Biocatalytic process optimization based on mechanistic modeling of cholic acid oxidation with cofactor regeneration

Abstract

Reduction and oxidation of steroids in the human gut are catalyzed by hydroxysteroid dehydrogenases of microorganisms. For the production of 12-ketochenodeoxycholic acid (12-Keto-CDCA) from cholic acid the biocatalytic application of the 12α-hydroxysteroid dehydrogenase of Clostridium group P, strain C 48-50 (HSDH) is an alternative to chemical synthesis. However, due to the intensive costs the necessary cofactor (NADP(+) ) has to be regenerated. The alcohol dehydrogenase of Thermoanaerobacter ethanolicus (ADH-TE) was applied to catalyze the reduction of acetone while regenerating NADP(+) . A mechanistic kinetic model was developed for the process development of cholic acid oxidation using HSDH and ADH-TE. The process model was derived by identifying the parameters for both enzymatic models separately using progress curve measurements of batch processes over a broad range of concentrations and considering the underlying ordered bi-bi mechanism. Both independently derived kinetic models were coupled via mass balances to predict the production of 12-Keto-CDCA with HSDH and integrated cofactor regeneration with ADH-TE and acetone as co-substrate. The prediction of the derived model was suitable to describe the dynamics of the preparative 12-Keto-CDCA batch production with different initial reactant and enzyme concentrations. These datasets were used again for parameter identification. This led to a combined model which excellently described the reaction dynamics of biocatalytic batch processes over broad concentration ranges. Based on the identified process model batch process optimization was successfully performed in silico to minimize enzyme costs. By using 0.1 mM NADP(+) the HSDH concentration can be reduced to 3-4 µM and the ADH concentration to 0.4-0.6 µM to reach the maximal possible conversion of 100 mM cholic acid within 48 h. In conclusion, the identified mechanistic model offers a powerful tool for a cost-efficient process design.

Kinetics based reaction optimization of enzyme catalyzed reduction of formaldehyde to methanol with synchronous cofactor regeneration

“…Process optimization of coupled reactions commonly requires laborious experimentation. Considering the underlying mechanism of kinetics of each reaction, kinetic models may however be developed to simulate the biocatalytic process and predict a defined optimal performance (Braun, Link, Liu, Schmid, & Weuster‐Botz, ).…”

Application of Chemical Engineering Methodology in Process Development: A Case Study of MenD-catalyzed Synthesis of 6-Cyano-4-oxohexanoic Acid

“…For further growth and development of biocatalysis, biochemical-engineering approaches can be very beneficial. They include the use of mo-deling techniques 10,11 , process optimization tools [12][13][14] , as well as the use of early-stage economic assessment tool 1,15 . A model-based methodology can be used to define the process design search-space within which the optimal solution can be found; it can save development time and money as simulations increase the process understanding 11 , and it can have a favorable impact on product quality 16 .…”

Rigorous Model-Based Design and Experimental Verification of Enzyme-Catalyzed Carboligation under Enzyme Inactivation

“…A traditional method for performing this optimization is by carrying out numerous experiments in the lab that are cost and time intensive. Therefore, computer aided model based approaches have been proposed to complement laboratory experiments [7,8]. As examples of the application of model based approaches to optimize enzyme catalyzed reactions, Stillger et al developed an enzyme-membrane continuous stirred tank reactor (CSTR) for the carboligation of benzaldehyde and acetaldehyde catalyzed by benzaldehyde lyase from Pseudomonas fluorescens (Pf BAL) to produce (R)-2-hydroxy-1-phenylpropanone (HPP) [9].…”

“…Their simulations also showed that controlling the pH could increase conversions and improve productivity. By performing simulations with coupled kinetic models and reactor design equations, Braun et al minimized the enzyme costs for the biocatalytic production of 12-ketochenodeoxycholic acid in a batch reactor [7]. Marpani et al [12] utilized a kinetic model and simulation approach to determine the optimal operating conditions for the biocatalytic conversion of formaldehyde to methanol in a batch reactor.…”