caa a22 4500 605502684 CHVBK 20210128100607.0 cr unu---uuuuu 210128e20151201xx s 000 0 eng 10.1007/s00253-015-6930-5 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6930-5 Stability engineering of the Geobacillus stearothermophilus alcohol dehydrogenase and application for the synthesis of a polyamide 12 precursor [Elektronische Daten] [Ludwig Kirmair, Daniel Seiler, Arne Skerra] The thermostable NAD+-dependent alcohol dehydrogenase from Geobacillus stearothermophilus (BsADH) was exploited with regard to the biocatalytic synthesis of ω-oxo lauric acid methyl ester (OLAMe), a key intermediate for biobased polyamide 12 production, from the corresponding long-chain alcohol. Recombinant BsADH was produced in Escherichia coli as a homogeneous tetrameric enzyme and showed high activity towards the industrially relevant substrate ω-hydroxy lauric acid methyl ester (HLAMe) with K M=86μM and 44Umg−1. The equilibrium constant for HLAMe oxidation to the aldehyde (OLAMe) with NAD+ was determined as 2.16×10−3 from the kinetic parameters of the BsADH-catalyzed forward and reverse reactions. Since BsADH displayed limited stability under oxidizing conditions, the predominant oxidation-prone residue Cys257 was mutated to Leu based on sequence homology with related enzymes and computational simulation. This substitution resulted in an improved BsADH variant exhibiting prolonged stability and an elevated inactivation temperature. Semi-preparative biocatalysis at 60°C using the stabilized enzyme, employing butyraldehyde for in situ cofactor regeneration with only catalytic amounts of NAD+, yielded up to 23% conversion of HLAMe to OLAMe after 30min. In contrast to other oxidoreductases, no overoxidation to the dodecanoic diacid monomethyl ester was detected. Thus, the mutated BsADH offers a promising biocatalyst for the selective oxidation of fatty alcohols to yield intermediates for industrial polymer production. Springer-Verlag Berlin Heidelberg, 2015 Biobased polymer nationallicence Biocatalysis nationallicence BsADH nationallicence Cofactor recycling nationallicence Enzyme engineering nationallicence Protein thermostability nationallicence Kirmair Ludwig Munich Center for Integrated Protein Science (CiPSM) and Lehrstuhl für Biologische Chemie, Technische Universität München, 85350, Freising-Weihenstephan, Germany aut Seiler Daniel Munich Center for Integrated Protein Science (CiPSM) and Lehrstuhl für Biologische Chemie, Technische Universität München, 85350, Freising-Weihenstephan, Germany aut Skerra Arne Munich Center for Integrated Protein Science (CiPSM) and Lehrstuhl für Biologische Chemie, Technische Universität München, 85350, Freising-Weihenstephan, Germany aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/24(2015-12-01), 10501-10513 0175-7598 99:24<10501 2015 99 253 https://doi.org/10.1007/s00253-015-6930-5 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6930-5 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Kirmair Ludwig Munich Center for Integrated Protein Science (CiPSM) and Lehrstuhl für Biologische Chemie, Technische Universität München, 85350, Freising-Weihenstephan, Germany aut NATIONALLICENCE 700 1- Seiler Daniel Munich Center for Integrated Protein Science (CiPSM) and Lehrstuhl für Biologische Chemie, Technische Universität München, 85350, Freising-Weihenstephan, Germany aut NATIONALLICENCE 700 1- Skerra Arne Munich Center for Integrated Protein Science (CiPSM) and Lehrstuhl für Biologische Chemie, Technische Universität München, 85350, Freising-Weihenstephan, Germany aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/24(2015-12-01), 10501-10513 0175-7598 99:24<10501 2015 99 253