A rapid procedure for the in situ assay of periplasmic, PQQ-dependent methanol dehydrogenase in intact single bacterial colonies

Vemuluri, Venkata Ramana; Shaw, Shreya; Autenrieth, Caroline; Ghosh, Robin

doi:10.1016/j.mimet.2017.03.013

Cited by 3 publications

(1 citation statement)

References 15 publications

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“…To engineer proteins for a desired function, suitable whole‐cell assays are highly beneficial, as they allow efficient screening of mutant libraries without prior purification of the different protein variants. Notably, two recent studies reported whole‐cell assays, allowing activity screens of native membrane‐associated PQQ‐ADHs using Gluconobacter and Methylobacterium strains (Peters et al ., ; Vemuluri et al ., ). However, to the best of our knowledge, a heterologous system suitable for efficient production, purification and engineering of soluble PQQ‐ADHs has not been developed thus far.…”

Section: Introductionmentioning

confidence: 97%

Engineering thermal stability and solvent tolerance of the soluble quinoprotein PedE from Pseudomonas putida KT2440 with a heterologous whole‐cell screening approach

Wehrmann

Klebensberger

2017

Microbial Biotechnology

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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).

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Section: Introductionmentioning

confidence: 97%

Engineering thermal stability and solvent tolerance of the soluble quinoprotein PedE from Pseudomonas putida KT2440 with a heterologous whole‐cell screening approach

Wehrmann

Klebensberger

2017

Microbial Biotechnology

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Dietary supplementation with pyrroloquinoline quinone promotes growth, relieves weaning stress, and regulates metabolism of piglets compared with adding zinc oxide

Shi,

Yu,

Wang

et al. 2023

Animal Nutrition

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Microbiological Enrichment

Batarshin

Semiokhin

Sotnikova

2020

IOP Conf. Ser.: Earth Environ. Sci.

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In the modern world, there is a problem of incomplete separation of concentrate from the enriched rock, which leads to losses of precious minerals and high costs at enterprises. This problem is not noticeable in the release of the concentrate of the rock, which is common. However, with the release of rare minerals, for example, such as gallium, cadmium or uranium, it becomes the main. This article will consider the process of enrichment of the rock, the way resorting to the help of mesophilic and thermophilic bacteria and allowing to allocate the maximum amount of concentrate.

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A rapid procedure for the in situ assay of periplasmic, PQQ-dependent methanol dehydrogenase in intact single bacterial colonies

Cited by 3 publications

References 15 publications

Engineering thermal stability and solvent tolerance of the soluble quinoprotein PedE from Pseudomonas putida KT2440 with a heterologous whole‐cell screening approach

Engineering thermal stability and solvent tolerance of the soluble quinoprotein PedE from Pseudomonas putida KT2440 with a heterologous whole‐cell screening approach

Dietary supplementation with pyrroloquinoline quinone promotes growth, relieves weaning stress, and regulates metabolism of piglets compared with adding zinc oxide

Microbiological Enrichment

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