22 research outputs found
Heterologous expression and characterisation of a biosynthetic thiolase from Clostridium butyricum DSM 10702
Central pathway engineering for enhanced succinate biosynthesis from acetate in Escherichia coli
Discovery of a novel thermostable Zn 2+ -dependent alcohol dehydrogenase from Chloroflexus aurantiacus through conserved domains mining
Combined heterogeneous bio- and chemo-catalysis for dynamic kinetic resolution of (rac)-benzoin
Dynamic kinetic resolution (DKR) of racemic starting material is a promising route to optically pure (S)-benzoin (2-hydroxy-1,2-di(phenyl)ethanone) and various symmetrical and unsymmetrical derivatives thereof. Here, this route was advanced towards technical scale synthesis using the basic (rac)-benzoin as model system. The reaction employed stereoselective transesterification of (S)-benzoin with lipase TL® from Pseudomonas stutzeri and racemization of (R)-benzoin with Metal-TUD-1, a metal-associated acidic meso-porous silicate, in pure organic solvent. Enzyme performance was improved by immobilization on Accurel MP1001 (yielding Acc-LipTL), and Zr-TUD-1 (Si/Zr = 25) was identified as most effective racemization catalyst. Compatibility in solvent and temperature dependency enabled performance in only one pot. DKR in toluene at 50 °C yielded conversions above 98% and an ee of >97% in only five hours. Stability of Acc-LipTL was further improved with polyethylene imine and the reaction system was then reused in five cycles, retaining a conversion of >99% and a product-ee of >98%. On a semi-preparative scale, the isolated yield of enantiopure (S)-benzoin butyrate was >98%. Thus, the system provides a good basis for synthesis of enantiopure benzoin, and potentially a broader range of aromatic α-hydroxy ketones
Reengineered carbonyl reductase for reducing methyl-substituted cyclohexanones
The carbonyl reductase from Candida parapsilosis (CPCR2) is a versatile biocatalyst for the production of optically pure alcohols from ketones. Prochiral ketones like 2-methyl cyclohexanone are, however, only poorly accepted, despite CPCR2's large substrate spectrum. The substrate spectrum of CPCR2 was investigated by selecting five amino positions (55, 92, 118, 119 and 262) and exploring them by single site-saturation mutagenesis. Screening of CPCR2 libraries with poor (14 compounds) and well-accepted (2 compounds) substrates showed that only position 55 and position 119 showed an influence on activity. Saturation of positions 92, 118 and 262 yielded only wild-type sequences for the two well-accepted substrates and no variant converted one of the 14 other compounds. Only the variant (L119M) showed a significantly improved activity (7-fold on 2-methyl cyclohexanone; vmax = 33.6 U/mg, Km = 9.7 mmol/l). The L119M substitution exhibited also significantly increased activity toward reduction of 3-methyl (>2-fold), 4-methyl (>5-fold) and non-substituted cyclohexanone (>4-fold). After docking 2-methyl cyclohexanone into the substrate-binding pocket of a CPCR2 homology model, we hypothesized that the flexible side chain of M119 provides more space for 2-methyl cyclohexanone than branched L119. This report represents the first study on CPCR2 engineering and provides first insights how to redesign CPCR2 toward a broadened substrate spectru
Using Hydrogel Microparticles To Transfer Hydrophilic Nanoparticles and Enzymes to Organic Media via Stepwise Solvent Exchange
Immobilization of lipase B within micron-sized poly-N-isopropylacrylamide hydrogel particles by solvent exchange
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.The aim of the present work is the use of a water soluble enzyme in an organic solvent, still with a pronounced catalytic activity. Therefore, lipase B from Candida antarctica (CalB) is immobilized within micron-sized thermosensitive p-NIPAM hydrogel particles using a solvent exchange from polar to organic solvents. The absorbed amount of CalB is investigated at different immobilization temperatures. Confocal laser scanning microscopy (CLSM) shows that CalB is homogeneously distributed within the polymer network. An enhanced specific activity of CalB in n-hexane is achieved after immobilization within the p-NIPAM microgels. In order to get information on the supply of the substrate depending on the temperature, the activity is determined at different reaction temperatures. Additionally, the system is stable in the organic solvent, namely n-hexane, and shows a good reusability.DFG, EXC 314, Unifying Concepts in Catalysi