He Dong, Luo Wen, Wang Zhiyuan, Lv Pengmei, Yuan Zhenhong, Huang Shaowei, Xv Jingliang
Key Laboratory of Renewable Energy, Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences, Guangzhou 510640, PR China; University of Chinese Academy of Sciences, Beijing 100049, PR China; Guangdong Key Laboratory of New and Renewable Energy Research and Development, Chinese Academy of Sciences, Guangzhou 510640, PR China.
Key Laboratory of Renewable Energy, Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences, Guangzhou 510640, PR China; South China Agricultural University, Guangzhou Guangdong 510642, PR China.
Enzyme Microb Technol. 2017 Jul;102:35-40. doi: 10.1016/j.enzmictec.2017.03.010. Epub 2017 Mar 25.
Directed evolution has been proved an effective way to improve the stability of proteins, but high throughput screening assays for directed evolution with simultaneous improvement of two or more properties are still rare. In this study, we aimed to establish a membrane-blot assay for use in the high-throughput screening of Rhizomucor miehei lipases (RMLs). With the assistance of the membrane-blot screening assay, a mutant E47K named G10 that showed improved thermal stability was detected in the first round of error-prone PCR. Using G10 as the parent, two variants G10-11 and G10-20 that showed improved thermal stability and methanol tolerance without loss of activity compared to the wild type RML were obtained. The T-value of G10-11 and G10-20 increased by 12°C and 6.5°C, respectively. After incubation for 1h, the remaining residual activity of G10-11 and G10-20 was 63.45% and 74.33%, respectively, in 50% methanol, and 15.98% and 30.22%, respectively, in 80% methanol. Thus, we successfully developed a membrane-blot assay that could be used for the high-throughput screening of RMLs with improved thermostability and methanol tolerance. Based on our findings, we believe that our newly developed membrane-blot assay will have potential applications in directed evolution in the future.
定向进化已被证明是提高蛋白质稳定性的有效方法，但用于同时改善两种或更多种特性的定向进化的高通量筛选测定仍然很少见。在本研究中，我们旨在建立一种用于米黑根毛霉脂肪酶（RMLs）高通量筛选的膜印迹测定法。借助膜印迹筛选测定法，在第一轮易错PCR中检测到一个名为G10的突变体E47K，其热稳定性有所提高。以G10为亲本，获得了两个变体G10-11和G10-20，与野生型RML相比，它们在不失活的情况下热稳定性和甲醇耐受性有所提高。G10-11和G10-20的T值分别提高了12°C和6.5°C。在50%甲醇中孵育1小时后，G10-11和G10-20的剩余残余活性分别为63.45%和74.33%，在80%甲醇中分别为15.98%和30.22%。因此，我们成功开发了一种可用于高通量筛选具有更高热稳定性和甲醇耐受性的RMLs的膜印迹测定法。基于我们的发现，我们相信我们新开发的膜印迹测定法在未来的定向进化中将具有潜在的应用。
