新型酰基转移酶Tri18能和乙酰CoA发生相互作用，发挥酰基转移的作用。本发明为解析单端孢霉烯的生物合成途径并促进酰基转移酶在生物催化中的应用奠定分子生物学基础，从而为解析露湿漆斑菌中单端孢霉烯的生物合成机制奠定了分子生物学基础，也为促进酰基转移酶在生物转化中的应用提供了物质基础。
附图说明
图1为重组载体pET30-Tri18的构建：A)为酰基转移酶基因Tri18的扩增；B)为酰基转移酶基因Tri18插入pET30a的菌液PCR验证；
图2为酰基转移酶Tri18蛋白的表达纯化图，S代表破碎后上清，P代表破碎后沉淀，F表示Ni柱穿过液，10％-100％表示不同浓度咪唑的洗脱液；
图3为酰基转移酶Tri18的酶学性质分析结果：A)为酰基转移酶Tri18的最适温度分析；B)酰基转移酶Tri18的最适pH分析；C)酰基转移酶Tri18的热稳定性分析；D)酰基转移酶Tri18的动力学分析。
具体实施方式
以下将参照附图，结合具体实施例对本发明进行进一步解释。但实施例本身对本发明不做任何形式的限定。
实施例1:露湿漆斑菌Myrothecium roridum A553中新型酰基转移酶Tri18的表达纯化
提取露湿漆斑菌Myrothecium roridum A553的基因组，用引物Tri18-F：CTTTAAGAAGGAGATATACAATGATGGCCTCCGTGGACCAAC和Tri18-R：TGGTGCTCGAGTGCGGCCGCACTCCTCGGCAAGAGAATCTGTG：扩增目的基因Tri18(图1A)，然后通过融合PCR去除内含子，从而获得tri18基因的完整片段(其编码核苷酸序列如SEQ ID NO.1所示，编码的酰基转移酶Tri18的氨基酸序列如SEQ ID NO.2所示)。将完整片段进行切胶回收，然后采用ClonExpress Ultra OneStep Cloning Kit(诺唯赞生物科技有限公司，南京)同源重组试剂盒将目的基因插入用限制性内切酶NdeI、HindⅢ双酶切的表达载体pET30a，反应体系如表1所示，50℃反应15min。
表1连接反应体系
连接产物转化大肠杆菌DH5α感受态细胞，用含有50μg/mLKana的LB平板筛选阳性克隆，采用菌液PCR扩增tri18基因，结果初步验证了基因Tri18成功插入至pET30a(见图1B)。通过测序结果进一步证实表达载体的成功构建。
将构建成功的重组质粒pET30a-Tri18转化至E.coli BL21(DE3)中，经IPTG诱导表达。结果表明：低温条件(18℃)下，0.1mM IPTG、200r/min过夜振荡诱导，经诱导原核表达的目的蛋白大小约为65.40kDa，而且蛋白主要以可溶形式表达(图2A)。
上清总蛋白经Ni柱的各部分洗脱液SDS-PAGE检测的结果如图2B。结果显示：在100mM咪唑的洗脱液中检测到纯度较高的酰基转移酶Tri18，经Bandscan 5.0分析，100mM咪唑洗脱液中酰基转移酶Tri18的纯度为97.8％。
实施例2：
新型酰基转移酶Tri18蛋白的酶学性质分析，具体步骤如下：
酰基转移酶活性检测参考Garvey等人的方法，配制BufferA：5mM acetyl CoA,0.1M PBS,pH 8.0；Buffer B：5g/L丁醇,0.1M PBS,pH 8.0；Buffer C：0.2mg/mL酰基转移酶Tri18；Buffer D：20mM DTNB,0.1M PBS,pH 8.0。
配制反应体系(300μL)，按体积比：10％A+10％B+10％C+1％D+69％PBS Buffer，同时配制一组不含酰基转移酶Tri18的空白对照，分别混匀，置于比色皿中，在反应进行0、5、10、15、20min时，用酶标仪分别测得其在412nm下的吸收值。常温下，每mL反应体系中每mg蛋白每分钟催化吸光值变化0.001个单位为一个酶活单位，以U/mg表示。计算公式为：
U/mg＝(△A测-△A空)/0.001/(Cpr*0.1mL)/T
Cpr-蛋白浓度，mg/mL；T-反应时间，min
温度会影响酶的催化活性，pH会影响酶的构象，还会影响酶与底物的解离状态，从而影响酶的活性和稳定性。
在pH7.0条件下测定不同温度下酰基转移酶Tri18对底物乙酰CoA的酶活力。
在温度为40℃条件下，测定不同pH值酰基转移酶Tri18对底物乙酰CoA的酶活力。
具体结果如下，酰基转移酶Tri18最适反应温度为40℃，但在反应体系为30-40℃温度时，保持了相近的酶活力(图3A)；最适反应的pH为6.0，在弱酸性条件下(pH＝4.0-5.0)酶活有较大丧失，在弱碱性条件下(pH＝7.0-8.0)酶活相对保留较好(图3B)。酰基转移酶Tri18的热稳定性不太理想，在40℃下孵育90min酶活保留40％左右(图3C)。通过改变反应体系中乙酰辅酶A的浓度(乙酰基受体仍假定为丁醇)，测得酰基转移酶Tri18的Vmax为307.7±10.91U/mg，Km为376.5±30.07μM，Kcat为1538.5S-1，Kcat/Km为245.18μM/min(图3D)。酰基转移酶Tri18在250μM乙酰辅酶A，pH＝8.0，室温反应条件下的酶活大概是115U/mg。
序列表
<110> 广东省微生物研究所（广东省微生物分析检测中心）
<120> 一种酰基转移酶及其编码基因和应用
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Claims (5)
1.一种酰基转移酶Tri18，其特征在于，其氨基酸序列如SEQ ID NO.2所示。
2.编码权利要求1所述的酰基转移酶Tri18的酰基转移酶基因。
3.根据权利要求2所述的酰基转移酶基因，其特征在于，其核苷酸序列如SEQ ID NO.1所示。
4.一种表达载体，其特征在于，含有权利要求2所述的酰基转移酶Tri18的酰基转移酶基因。
5.权利要求1所述的酰基转移酶Tri18在催化酰基转移形成酯中的应用，所述的催化，其供体是乙酰辅酶A，受体是丁醇。
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