Oliveira Gabriela, Viviani Vadim R
Laboratory of Biochemistry and Biotechnology of Bioluminescence, Graduate Program of Biotechnology and Environmental Monitoring, Department of Chemistry, Physics and Mathematics, Federal University of São Carlos (UFSCar), Campus of Sorocaba, Sorocaba, SP, Brazil.
Department of Evolutive Genetics and Molecular Biology, Federal University of São Carlos (UFSCar), São Carlos, SP, Brazil.
Luminescence. 2018 Mar;33(2):282-288. doi: 10.1002/bio.3411. Epub 2017 Nov 2.
Firefly luciferases have been used extensively as bioanalytical reagents and their cDNAs as reporter genes for biosensors and bioimaging, but they are in general unstable at temperatures above 30°C. In the past few years, efforts have been made to stabilize some firefly luciferases for better application as analytical reagents. Novel luciferases from different beetle families, displaying distinct bioluminescence colours and kinetics, may offer desirable alternatives to extend the range of applications. In the past years, our group has cloned the largest variety of luciferases from the three main families of bioluminescent beetles (Elateridae: P. termitilluminans, F. bruchi, P. angustus; Phengodidae: P. hirtus, P. vivianii; and Lampyridae: A. vivianii, C. distinctus and Macrolampis sp2) occurring in Brazilian biomes. We compared the thermostability of these recombinant luciferases and investigated their relationships with bioluminescence spectra and kinetics. The most thermostable luciferases were those of Pyrearinus termitilluminans larval click beetle (534 nm), Amydetes vivianii firefly (539 nm) and Phrixotrix vivianii railroad worm (546 nm), which are the most blue-shifted examples in each family, confirming the trend that the most blue-shifted emitting luciferases are also the most thermostable. Comparatively, commercial P. pyralis firefly luciferase was less thermostable than P. termitilluminans click beetle and A. vivianii firefly luciferases. The higher thermostability in these luciferases could be related to higher degree of hydrophobic packing and disulfide bond content (for firefly luciferases).
萤火虫荧光素酶已被广泛用作生物分析试剂，其cDNA作为生物传感器和生物成像的报告基因，但它们通常在30°C以上的温度下不稳定。在过去几年中，人们努力使一些萤火虫荧光素酶稳定化，以便更好地用作分析试剂。来自不同甲虫科的新型荧光素酶，具有独特的生物发光颜色和动力学特性，可能为扩展应用范围提供理想的替代方案。在过去几年中，我们的团队从巴西生物群落中出现的三个主要发光甲虫科（叩甲科：白蚁发光叩甲、布鲁氏萤叩甲、狭翅萤叩甲；光萤科：多毛光萤、维氏光萤；萤科：维氏萤、显萤、大萤属sp2）中克隆了种类最多的荧光素酶。我们比较了这些重组荧光素酶的热稳定性，并研究了它们与生物发光光谱和动力学的关系。热稳定性最高的荧光素酶是白蚁发光叩甲幼虫（534nm）、维氏萤萤火虫（539nm）和维氏铁路虫（546nm）的荧光素酶，它们是每个科中蓝移最大的例子，证实了蓝移最大的发光荧光素酶也是热稳定性最高的这一趋势。相比之下，商业上的萤火虫荧光素酶比白蚁发光叩甲和维氏萤萤火虫荧光素酶的热稳定性更低。这些荧光素酶较高的热稳定性可能与更高程度的疏水堆积和二硫键含量（对于萤火虫荧光素酶而言）有关。
