CN104059893B - Cyp119‑t213g酶及其纯化方法 - Google Patents
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Abstract
本发明属于生物工程技术领域,具体涉及CYP119‑T213G酶及其纯化方法。本发明要解决的技术问题是提供一种催化效率更高的CYP119酶。本发明的技术方案是一种CYP119‑T213G酶,其氨基酸序列如SEQ ID No.2所示。本发明还提供了表达CYP119‑T213G酶的载体。本发明还提供了CYP119‑T213G酶的纯化方法。本发明提供了一种催化效率更高的CYP119酶,具有广泛的应用前景。
Description
技术领域
本发明属于生物工程技术领域,具体涉及CYP119-T213G酶及其纯化方法。
背景技术
CYP119酶(cytochrome P450 119)来自黄石公园火山口分离的嗜酸嗜热硫矿硫叶菌(Sulfolobus solfataricus),因此该酶具耐酸抗高温等作用。CYP119酶能催化月桂酸和苯乙烯等底物发生环氧化反应,该反应绿色环保具有较高应用前景。目前文献报道CYP119酶在过氧化氢或假单孢氧还蛋白-还原酶(Pd/PdR)和辅酶NADPH条件下能催化月桂酸羟基化和苯乙烯环氧化的反应。在常温下CYP119酶催化苯乙烯环氧化反应的过氧化氢酶途径中,催化常数为Kcat=0.6min-1(Laura S.Koo et al.2000)。R和S构型的环氧苯乙烷之比约为1:3。后来Rabe KS等,考察了CYP119酶催化苯乙烯环氧化反应的最佳温度和pH值,结果表明,温度为70℃,叔丁基过氧化氢(TBHP)为辅助氧化剂,pH8.5的甘氨酸缓冲液为野生型CYP119酶催化苯乙烯环氧化反应的最佳反应条件。通过动力学研究得到了其kcat=78.2±20.6min-1,Km=9.2±4.3Mm,比常温下提高了100多倍,但是随着温度的升高,其对应选择性降低。为改善酶的催化效率,Laura S.Koo(2000)小组将野生型酶进行突变,发现突变体T214V能明显改善与苯乙烯的结合能力,过氧化氢途径下的苯乙烯环氧化反应速率比野生型提高了近3倍,对应选择性未发生改变。该小组发现213位是该酶的活性中心,而214位在电子传递中起到关键作用(Laura S.K et al.2002)。在213位进行了A、V、S、F、W几种氨基酸的突变,仅T213A的催化效率接近野生型酶,其它几种突变体催化效率明显降低。
发明内容
本发明要解决的技术问题是提供一种催化效率更高的CYP119酶。
本发明的技术方案是一种CYP119-T213G酶,其氨基酸序列如SEQ ID No.2所示。
进一步的,编码CYP119-T213G酶的核苷酸序列如SEQ ID No.1所示。
本发明还提供了表达CYP119-T213G酶的载体。
进一步的,所述的载体为质粒。
本发明还提供了包含所述载体的宿主。
进一步的,所述的宿主为大肠杆菌。
本发明还提供了CYP119-T213G酶的纯化方法,包括如下步骤:
a、取发酵后的菌液离心收集菌体,PBS悬浮菌体,破碎细胞,离心,收集上清液,获得粗酶液;
b、0.22μm过滤膜过滤粗酶液,利用Ni-NTA柱进行纯化,收集洗脱液;
c、对洗脱液用超滤膜浓缩,再用50mM pH7.4的PBS置换洗脱液3次,浓缩至酶液呈现鲜红色或红黑色,得到纯品。
具体的,步骤b中的洗脱条件为:平衡液I洗脱5个柱体积;将0.22μm过滤膜过滤后的粗酶液上柱;平衡液I洗5个柱体积;平衡液II洗5个柱体积;洗脱液I洗6个柱体积;洗脱液II洗5个柱体积,收集洗脱液;平衡液I为50mM PBS,pH7.4,5mM咪唑;平衡液II为50mM PBS,0.5M NaCl,pH7.4,5mM咪唑;洗脱液I为50mM PBS,pH7.4,20mM咪唑;洗脱液II为50mM PBS,pH7.4,80mM咪唑。
本发明提供了一种催化效率更高的CYP119酶,是在213位将苏氨酸突变为甘氨酸得到的突变体。在常温下,CYP119-T213G酶催化苯乙烯环氧化反应的催化能力提高了近80倍,且对应选择性也比野生型酶高,R/S=1:5,具有出人意料的技术效果。以上结果说明突变后的CYP119-T213G酶能提高酶对苯乙烯的环氧化反应,且具有较高的对应选择性,具有广泛的应用价值。
附图说明
图1 CYP119-T213G催化苯乙烯环氧化反应米氏方程
具体实施方式
实施例1CYP119-T213G酶表达载体构建
CYP119酶的突变:根据pET30a-CYP119-T213G载体设计突变位点的引物序列为:SEQ ID No.3和SEQ ID No.4。利用QuickChange Lighting Site-directed MutagenesisKit定点突变试剂盒进行定点突变,转化大肠杆菌DH5α。挑选阳性克隆测序,确认是否完成突变及突变后位点的准确性。
SEQ ID No.3:扩增CYP119-T213G酶的上游引物
5'-TTCTCATAGCGGGTAATGAGACAACTAACTTAATATCAAA-3',加粗下划位点为突变位点。
SEQ ID No.4:扩增CYP119-T213G酶的下游引物
5'-TTTGATATTAAGTTAGTTGTCTCATTACCCGCTATGAGAA-3',加粗下划位点为突变位点。
实施例2CYP119-T213G酶的大量表达
将pET30a-CYP119-T213G质粒转化BL21(DE3)plysS大肠杆菌感受态细胞。挑选阳性 单菌落,接种于5mL双抗LB液体培养基中,37℃震荡培养过夜。取2mL过夜培养的菌液于1L双抗的TB培养基培养基。加入250μl/L Trace Element,37℃,震荡培养至OD0.6。加入0.4mM IPTG至终浓度0.4mM,32℃,诱导45h。
实施例3CYP119-T213G酶的纯化
取实施例2发酵好的菌液,12000rpm,4℃离心10min,弃上清,加入20mL pH7.4,50mMPBS溶液充分悬浮菌体。将样品置于冰水上,超声破胞仪,50%功率,3s-3s,20min分两次破完。破胞后在55℃水浴中加热15min,12000rpm,4℃,离心40min,取上清液即为粗酶液。
然后将粗酶液进行纯化,平衡液I洗脱5个柱体积;将0.22μm过滤膜过滤后的粗酶液上柱;平衡液I洗5个柱体积;平衡液II洗5个柱体积;洗脱液I洗6个柱体积;洗脱液II洗5个柱体积,收集洗脱液。
将收集的洗脱液II用超滤膜( Ultra-1510K)浓缩至800μL左右,再用50mMPBS,pH7.4置换缓冲液3次,浓缩至600μL左右,酶液呈现鲜红色或红黑色。将浓缩酶稀释201倍后,紫外分光光度计测得A415=0.4183,据此算出酶量为20.85mg/L。-80℃保存。
经过此纯化后,蛋白的纯度可达90%以上,能满足后续工作的要求。
纯化所需试剂配方:
平衡液I:50mM PBS,pH7.4,5mM咪唑
平衡液II:50mM PBS,0.5M NaCl,pH7.4,5mM咪唑
洗脱液I:50mM PBS,pH7.4,20mM咪唑
洗脱液II:50mM PBS,pH7.4,80mM咪唑。
实施例4CYP119-T213G酶的性质研究
1、米氏方程
利用CYP119-T213G酶催化苯乙烯环氧化反应,根据米氏方程得到此突变酶的Km,Vmax和Kcat等酶动力学常数。
叔丁基过氧化氢(TBHP)为辅助氧化剂,苯乙烯溶解在乙腈中,反应温度为35℃。反应缓冲体系为50mM bis-Tris buffer,pH7.4。总体系80μL。反应完成后用720μL乙腈淬灭反应后,于HPLC检测产物。液相色谱检测条件:戴安(Dionex U-3000),C18柱,30%ddH2O,70%乙腈作为流动相,检测波长220nm,流速:1mL/min。苯乙烯保留时间4.9min,环氧苯乙烷2.5min。
反应体系见表1。每个浓度梯度重复三次反应,求得产物(环氧苯乙烷)的平均值。根据环氧苯乙烷生成速率的倒数和苯乙烯浓度的倒数作图,获得米氏方程,见图1。
表1 反应体系
苯乙烯浓度(mM) | TBHP浓度(mM) | CYP119-T213G酶浓度(μM) | 反应时间(s) |
3 | 3 | 12.5 | 30 |
3.5 | 3.5 | 12.5 | 30 |
4 | 4 | 12.5 | 30 |
4.3 | 4.3 | 12.5 | 30 |
5 | 5 | 12.5 | 30 |
5.3 | 5.3 | 12.5 | 30 |
6.5 | 6.5 | 12.5 | 30 |
7 | 7 | 12.5 | 30 |
根据米氏方程:
Km=14.1mM,Vmax=0.64mmol·L·min-1,Kcat=51.2min-1
2、对应选择性
利用GC检测CYP119-T213G酶催化苯乙烯环氧化反应的对应选择性。反应体系及条件:TBHP为辅助氧化剂浓度为8mM,苯乙烯浓度为8mM,CYP119-T213G酶12.5μM反应缓冲体系为50mM bis-Tris buffer,pH6.0,反应总体系100μL,温度为30℃,反应10min后100μLCH2CL2淬灭反应,等体积萃取3次。每次反应重复三次,求平均值。GC检测条件:安捷伦7890A,柱子HP-5,条件:程序升温:100℃,10min;100-190℃,20℃/min升高;190~250℃,20℃/min。苯乙烯保留时间4.27min,R型环氧苯乙烷10.93min,S型环氧苯乙烷11.41min。结果为R构型与S构型环氧苯乙烷峰面积之比约为1:5。说明该酶在此条件下催化苯乙烯环氧化反应的对应选择性较好。
参考文献
[1]Koo L.S.,Tschirret-Guth R.A.,Straub W.E.,et al.J.Biol.Chem.2000,275,14112–14123.
[2]Koo L.S.,Ortiz de Montellano.J.Biol.Chem.,2002,124,5684-5691.
[3]Rabe KS,Kiko K,Niemeyer CM.Chemical biology chemical.2008,9,420-425。
Claims (8)
1.一种CYP119-T213G酶,其特征在于:其氨基酸序列如SEQ ID No.2所示。
2.如权利要求1所述的CYP119-T213G酶,其特征在于:编码CYP119-T213G酶的核苷酸序列如SEQ ID No.1所示。
3.表达权利要求1所述的CYP119-T213G酶的载体。
4.如权利要求3所述的载体,其特征在于:所述的载体为质粒。
5.包含权利要求3或4所述载体的宿主。
6.如权利要求5所述的宿主,其特征在于:所述的宿主为大肠杆菌。
7.CYP119-T213G酶的纯化方法,其特征在于:包括如下步骤:
a、取发酵后的权利要求6所述的宿主大肠杆菌的菌液离心收集菌体,PBS悬浮菌体,破碎细胞,离心,收集上清液,获得粗酶液;
b、0.22μm过滤膜过滤粗酶液,利用Ni-NTA柱进行纯化,收集洗脱液;
c、对洗脱液用超滤膜浓缩,再用50mM pH 7.4的PBS置换洗脱液3次,浓缩至酶液呈现鲜红色或红黑色,得到纯品。
8.如权利要求7所述的方法,其特征在于:步骤b中的洗脱条件为:平衡液I洗脱5个柱体积;将0.22μm过滤膜过滤后的粗酶液上柱;平衡液I洗5个柱体积;平衡液II洗5个柱体积;洗脱液I洗6个柱体积;洗脱液II洗5个柱体积,收集洗脱液;平衡液I为50mM PBS,pH 7.4,5mM咪唑;平衡液II为50mM PBS,0.5M NaCl,pH 7.4,5mM咪唑;洗脱液I为50mM PBS,pH 7.4,20mM咪唑;洗脱液II为50mM PBS,pH 7.4,80mM咪唑。
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细胞色素P450 119酶及其突变酶催化活性的探究;李静等;《第八届全国化学生物学学术会议论文摘要集》;20130915;235 * |
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