Planchestainer Matteo, Hegarty Eimear, Heckmann Christian M, Gourlay Louise J, Paradisi Francesca
University of Nottingham , School of Chemistry , University Park , Nottingham NG7 2RD , United Kingdom.
Structural Biology Unit , Dep. Biosciences , Università degli Studi di Milano , Via Celoria 26 , 20133 Milano , Italy.
Chem Sci. 2019 May 9;10(23):5952-5958. doi: 10.1039/c8sc05712e. eCollection 2019 Jun 21.
Directed evolution of transaminases is a widespread technique in the development of highly sought-after biocatalysts for industrial applications. This process, however, is challenged by the limited availability of effective high-throughput protocols to evaluate mutant libraries. Here we report a rapid, reliable, and widely applicable background depletion method for solid-phase screening of transaminase variants, which was successfully applied to a transaminase from (HEWT), evolved through rounds of random mutagenesis towards a series of diverse prochiral ketones. This approach enabled the identification of transaminase variants in viable cells with significantly improved activity towards para-substituted acetophenones (up to 60-fold), as well as tetrahydrothiophen-3-one and related substrates. Rationalisation of the mutants was assisted by determination of the high-resolution wild-type HEWT crystal structure presented herein.
转氨酶的定向进化是开发备受青睐的工业应用生物催化剂的一种广泛应用的技术。然而，这一过程受到用于评估突变文库的有效高通量方案有限的挑战。在此，我们报告了一种用于转氨酶变体固相筛选的快速、可靠且广泛适用的背景消除方法，该方法已成功应用于来自（HEWT）的一种转氨酶，该转氨酶通过多轮随机诱变朝着一系列不同的前手性酮进化。这种方法能够在活细胞中鉴定出对对位取代苯乙酮（活性提高高达60倍）以及四氢噻吩-3-酮和相关底物具有显著改善活性的转氨酶变体。本文通过测定高分辨率的野生型HEWT晶体结构辅助了对突变体的合理化分析。
