Luo Liu

Cloning, expression, and characterization of a self-sufficient cytochrome P450 monooxygenase from Rhodococcus ruber DSM 44319

A new member of class IV of cytochrome P450 monooxygenases was identified in Rhodococcus ruber strain DSM 44319. As the genome of R. ruber has not been sequenced, a P450-like gene fragment was amplified using degenerated primers. The flanking regions of the P450-like DNA fragment were identified by directional genome walking using polymerase chain reaction. The primary protein structure suggests a natural self-sufficient fusion protein consisting of ferredoxin, flavin-containing reductase, and P450 monooxygenase. The only flavin found within the enzyme was riboflavin 5′-monophosphate. The enzyme was successfully expressed in Escherichia coli, purified and characterized. In the presence of NADPH, the P450 monooxygenase showed hydroxylation activity towards polycyclic aromatic hydrocarbons naphthalene, indene, acenaphthene, toluene, fluorene, m-xylene, and ethyl benzene. The conversion of naphthalene, acenaphthene, and fluorene resulted in respective ring monohydroxylated metabolites. Alkyl aromatics like toluene, m-xylene, and ethyl benzene were hydroxylated exclusively at the side chains. The new enzyme’s ability to oxidize such compounds makes it a potential candidate for biodegradation of pollutants and an attractive biocatalyst for synthesis.

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

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. Biotechnol. Bioeng. 2011; 108:1307–1317.