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Optimization for Sequencing and Analysis of Degraded FFPE-RNA Samples

Optimization for Sequencing and Analysis of Degraded FFPE-RNA Samples

Published on: June 8, 2020

Sequence-Based

Stefan Velikogne¹, Verena Resch¹, Carina Dertnig¹

¹University of Graz Institute of Chemistry NAWI Graz, BioTechMed Graz Heinrichstrasse 28 8010 Graz Austria.

|September 11, 2018

Summary

Novel imine reductases (IREDs) were discovered using in silico methods and demonstrated high activity and stereoselectivity for cyclic imine reduction. Whole-cell biocatalysts efficiently produced chiral amines, showcasing their potential in biocatalysis.

Keywords:

Amines Asymmetric Catalysis Biocatalysis Imine Reductases Oxidoreductases

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Area of Science:

Biocatalysis and enzyme engineering
Organic chemistry
Synthetic biology

Background:

Imine reductases (IREDs) are emerging biocatalysts for amine synthesis.
IREDs complement existing amine-forming enzymes like transaminases and amine dehydrogenases.
In silico enzyme discovery is a powerful tool for identifying novel biocatalysts.

Purpose of the Study:

To discover and characterize novel IRED biocatalysts through sequence-based in silico methods.
To evaluate the activity and stereoselectivity of these IREDs with diverse cyclic imine substrates.
To develop efficient whole-cell biocatalysts for preparative chiral amine production.

Main Methods:

In silico sequence-based enzyme discovery for IRED identification.
Biocatalyst characterization including activity and stereoselectivity assays.
Co-expression of IREDs with alcohol dehydrogenase for cofactor regeneration in whole-cell systems.
Gram-scale preparative biotransformations of cyclic imines.

Main Results:

Discovery of novel IRED biocatalysts with specific activities exceeding 1 U/mg.
High stereoselectivities (ee > 99%) achieved for many cyclic imine reductions.
Demonstrated tolerance of IREDs to high substrate loadings.
Development of efficient whole-cell biocatalysts reducing 100 mM imines without exogenous cofactor.
Gram-scale production of chiral amines in high yield and optical purity.

Conclusions:

Novel IREDs identified via in silico methods exhibit significant potential for biocatalytic amine synthesis.
Engineered whole-cell biocatalysts offer a sustainable and efficient platform for producing chiral amines.
These findings advance the application of IREDs in green chemistry and pharmaceutical manufacturing.