Abstract
SummaryDue to their ability for direct electron transfer to electrodes, the utilization of rare earth metals as cofactor, and their periplasmic localization, pyrroloquinoline quinone‐dependent alcohol dehydrogenases (PQQ‐ADHs) represent an interesting class of biocatalysts for various biotechnological applications. For most biocatalysts protein stability is crucial, either to increase the performance of the protein under a given process condition or to maximize robustness of the protein towards mutational manipulations, which are often needed to enhance or introduce a functionality of interest. In this study, we describe a whole‐cell screening assay, suitable for probing PQQ‐ADH activities in Escherichia coli BL21(DE3) cells, and use this assay to screen smart mutant libraries for increased thermal stability of the PQQ‐ADH PedE (PP_2674) from Pseudomonas putida KT2440. Upon three consecutive rounds of screening, we identified three different amino acid positions, which significantly improve enzyme stability. The subsequent combination of the beneficial mutations finally results in the triple mutant R91D/E408P/N410K, which not only exhibits a 7°C increase in thermal stability but also a twofold increase in residual activity upon incubation with up to 50% dimethyl sulfoxide (DMSO), while showing no significant difference in enzymatic efficiency (k cat/KM).
Highlights
Dehydrogenases are important industrial enzymes with biotechnological applications ranging from the selective oxidation of alcohols and aldehydes to the regioselective oxidation of polyols, the production of enantiopure alcohols via kinetic resolution or the oxidation of prochiral compounds (Patel, 2004; Liese and Seelbach, 2006; Yakushi and Matsushita, 2010; Hollmann et al, 2011; Romano et al, 2012)
Among alcohol dehydrogenases (ADHs), the pyrroloquinoline quinone (PQQ)-dependent ADHs are of particular interest for several reasons
By determining the T5060 values, representing the temperature required to reduce the initial activity of the enzyme to 50% upon 1 h of incubation, we revealed that for the R91D/E408P/ N410K variant this value was increased by 7°C from 58°C to 65°C compared with the wild-type allele (Fig. 4A)
Summary
Dehydrogenases are important industrial enzymes with biotechnological applications ranging from the selective oxidation of alcohols and aldehydes to the regioselective oxidation of polyols, the production of enantiopure alcohols via kinetic resolution or the oxidation of prochiral compounds (Patel, 2004; Liese and Seelbach, 2006; Yakushi and Matsushita, 2010; Hollmann et al, 2011; Romano et al, 2012). Among alcohol dehydrogenases (ADHs), the pyrroloquinoline quinone (PQQ)-dependent ADHs are of particular interest for several reasons They can be coupled to electrodes, rendering them suitable for biosensor and fuel-cell applications (Igarashi et al, 2004; Treu and Minteer, 2008; Takeda et al, 2013; Scherbahn et al, 2014; Guo et al, 2016). Together with the irreversible nature of the catalyzed reaction, this leads to unhindered accumulation of oxidized products outside of the cells For living biocatalysts, this can be of great advantage as it minimizes toxic effects of substrates and corresponding oxidation products (Acharya and Manning, 1983; Huang et al, 2005). The recent identification of specific PQQ-ADHs which utilize lanthanides as metal cofactor has fuelled the idea of using these enzymes or the corresponding bacterial hosts in biomining and recycling processes of rare earth metals (Skovran and Martinez-Gomez, 2015; Martinez-Gomez et al, 2016; Wehrmann et al, 2017)
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