酶的活性
酶热失活
什么是热失活？
热失活，英文是Heat inactivation，也叫热灭活，是一种化学现象，特别是在蛋白质和酶的领域中较为常见。它指的是蛋白质或酶在高温条件下失去其活性或功能的过程。这
种失活通常发生在蛋白质或酶的最适反应温度以上。
热失活是停止限制性核酸内切酶反应的便捷方法。例如，在65°C下孵育20分钟，可以使大多数**孵育温度为37°C的限制性核酸内切酶失活。在65°C下不能失活的酶通常可以通过在80°C下孵育20分钟而失活。下表显示了酶是否可以被热灭活以及所需的温度。
对于在65°C或80°C下无法热失活的酶，我们建议使用色谱柱进行清除（例如Monarch®PCR＆DNA清除试剂盒），或在琼脂糖凝胶上进行反应，然后提取DNA（建议使用Monarch凝胶提取试剂盒），或进行苯酚/氯仿提取。
下面是一个类似热灭活的典型实验案例。
将含有适当NEBuffer，0.5µg小牛胸腺DNA和5或10µl限制性核酸内切酶的50µl反应混合物在37°C孵育60分钟，然后在65°C或80°C孵育20分钟。将0.5µg底物DNA（通常为λ）添加到反应混合物中，并在酶的**反应温度下孵育60分钟。
利用琼脂糖凝胶电泳观察，如果第二次孵育后底物DNA出现任何的消化（包括完全或部分），均可以认为是不完全热灭活。
酶一般在什么温度下失活？
酶的失活温度和时间大全
| 酶的名称 | 热失活温度 | 热失活所需时间 |
| AatII | 80°C | 20 minutes |
| Acc65I | 65°C | 20 minutes |
| AccI | 80°C | 20 minutes |
| AciI | 65°C | 20 minutes |
| AclI | No | -- |
| AcuI | 65°C | 20 minutes |
| AfeI | 65°C | 20 minutes |
| AflII | 65°C | 20 minutes |
| AflIII | 80°C | 20 minutes |
| AgeI | 65°C | 20 minutes |
| AhdI | 65°C | 20 minutes |
| AleI | 80°C | 20 minutes |
| AleI-v2 | 65°C | 20 minutes |
| AluI | 80°C | 20 minutes |
| AlwI | No | -- |
| AlwNI | 80°C | 20 minutes |
| ApaI | 65°C | 20 minutes |
| ApaLI | No | -- |
| ApeKI | No | -- |
| ApoI | 80°C | 20 minutes |
| AscI | 80°C | 20 minutes |
| AseI | 65°C | 20 minutes |
| AsiSI | 80°C | 20 minutes |
| AvaI | 80°C | 20 minutes |
| AvaII | 80°C | 20 minutes |
| AvrII | No | -- |
| BaeGI | 80°C | 20 minutes |
| BaeI | 65°C | 20 minutes |
| BamHI | No | -- |
| BamHI | No | -- |
| BanI | 65°C | 20 minutes |
| BanII | 80°C | 20 minutes |
| BbsI | 65°C | 20 minutes |
| BbsI | 65°C | 20 minutes |
| BbvCI | No | -- |
| BbvI | 65°C | 20 minutes |
| BccI | 65°C | 20 minutes |
| BceAI | 65°C | 20 minutes |
| BcgI | 65°C | 20 minutes |
| BciVI | 80°C | 20 minutes |
| BclI | No | -- |
| BclI | 65°C | 20 minutes |
| BcoDI | No | -- |
| BfaI | 80°C | 20 minutes |
| BfuAI | 65°C | 20 minutes |
| BfuCI | 80°C | 20 minutes |
| BglI | 65°C | 20 minutes |
| BglII | No | -- |
| BlpI | No | -- |
| BmgBI | 65°C | 20 minutes |
| BmrI | 65°C | 20 minutes |
| BmtI | 65°C | 20 minutes |
| BpmI | 65°C | 20 minutes |
| Bpu10I | 80°C | 20 minutes |
| BpuEI | 65°C | 20 minutes |
| BsaAI | No | -- |
| BsaBI | 80°C | 20 minutes |
| BsaHI | 80°C | 20 minutes |
| BsaI | 65°C | 20 minutes |
| BsaIv2 | 80°C | 20 minutes |
| BsaJI | 80°C | 20 minutes |
| BsaWI | 80°C | 20 minutes |
| BsaXI | No | -- |
| BseRI | 80°C | 20 minutes |
| BseYI | 80°C | 20 minutes |
| BsgI | 65°C | 20 minutes |
| BsiEI | No | -- |
| BsiHKAI | No | -- |
| BsiWI | 80°C | 20 minutes |
| BsiWI | 80°C | 20 minutes |
| BslI | No | -- |
| BsmAI | No | -- |
| BsmBI-v2 | 80°C | 20 minutes |
| BsmFI | 80°C | 20 minutes |
| BsmI | 80°C | 20 minutes |
| BsoBI | 80°C | 20 minutes |
| Bsp1286I | 65°C | 20 minutes |
| BspCNI | 80°C | 20 minutes |
| BspDI | 80°C | 20 minutes |
| BspEI | 80°C | 20 minutes |
| BspHI | 80°C | 20 minutes |
| BspMI | 65°C | 20 minutes |
| BspQI | 80°C | 20 minutes |
| BsrBI | 80°C | 20 minutes |
| BsrDI | 80°C | 20 minutes |
| BsrFI | No | -- |
| BsrFI-v2 | No | -- |
| BsrGI | 80°C | 20 minutes |
| BsrI | 80°C | 20 minutes |
| BssHII | 65°C | 20 minutes |
| BssKI | 80°C | 20 minutes |
| BssSI | 80°C | 20 minutes |
| BssSI-v2 | No | -- |
| BstAPI | 80°C | 20 minutes |
| BstBI | No | -- |
| BstEII | No | -- |
| BstNI | No | -- |
| BstUI | No | -- |
| BstXI | 80°C | 20 minutes |
| BstYI | No | -- |
| BstZ17I | No | -- |
| BstZ17I | No | -- |
| Bsu36I | 80°C | 20 minutes |
| BtgI | 80°C | 20 minutes |
| BtgZI | 80°C | 20 minutes |
| BtsCI | 80°C | 20 minutes |
| BtsI | 80°C | 20 minutes |
| BtsI-v2 | No | -- |
| BtsIMutI | 80°C | 20 minutes |
| Cac8I | 65°C | 20 minutes |
| ClaI | 65°C | 20 minutes |
| CspCI | 65°C | 20 minutes |
| CviKI-1 | No | -- |
| CviQI | No | -- |
| DdeI | 65°C | 20 minutes |
| DpnI | 80°C | 20 minutes |
| DpnII | 65°C | 20 minutes |
| DraI | 65°C | 20 minutes |
| DraIII | 65°C | 20 minutes |
| DraIII | No | -- |
| DrdI | 65°C | 20 minutes |
| EaeI | 65°C | 20 minutes |
| EagI | 65°C | 20 minutes |
| EarI | 65°C | 20 minutes |
| EciI | 65°C | 20 minutes |
| Eco53kI | 65°C | 20 minutes |
| EcoNI | 65°C | 20 minutes |
| EcoO109I | 65°C | 20 minutes |
| EcoP15I | 65°C | 20 minutes |
| EcoRI | 65°C | 20 minutes |
| EcoRI | 65°C | 20 minutes |
| EcoRV | 80°C | 20 minutes |
| EcoRV | 65°C | 20 minutes |
| Esp3I | 65°C | 20 minutes |
| FatI | 80°C | 20 minutes |
| FauI | 65°C | 20 minutes |
| Fnu4HI | No | -- |
| FokI | 65°C | 20 minutes |
| FseI | 65°C | 20 minutes |
| FspI | No | -- |
| HaeII | 80°C | 20 minutes |
| HaeIII | 80°C | 20 minutes |
| HgaI | 65°C | 20 minutes |
| HhaI | 65°C | 20 minutes |
| HinP1I | 65°C | 20 minutes |
| HincII | 65°C | 20 minutes |
| HindIII | 80°C | 20 minutes |
| HindIII | 80°C | 20 minutes |
| HinfI | 80°C | 20 minutes |
| HpaI | No | -- |
| HpaII | 80°C | 20 minutes |
| HphI | 65°C | 20 minutes |
| Hpy166II | 65°C | 20 minutes |
| Hpy188I | 65°C | 20 minutes |
| Hpy188III | 65°C | 20 minutes |
| Hpy99I | 65°C | 20 minutes |
| HpyAV | 65°C | 20 minutes |
| HpyCH4III | 65°C | 20 minutes |
| HpyCH4IV | 65°C | 20 minutes |
| HpyCH4V | 65°C | 20 minutes |
| I-CeuI | 65°C | 20 minutes |
| I-SceI | 65°C | 20 minutes |
| KasI | 65°C | 20 minutes |
| KpnI | No | -- |
| MboI | 65°C | 20 minutes |
| MboII | 65°C | 20 minutes |
| MfeI | No | -- |
| MluCI | No | -- |
| MluI | No | -- |
| MlyI | 65°C | 20 minutes |
| MmeI | 65°C | 20 minutes |
| MnlI | 65°C | 20 minutes |
| MscI | 80°C | 20 minutes |
| MseI | 65°C | 20 minutes |
| MslI | 80°C | 20 minutes |
| MspA1I | 65°C | 20 minutes |
| MspI | No | -- |
| MspJI | 65°C | 20 minutes |
| MwoI | No | -- |
| NaeI | No | -- |
| NarI | 65°C | 20 minutes |
| Nb.BbvCI | 80°C | 20 minutes |
| Nb.BsmI | 80°C | 20 minutes |
| Nb.BsrDI | 80°C | 20 minutes |
| Nb.BssSI | No | -- |
| Nb.BtsI | 80°C | 20 minutes |
| NciI | No | -- |
| NcoI | 80°C | 20 minutes |
| NcoI | 80°C | 20 minutes |
| NdeI | 65°C | 20 minutes |
| NgoMIV | No | -- |
| NheI | 80°C | 20 minutes |
| NlaIII | 65°C | 20 minutes |
| NlaIV | 65°C | 20 minutes |
| NmeAIII | 65°C | 20 minutes |
| NotI | 65°C | 20 minutes |
| NotI | 65°C | 20 minutes |
| NruI | No | -- |
| NsiI | 65°C | 20 minutes |
| NsiI | 80°C | 20 minutes |
| NspI | 65°C | 20 minutes |
| Nt.AlwI | 80°C | 20 minutes |
| Nt.BbvCI | 80°C | 20 minutes |
| Nt.BsmAI | 65°C | 20 minutes |
| Nt.BspQI | 80°C | 20 minutes |
| Nt.BstNBI | 80°C | 20 minutes |
| Nt.CviPII | 65°C | 20 minutes |
| PI-PspI | No | -- |
| PI-SceI | 65°C | 20 minutes |
| PacI | 65°C | 20 minutes |
| PaeR7I | No | -- |
| PaqCI | 65°C | 20 minutes |
| PciI | 80°C | 20 minutes |
| PflFI | 65°C | 20 minutes |
| PflMI | 65°C | 20 minutes |
| PhoI | No | -- |
| PleI | 65°C | 20 minutes |
| PluTI | 65°C | 20 minutes |
| PmeI | 65°C | 20 minutes |
| PmlI | 65°C | 20 minutes |
| PpuMI | No | -- |
| PshAI | 65°C | 20 minutes |
| PsiI | 65°C | 20 minutes |
| PsiI-v2 | 65°C | 20 minutes |
| PspGI | No | -- |
| PspOMI | 65°C | 20 minutes |
| PspXI | No | -- |
| PstI | 80°C | 20 minutes |
| PstI | No | -- |
| PvuI | No | -- |
| PvuII | No | -- |
| PvuII | No | -- |
| RsaI | No | -- |
| RsrII | 65°C | 20 minutes |
| SacI | 65°C | 20 minutes |
| SacII | 65°C | 20 minutes |
| SalI | 65°C | 20 minutes |
| SalI | 65°C | 20 minutes |
| SapI | 65°C | 20 minutes |
| Sau3AI | 65°C | 20 minutes |
| Sau96I | 65°C | 20 minutes |
| SbfI | 80°C | 20 minutes |
| ScaI | 80°C | 20 minutes |
| ScaI | 80°C | 20 minutes |
| ScrFI | 65°C | 20 minutes |
| SexAI | 65°C | 20 minutes |
| SfaNI | 65°C | 20 minutes |
| SfcI | 65°C | 20 minutes |
| SfiI | No | -- |
| SfoI | No | -- |
| SgrAI | 65°C | 20 minutes |
| SmaI | 65°C | 20 minutes |
| SmlI | No | -- |
| SnaBI | 80°C | 20 minutes |
| SpeI | 80°C | 20 minutes |
| SphI | 65°C | 20 minutes |
| SphI | 65°C | 20 minutes |
| SrfI | 65°C | 20 minutes |
| SspI | 65°C | 20 minutes |
| StuI | No | -- |
| StyD4I | 65°C | 20 minutes |
| StyI | 65°C | 20 minutes |
| SwaI | 65°C | 20 minutes |
| TaqI-v2 | No | -- |
| TfiI | No | -- |
| TliI | No | -- |
| TseI | No | -- |
| Tsp45I | No | -- |
| Tsp509I | No | -- |
| TspMI | No | -- |
| TspRI | No | -- |
| Tth111I | No | -- |
| WarmStart | 80°C | 20 minutes |
| XbaI | 65°C | 20 minutes |
| XcmI | 65°C | 20 minutes |
| XhoI | 65°C | 20 minutes |
| XmaI | 65°C | 20 minutes |
| XmnI | 65°C | 20 minutes |
| ZraI | 80°C | 20 minutes |
表1. 酶的失活温度和时间 (美格生物)
扩展阅读
如何找到**的PCR反应体系？PCR技术所需要的酶和原料的调整
