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Applied Microbiology and Biotechnology

, Volume 99, Issue 24, pp 10501–10513 | Cite as

Stability engineering of the Geobacillus stearothermophilus alcohol dehydrogenase and application for the synthesis of a polyamide 12 precursor

  • Ludwig Kirmair
  • Daniel Leonard Seiler
  • Arne Skerra
Biotechnologically relevant enzymes and proteins

Abstract

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 = 86 μM and 44 U mg−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 30 min. 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.

Keywords

Biobased polymer Biocatalysis BsADH Cofactor recycling Enzyme engineering Protein thermostability 

Notes

Acknowledgments

The authors are grateful to Evonik Industries AG, Marl, Germany, for materials and equipment. L.K. wishes to thank Stefan Achatz for help with automated liquid handling, Irmgard Neumaier for technical assistance, Andreas Reichert for ESI-MS measurements, and Dr. Lukas Eisoldt for discussions.

Funding

This work was financially supported through the German Bundesministerium für Bildung und Forschung in frame of the project “BISON” (Grant No. 0316044) as well as the Federal State of Bavaria and the Deutsche Forschungsgemeinschaft by providing the robotic screening platform in frame of their Major Research Instrumentation Programme (Grant No. INST 95/1031-1).

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Ludwig Kirmair
    • 1
  • Daniel Leonard Seiler
    • 1
  • Arne Skerra
    • 1
  1. 1.Munich Center for Integrated Protein Science (CiPSM) and Lehrstuhl für Biologische ChemieTechnische Universität MünchenFreising-WeihenstephanGermany

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