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.
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References
Alberty RA (1953) The relationship between Michaelis constants, maximum velocities and the equilibrium constant for an enzyme-catalyzed reaction. J Am Chem Soc 75:1928–1932
Altschul SF, Gish W, Miller W, Meyers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410
Auld DS, Bergman T (2008) Medium- and short-chain dehydrogenase/reductase gene and protein families. Cell Mol Life Sci 65:3961–3970
Benson S, Shapiro J (1976) Plasmid-determined alcohol dehydrogenase activity in alkane-utilizing strains of Pseudomonas putida. J Bacteriol 126:794–798
Bommarius AS, Paye MF (2013) Stabilizing biocatalysts. Chem Soc Rev 42:6534–6565
Cannio R, Rossi M, Bartolucci S (1994) A few amino acid substitutions are responsible for the higher thermostability of a novel NAD+-dependent bacillar alcohol dehydrogenase. Eur J Biochem 222:345–352
Cassimjee KE, Branneby C, Abedi V, Wells A, Berglund P (2010) Transaminations with isopropyl amine: equilibrium displacement with yeast alcohol dehydrogenase coupled to in situ cofactor regeneration. Chem Commun 46:5569–5571
Ceccarelli C, Liang Z-X, Strickler M, Prehna G, Goldstein BM, Klinman JP, Bahnson BJ (2004) Crystal structure and amide H/D exchange of binary complexes of alcohol dehydrogenase from Bacillus stearothermophilus: insight into thermostability and cofactor binding. Biochemistry 43:5266–5277
Chao C-C, Ma Y-S, Stadtman ER (1997) Modification of protein surface hydrophobicity and methionine oxidation by oxidative systems. Proc Natl Acad Sci U S A 94:2969–2974
DeLano WL (2002) The PyMOL Molecular Graphics System. DeLano Scientific, Palo Alto, CA
Fiorentino G, Cannio R, Rossi M, Bartolucci S (1998) Decreasing the stability and changing the substrate specificity of the Bacillus stearothermophilus alcohol dehydrogenase by single amino acid replacements. Protein Eng 11:925–930
Gasteiger E, Gattiker A, Hoogland C, Ivanyi I, Appel RD, Bairoch A (2003) ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res 31:3784–3788
Geueke B, Riebel B, Hummel W (2003) NADH oxidase from Lactobacillus brevis: a new catalyst for the regeneration of NAD. Enzyme Microb Tech 32:205–211
Grant C, Woodley JM, Baganz F (2011) Whole-cell bio-oxidation of n-dodecane using the alkane hydroxylase system of P. putida GPo1 expressed in E. coli. Enzym Microb Tech 48:480–486
Grant C, da Silva Damas Pinto AC, Lui H-P, Woodley JM, Baganz F (2012) Tools for characterizing the whole-cell bio-oxidation of alkanes at microscale. Biotechnol Bioeng 109:2179–2189
Gröger H (2014) Hydroxy functionalization of non-activated C–H and C=C bonds: new perspectives for the synthesis of alcohols through biocatalytic processes. Angew Chem Int Ed Engl 53:3067–3069
Guagliardi A, Matilde M, Ingram I, De Rosa M, Rossi M, Bartolucci S (1996) Purification and characterization of the alcohol dehydrogenase from a novel strain of Bacillus stearothermophilus growing at 70°C. Int J Biochem Cell Biol 28:239–246
Guterl JK, Garbe D, Carsten J, Steffler F, Sommer B, Reisse S, Philipp A, Haack M, Ruhmann B, Koltermann A, Kettling U, Bruck T, Sieber V (2012) Cell-free metabolic engineering: production of chemicals by minimized reaction cascades. ChemSusChem 5:2165–2172
Hanekom AJ, Hofmeyr JHS, Snoep JL, Rohwer JM (2006) Experimental evidence for allosteric modifier saturation as predicted by the bi-substrate Hill equation. Syst Biol (Stevenage) 153:342–345
Henry CS, Broadbelt LJ, Hatzimanikatis V (2007) Thermodynamics-based metabolic flux analysis. Biophys J 92:1792–1805
Kara S, Schrittwieser JH, Hollmann F, Ansorge-Schumacher MB (2014) Recent trends and novel concepts in cofactor-dependent biotransformations. Appl Microbiol Biotechnol 98:1517–1529
Keinan E, Hafeli EK, Seth KK, Lamed R (1986) Thermostable enzymes in organic synthesis. 2. Asymmetric reduction of ketones with alcohol dehydrogenase from Thermoanaerobium brockii. J Am Chem Soc 108:162–169
Kirmair L, Skerra A (2014) Biochemical analysis of recombinant AlkJ from Pseudomonas putida reveals a membrane-associated, flavin adenine dinucleotide-dependent dehydrogenase suitable for the biosynthetic production of aliphatic aldehydes. Appl Environ Microbiol 80:2468–2477
Koch DJ, Chen MM, van Beilen JB, Arnold FH (2009) In vivo evolution of butane oxidation by terminal alkane hydroxylases AlkB and CYP153A6. Appl Environ Microbiol 75:337–344
Lerchner A, Achatz S, Rausch C, Haas T, Skerra A (2013) Coupled enzymatic alcohol-to-amine conversion of isosorbide using engineered transaminases and dehydrogenases. ChemCatChem 5:3374–3383
Li S, Schöneich C, Borchardt RT (1995) Chemical instability of protein pharmaceuticals: mechanisms of oxidation and strategies for stabilization. Biotechnol Bioeng 48:490–500
Littlechild JA, Guy MN, Isupov MN (2004) Hyperthermophilic dehydrogenase enzymes. Biochem Soc Trans 32:255–258
Marshall SA, Lazar GA, Chirino AJ, Desjarlais JR (2003) Rational design and engineering of therapeutic proteins. Drug Discov Today 8:212–221
May SW, Katopodis AG (1986) Oxygenation of alcohol and sulphide substrates by a prototypical non-haem iron monooxygenase: catalysis and biotechnological potential. Enzym Microb Tech 8:17–21
Metzger JO, Bornscheuer U (2006) Lipids as renewable resources: current state of chemical and biotechnological conversion and diversification. Appl Microbiol Biotechnol 71:13–22
Olaofe O, Fenner C, Gudiminchi RK, Smit M, Harrison S (2013) The influence of microbial physiology on biocatalyst activity and efficiency in the terminal hydroxylation of n-octane using Escherichia coli expressing the alkane hydroxylase, CYP153A6. Microb Cell Factories 12:8
Opperman DJ, Reetz MT (2010) Towards practical Baeyer-Villiger-monooxygenases: design of cyclohexanone monooxygenase mutants with enhanced oxidative stability. ChemBioChem 11:2589–2596
Otte KB, Kirtz M, Nestl BM, Hauer B (2013) Synthesis of 9-oxononanoic acid, a precursor for biopolymers. ChemSusChem 6:2149–2156
Pennacchio A, Giordano A, Rossi M, Raia CA (2011) Asymmetric reduction of α-keto esters with Thermus thermophilus NADH-dependent carbonyl reductase using glucose dehydrogenase and alcohol dehydrogenase for cofactor regeneration. Eur J Org Chem 2011:4361–4366
Pennacchio A, Rossi M, Raia CA (2013) Synthesis of cinnamyl alcohol from cinnamaldehyde with Bacillus stearothermophilus alcohol dehydrogenase as the isolated enzyme and in recombinant E. coli cells. Appl Biochem Biotechnol 170:1482–1490
Peretz M, Weiner LM, Burstein Y (1997) Cysteine reactivity in Thermoanaerobacter brockii alcohol dehydrogenase. Protein Sci 6:1074–1083
Perry LJ, Wetzel R (1987) The role of cysteine oxidation in the thermal inactivation of T4 lysozyme. Protein Eng 1:101–105
Sambrook J and Russell DW (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Sattler JH, Fuchs M, Tauber K, Mutti FG, Faber K, Pfeffer J, Haas T, Kroutil W (2012) Redox self-sufficient biocatalyst network for the amination of primary alcohols. Angew Chem Int Ed 51:9156–9159
Schaffer S, Haas T (2014) Biocatalytic and fermentative production of α,ω-bifunctional polymer precursors. Org Process Res Dev 18:752–766
Scheps D, Malca SH, Richter SM, Marisch K, Nestl BM, Hauer B (2013) Synthesis of ω-hydroxy dodecanoic acid based on an engineered CYP153A fusion construct. Microb Biotechnol 6:694–707
Schmidt TG, Skerra A (2007) The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins. Nat Protoc 2:1528–1535
Schmitt J, Hess H, Stunnenberg HG (1993) Affinity purification of histidine-tagged proteins. Mol Biol Rep 18:223–230
Schrewe M, Magnusson AO, Willrodt C, Bühler B, Schmid A (2011) Kinetic analysis of terminal and unactivated C-H bond oxyfunctionalization in fatty acid methyl esters by monooxygenase-based whole-cell biocatalysis. Adv Synth Catal 353:3485–3495
Schrewe M, Ladkau N, Bühler B, Schmid A (2013) Direct terminal alkylamino-functionalization via multistep biocatalysis in one recombinant whole-cell catalyst. Adv Synth Catal 355:1693–1697
Schrewe M, Julsing MK, Lange K, Czarnotta E, Schmid A, Bühler B (2014) Reaction and catalyst engineering to exploit kinetically controlled whole-cell multistep biocatalysis for terminal FAME oxyfunctionalization. Biotechnol Bioeng 111:1820–1830
Shim E-J, Sang-Hoon J, Kwang-Hoon K (2003) Overexpression, purification and biochemical characterization of the thermostable NAD-dependent alcohol dehydrogenase from Bacillus stearo- thermophilus. J Microbiol Biotechnol 13:738–744
Skerra A (1994) Use of the tetracycline promoter for the tightly regulated production of a murine antibody fragment in Escherichia coli. Gene 151:131–135
Slavica A, Dib I, Nidetzky B (2005) Single-site oxidation, cysteine 108 to cysteine sulfinic acid, in d-amino acid oxidase from Trigonopsis variabilis and its structural and functional consequences. Appl Environ Microbiol 71:8061–8068
Song J-W, Lee J-H, Bornscheuer UT, Park J-B (2014) Microbial synthesis of medium-chain α,ω-dicarboxylic acids and ω-aminocarboxylic acids from renewable long-chain fatty acids. Adv Synth Catal 356:1782–1788
Tiwari A, Bhat R (2006) Stabilization of yeast hexokinase A by polyol osmolytes: correlation with the physicochemical properties of aqueous solutions. Biophys Chem 124:90–99
Tokuriki N, Stricher F, Serrano L and Tawfik DS (2008) How protein stability and new functions trade off. PLoS Comput Biol 4:e1000002.
Tufvesson P, Lima-Ramos J, Nordblad M, Woodley JM (2010) Guidelines and cost analysis for catalyst production in biocatalytic processes. Org Process Res Dev 15:266–274
Vagenende V, Yap MGS, Trout BL (2009) Mechanisms of protein stabilization and prevention of protein aggregation by glycerol. Biochemistry 48:11084–11096
van Beilen JB, Duetz WA, Schmid A, Witholt B (2003) Practical issues in the application of oxygenases. Trends Biotechnol 21:170–177
van der Donk WA, Zhao H (2003) Recent developments in pyridine nucleotide regeneration. Curr Opin Biotechnol 14:421–426
Vogt W (1995) Oxidation of methionyl residues in proteins: tools, targets, and reversal. Free Radic Biol Med 18:93–105
Wu H, Tian C, Song X, Liu C, Yang D, Jiang Z (2013) Methods for the regeneration of nicotinamide coenzymes. Green Chem 1773-1789
Xia X, Longo LM, Blaber M (2015) Mutation choice to eliminate buried free cysteines in protein therapeutics. J Pharm Sci 104:566–576
Xie M, Alonso H, Roujeinikova A (2011) An improved procedure for the purification of catalytically active alkane hydroxylase from Pseudomonas putida GPo1. Appl Biochem Biotechnol 165:823–831
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).
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The authors declare that they have no competing interests.
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This article does not contain any studies with human participants or animals performed by any of the authors.
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Kirmair, L., Seiler, D.L. & Skerra, A. Stability engineering of the Geobacillus stearothermophilus alcohol dehydrogenase and application for the synthesis of a polyamide 12 precursor. Appl Microbiol Biotechnol 99, 10501–10513 (2015). https://doi.org/10.1007/s00253-015-6930-5
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DOI: https://doi.org/10.1007/s00253-015-6930-5