2549-57-7Relevant academic research and scientific papers
High performing immobilized Baeyer-Villiger monooxygenase and glucose dehydrogenase for the synthesis of ε-caprolactone derivative
Delgove, Marie A.F.,Valencia, Daniela,Solé, Jordi,Bernaerts, Katrien V.,De Wildeman, Stefaan M.A.,Guillén, Marina,álvaro, Gregorio
, p. 134 - 141 (2019/01/09)
The industrial application of Baeyer-Villiger monooxygenases (BVMOs) is typically hindered by stability and cofactor regeneration considerations. The stability of biocatalysts can be improved by immobilization. The goal of this study was to evaluate the (co)-immobilization of a thermostable cyclohexanone monooxygenase from Thermocrispum municipale (TmCHMO) with a glucose dehydrogenase (GDH) from Thermoplasma acidophilum for NADPH cofactor regeneration. Both enzymes were immobilized on an amino-functionalized agarose-based support (MANA-agarose). They were applied to the oxidation of 3,3,5-trimethylcyclohexanone for the synthesis of ε-caprolactone derivatives which are precursors of polyesters. The performances of the immobilized biocatalysts were evaluated in reutilization reactions with as many as 15 cycles and compared to the corresponding soluble enzymes. Co-immobilization proved to provide the most efficient biocatalyst with an average conversion of 83% over 15 reutilization cycles leading to a 50-fold increase of the biocatalyst yield compared to the use of soluble enzymes which were applied in a fed-batch strategy. TmCHMO was immobilized for the first time in this work, with very good retention of the activity throughout reutilization cycles. This immobilized biocatalyst contributes to the application of BVMOs in up-scaled biooxidation processes.
Enzymatic Synthesis of Trimethyl-?-caprolactone: Process Intensification and Demonstration on a 100 L Scale
Solé, Jordi,Brummund, Jan,Caminal, Gloria,álvaro, Gregorio,Schürmann, Martin,Guillén, Marina
, p. 2336 - 2344 (2019/10/14)
Optimization and scaling up of the Baeyer-Villiger oxidation of 3,3,5-trimethyl-cyclohexanone to trimethyl-?-caprolactones (CHLs) were studied to demonstrate this technology on a 100 L pilot plant scale. The reaction was catalyzed by a cyclohexanone monooxygenase from Thermocrispum municipale that utilizes the costly redox cofactor nicotinamide adenine dinucleotide phosphate (reduced form), which was regenerated by a glucose dehydrogenase (GDH). As a first stage, different cyclohexanone monooxygenase formulations were tested: Cell-free extract, whole cells, fermentation broth, and sonicated fermentation broth. Using broth resulted in the highest yield (63%) and required the least biocatalyst preparation effort. Two commercial glucose dehydrogenases (GDH-105 and GDH-01) were evaluated, resulting in similar performances. Substrate dosing rates and biocatalyst loadings were optimized. On a 30 mL scale, the best conditions were found when 30 mM h-1 dosing rate, 10% (v/v) cyclohexanone monooxygenase broth, and 0.05% (v/v) of glucose dehydrogenase (GDH-01) liquid enzyme formulation were applied. These same conditions (with oxygen instead of air) were applied on a 1 L scale with 92% conversion, achieving a specific activity of 13.3 U gcell wet weight (cww) -1, a space time yield of 3.4 gCHL L-1 h-1, and a biocatalyst yield of 0.83 gCHL gcww -1. A final 100 L demonstration was performed in a pilot plant facility. After 9 h, the reaction reached 85% conversion, 12.8 U gcww -1, a space time yield of 2.7 g L-1 h-1, and a biocatalyst yield of 0.60 gCHL gcww -1. The extraction of product resulted in 2.58 kg of isolated final product. The overall isolated CHL yield was 76% (distal lactone 47% and proximal lactone 53%).
Toward Upscaled Biocatalytic Preparation of Lactone Building Blocks for Polymer Applications
Delgove, Marie A. F.,Elford, Matthew T.,Bernaerts, Katrien V.,Wildeman, Stefaan M. A. De
supporting information, p. 803 - 812 (2018/06/19)
Although Baeyer-Villiger monooxygenases (BVMOs) have gained attention in recent years, there are few cases of their upscaled application for lactone synthesis. A thermostable cyclohexanone monooxygenase from Thermocrispum municipale (TmCHMO) was applied to the oxidation of 3,3,5-trimethylcyclohexanone using a glucose dehydrogenase (GDH) for cofactor regeneration. The reaction progress was improved by optimizing the biocatalyst loading, with investigation into oxygen limitations. The product concentration and productivity were increased by keeping the substrate concentration below the inhibitory level via continuous substrate feeding (CSF). This substrate feeding strategy was evaluated against two biphasic reactions using either toluene or n-butyl acetate as immiscible organic solvents. A product concentration of 38 g L-1 and a space-time yield of 1.35 g L-1 h-1 were achieved during the gram-scale synthesis of the two regioisomeric lactones by applying the CSF strategy. These improvements contribute to the large-scale application of BVMOs in the synthesis of branched building blocks for polymer applications.
Polycyclic ketone monooxygenase from the thermophilic fungus Thermothelomyces thermophila: A structurally distinct biocatalyst for bulky substrates
Fürst, Maximilian J.L.J.,Savino, Simone,Dudek, Hanna M.,Castellanos, J. Rúben Gómez,De Souza, Cora Gutiérrez,Rovida, Stefano,Fraaije, Marco W.,Mattevi, Andrea
supporting information, p. 627 - 630 (2017/05/15)
Regio- and stereoselective Baeyer-Villiger oxidations are difficult to achieve by classical chemical means, particularly when large, functionalized molecules are to be converted. Biocatalysis using flavin-containing Baeyer-Villiger monooxygenases (BVMOs) is a wellestablished tool to address these challenges, but known BVMOs have shortcomings in either stability or substrate selectivity. We characterized a novel BVMO from the thermophilic fungus Thermothelomyces thermophila, determined its three-dimensional structure, and demonstrated its use as a promising biocatalyst. This fungal enzyme displays excellent enantioselectivity, acts on various ketones, and is particularly active on polycyclic molecules. Most notably we observed that the enzyme can perform oxidations on both the A and D ring when converting steroids. These functional properties can be linked to unique structural features, which identify enzymes acting on bulky substrates as a distinct subgroup of the BVMO class.
Lactones 34 [1]. Application of alcohol dehydrogenase from horse liver (HLADH) in enantioselective synthesis of δ- and ε-lactones
Boratynski, Filip,Kielbowicz, Grzegorz,Wawrzenczyk, Czeslaw
experimental part, p. 30 - 36 (2010/12/18)
The ability of horse liver alcohol dehydrogenase (HLADH) to the enantioselective oxidation of primary-primary, primary-secondary and primary-tertiary aliphatic 1,5- and 1,6-diols 1a-i was studied. No enantioselectivity of the transformations of primary-primary 1,6-diols 1a-d to ε-lactones 4a-d was observed. Regioselective oxidation of primary-secondary 1,6-diols 1e,f and 1,5-diols 1h,i afforded enantiomerically enriched ε-lactones 4e,f and δ-lactones 4h,i. ε-Lactones 4e,f were formed with higher enantiomeric excesses (e.e. = 85-99%). Enzymatic oxidation of primary-tertiary 1,6-diol 1g did not give lactone product.
Crystal structures of cyclohexanone monooxygenase reveal complex domain movements and a sliding cofactor
Mirza, I. Ahmad,Yachnin, Brahm J.,Wang, Shaozhao,Grosse, Stephan,Bergeron, Helene,Imura, Akihiro,Iwaki, Hiroaki,Hasegawa, Yoshie,Lau, Peter C. K.,Berghuis, Albert M.
experimental part, p. 8848 - 8854 (2009/12/04)
Cyclohexanone monooxygenase (CHMO) is a flavoprotein that carries out the archetypical Baeyer-Villiger oxidation of a variety of cyclic ketones into lactones. Using NADPH and O2 as cosubstrates, the enzyme inserts one atom of oxygen into the substrate in a complex catalytic mechanism that involves the formation of a flavin-peroxide and Criegee intermediate. We present here the atomic structures of CHMO from an environmental Rhodococcus strain bound with FAD and NADP+ in two distinct states, to resolutions of 2.3 and 2.2 A. The two conformations reveal domain shifts around multiple linkers and loop movements, involving conserved arginine 329 and tryptophan 492, which effect a translation of the nicotinamide resulting in a sliding cofactor. Consequently, the cofactor is ideally situated and subsequently repositioned during the catalytic cycle to first reduce the flavin and later stabilize formation of the Criegee intermediate. Concurrent movements of a loop adjacent to the active site demonstrate how this protein can effect large changes in the size and shape of the substrate binding pocket to accommodate a diverse range of substrates. Finally, the previously identified BVMO signature sequence is highlighted for its role in coordinating domain movements. Taken together, these structures provide mechanistic insights into CHMO-catalyzed Baeyer-Villiger oxidation.
