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Coupled Enzymatic Alcohol‐to‐Amine Conversion of Isosorbide using Engineered Transaminases and Dehydrogenases
Author(s) -
Lerchner Alexandra,
Achatz Stefan,
Rausch Christian,
Haas Thomas,
Skerra Arne
Publication year - 2013
Publication title -
chemcatchem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.497
H-Index - 106
eISSN - 1867-3899
pISSN - 1867-3880
DOI - 10.1002/cctc.201300284
Subject(s) - chemistry , transamination , cofactor , enzyme , biocatalysis , transaminase , substrate (aquarium) , enzyme catalysis , amine gas treating , alcohol dehydrogenase , cascade reaction , combinatorial chemistry , biochemistry , stereochemistry , catalysis , organic chemistry , reaction mechanism , biology , ecology
A matching dehydrogenase and transaminase pair was engineered with regard to substrate recognition, catalytic activity, and cofactor specificity with the final goal to convert the bicyclic dialcohol isosorbide into a diamine by a multistep biocatalytic process. Individual catalytic turnover rates as well as coupled conversion to the amine were investigated for the enzymes in analytical assays that used the substrate isosorbide and different intermediates along the multiple pathway reaction cascade, in particular, stereoisomers of hydroxy ketones, amino alcohols, and amino ketones. For parallel screening and evaluation of mutant enzymes with regard to catalytic activities and optimal reaction conditions, a robotic platform was established that comprised all steps from bacterial protein expression to the enzymatic assay. As a result, we present a three‐enzyme system composed of L. aquatica levodione reductase, an engineered P. denitrificans ω‐aminotransferase, and B. subtilis alanine dehydrogenase that catalyzes formation of the isosorbide monoamine with a yield of up to ≈7 % under our analytical assay conditions. After further optimization and adaptation to whole‐cell catalysis, this enzyme system may open a biotechnologically attractive route to a structurally rigid diamine for the production of biosynthetic polymer materials.