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Your search keyword '"Yannik Brack"' showing total 2 results
Start Over Author "Yannik Brack" Topic biotechnology
2 results on '"Yannik Brack"'

1. NADPH biosensor-based identification of an alcohol dehydrogenase variant with improved catalytic properties caused by a single charge reversal at the protein surface

Author

Alina Spielmann, Yannik Brack, Hugo van Beek, Lion Flachbart, Lea Sundermeyer, Meike Baumgart, and Michael Bott

Subjects
NADPH biosensor, Lactobacillus brevis, NADPH-dependent alcohol dehydrogenase, Enzyme optimization, Fluorescence-activated cell sorting, Random mutagenesis, Biotechnology, TP248.13-248.65, Microbiology, QR1-502
Abstract

Abstract Alcohol dehydrogenases (ADHs) are used in reductive biotransformations for the production of valuable chiral alcohols. In this study, we used a high-throughput screening approach based on the NADPH biosensor pSenSox and fluorescence-activated cell sorting (FACS) to search for variants of the NADPH-dependent ADH of Lactobacillus brevis (LbADH) with improved activity for the reduction of 2,5-hexanedione to (2R,5R)-hexanediol. In a library of approx. 1.4 × 106 clones created by random mutagenesis we identified the variant LbADHK71E. Kinetic analysis of the purified enzyme revealed that LbADHK71E had a ~ 16% lowered KM value and a 17% higher Vmax for 2,5-hexanedione compared to the wild-type LbADH. Higher activities were also observed for the alternative substrates acetophenone, acetylpyridine, 2-hexanone, 4-hydroxy-2-butanone, and methyl acetoacetate. K71 is solvent-exposed on the surface of LbADH and not located within or close to the active site. Therefore, K71 is not an obvious target for rational protein engineering. The study demonstrates that high-throughput screening using the NADPH biosensor pSenSox represents a powerful method to find unexpected beneficial mutations in NADPH-dependent alcohol dehydrogenases that can be favorable in industrial biotransformations.

Published
2020
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2. Two novel cyanobacterial α-dioxygenases for the biosynthesis of fatty aldehydes

Author

In Jung Kim, Yannik Brack, Thomas Bayer, and Uwe T. Bornscheuer

Subjects
Aldehydes, Fatty Acids, General Medicine, Cyanobacteria, Fatty acid, Applied Microbiology and Biotechnology, Dioxygenases, Fatty aldehyde, Aroma compounds, polycyclic compounds, Escherichia coli, α-Dioxygenase, Biotechnologically Relevant Enzymes and Proteins, Biotechnology
Abstract

α-Dioxygenases (α-DOXs) are known as plant enzymes involved in the α-oxidation of fatty acids through which fatty aldehydes, with a high commercial value as flavor and fragrance compounds, are synthesized as products. Currently, little is known about α-DOXs from non-plant organisms. The phylogenic analysis reported here identified a substantial number of α-DOX enzymes across various taxa. Here, we report the functional characterization and Escherichia coli whole-cell application of two novel α-DOXs identified from cyanobacteria: CalDOX from Calothrix parietina and LepDOX from Leptolyngbya sp. The catalytic behavior of the recombinantly expressed CalDOX and LepDOX revealed that they are heme-dependent like plant α-DOXs but exhibit activities toward medium carbon fatty acids ranging from C10 to C14 unlike plant α-DOXs. The in-depth molecular investigation of cyanobacterial α-DOXs and their application in an E. coli whole system employed in this study is useful not only for the understanding of the molecular function of α-DOXs, but also for their industrial utilization in fatty aldehyde biosynthesis.Key points• Two novel α-dioxygenases from Cyanobacteria are reported• Both enzymes prefer medium-chain fatty acids• Both enzymes are useful for fatty aldehyde biosynthesis Graphical abstract

Published
2021
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