Thermodynamic and kinetic phenomena in the enzymatic conversion of acetophenone to 1-(R)-phenylethanol in a continuous gas/solid reactor

  • Thermodynamische und kinetische Phänomene in der enzymatischen Konversion von Acetophenon zu 1-(R)-Phenylethanol in einem kontinuierlichen Gas/Feststoff-Reaktor

Dimoula, Kerasina; Büchs, Jochen (Thesis advisor)

Aachen : Publikationsserver der RWTH Aachen University (2009)
Dissertation / PhD Thesis

Aachen, Techn. Hochsch., Diss., 2009

Abstract

Nowadays, the Gas/Solid Biocatalytic Systems gain continuously ground within the area of the Non-Conventional Biocatalysis. In order to use the gas/solid catalysis for analytical and synthetical purposes, the respective process has to be understood in detail. Therefore, the construction and characterization of a continuous gas/solid reactor was here demonstrated. The overlaying thermodynamic and kinetic phenomena taking place during the reduction of acetophenone to 1-(R)-phenylethanol with the concomitant oxidation of 2-propanol to acetone catalyzed by deposited wild type LBADH were described. The operation of the reactor set-up was validated and the achievement of thermodynamic control was proven. At the validated system, the mass transfer along the packed bed was investigated and axial dispersion of the reacting compounds along the packed-bed was predicted. Nevertheless, the dispersion over the packed-bed was overlaid by a pronounced adsorption of acetophenone, reaching up to 6 mgacetophenone/mgprotein. The role of water was investigated through adsorption studies and was proven to be central, influencing and interconnecting the separately studied phenomena. The hydration of the deposited enzyme was described by a BET-like isotherm. It was shown that the presence of sucrose in the enzyme preparation is significant for water activities exceeding 0.5 and reaches a factor of 2 with respect to bead mass at a water activity of 0.9. Significant hysteresis during water desorption was identified, resulting in up to 0.6 mgwater/mgprotein for lyophilized enzyme and up to 10 mgwater/mgprotein for deposited enzyme preparation. The minimal water activity at which measurable conversion at the reactor was achieved was shown to be between 0.2 and 0.25, corresponding to approximately 5 mgwater/mgprotein of adsorbed water, whereas further increase of the water activity of the reaction mixture led to an almost exponential increase of the conversion. A kinetic study was performed, under strictly reaction rate limiting conditions that demonstrated a first order kinetic pattern with respect to acetophenone (vmax/KM=0.0046 µmol/min/IU) and a Michaelis-Menten pattern with respect to 2-propanol (vmax=0.0046 µmol/min/IU and KM=0.105). The stability of the deposited wild type LBADH without sucrose was investigated under standard operating conditions and the significant role of water activity was underlined. The purification state of the enzyme prior to deposition and the handling of the deposited enzyme preparation were highlighted as decisive factors for the operational stability. A comparative study of the operational stability of the wild type LBADH and the G37D NADH-dependent mutant demonstrated that the prediction of enzyme properties from data obtained in solution is not straightforward. The enantioselectivity of the enzyme in the studied reaction system was also investigated. The acetophenone conversion was performed with high enantioselectivity in the gas/solid reactor, yielding enantiomeric excess values approaching 99.5 %, at all commonly used operating conditions. The most significant parameter influencing the enantioselectivity was found to be the water activity of the reaction mixture. Throughout the overall project the performance of the reactor set-up as well as of the deposited enzyme preparation was tested and challenged. Several weaknesses of the immobilization method of choice were revealed: pronounced diffusional limitation at high specific activities of deposited enzyme preparation with sucrose, leaching of the enzyme from its support at higher humidity levels, effect of the enzyme preparation handling on the measurement reproducibility and others. Those effects suggested the choice of a stronger, probably covalent immobilization method in the future and/or use of porous carriers.

Identifier