Jin Byung Park.

Post-Doctorate Researcher
University of California, Berkeley

Ph.D. Biocatalysis and Biotransformation, 2004
Swiss Federal Institute of Technology (ETH), Zurich

M.S. Biochemical Engineering, 1995
Seoul National University, Korea

B.S. Food Science and Technology, 1993
Seoul National University, Korea


Biocatalysis

The biocatalyst is one of the most useful tools for asymmetric organic synthesis. The enzyme catalyzes a variety of reactions with high regio- and enantioselectivity under mild reaction conditions. Biocatalysis and biotransformation could account for 30% of the chemicals business by the year 2050 (OECD annual report, 1998). Of those, the hydroxylation of unactivated centers in hydrocarbons has been called potentially the most useful of all biotransformations.

Oxygenases and peroxidases are often used for asymmetric oxidation of hydrocarbons that is not accessible by chemical routes. Particularly, peroxidases (e.g. chloroperoxidase) do not require costly cofactors, and are stable outside cells. Yet, the enzymes can be irreversibly deactivated by oxidants (e.g. H2O2) and hydrophobic organic substrates and products during biotransformations. The goal of my research is to develop a reaction system to allow high stability of chloroperoxidase under process conditions. Salt-activation of the enzyme and the use of two-liquid phase system are investigated with bioconversion of styrene into (R)-styrene oxide as a model system.

Recently, we developed a new reaction system based on an organic/aqueous biphasic medium. This system could attenuate not only toxicity of the substrate and product but also toxicity of oxidants. Styrene oxide was produced to a concentration of 26mM in the organic phase within 2.5h under process conditions, indicating an average turnover frequency and a total turnover number of 4.2s-1 and 8,000 mol product/mol enzyme. These values correspond to the maximal and 5-fold higher than previously reported, respectively, for styrene epoxidation by CPO.

Summary of the PhD thesis

As compared to peroxidases, oxygenases mediate various reactions with high regio- and enantioselectivity. The enzymes have high substrate specificity yielding little byproducts. The molecular oxygen, which has no toxic effects on the biocatalysts, is used as oxidant. However, the enzymes are usually cofactor dependent and composed of multi-components, which are often unstable outside cells. Thus, living whole-cell cultures are favored in practical applications over the use of isolated enzymes as catalysts.

The investigation of oxygenase-based whole-cell biocatalysts lead to steadily increasing catalytic efficiencies. Yet, turnover rates of biocatalysts are frequently unstable. In long-term biotransformations, they are often lower than in short-term activity assays. In this study, we investigated factors influencing the biocatalytic efficiency of Escherichia coli JM101 (pSPZ10) expressing the styrene monooxygenase genes styAB of Pseudomonas sp. VLB120 in a bis(2-ethylhexyl)phthalate (BEHP)-based two-liquid phase system. The epoxidation of styrene to (S)-styrene oxide was not limited by cellular enzyme activity and mass transport of the reaction substrates styrene and oxygen to the biocatalyst. It was influenced by product toxicity and the availability of a carbon source needed for cofactor regeneration.

An epoxidation process based on fed-batch cultivation was further characterized. The optimization of biocatalyst concentration in the reaction medium, carbon feed rate and composition, and biotransformation conditions such as pH and dissolved oxygen tension resulted in average and maximal volumetric productivities higher than 800U/L and 1800U/L, respectively. Product concentrations above 500mM in the organic phase (60g/Lorg) were reached. The efficiency of reduction equivalent transfer from the carbon source to bioconversion increased from 10.4 to 13.8%.

One approach to increase the oxidation efficiency of the whole-cell biocatalyst may be the use of solvent tolerant strains as biocatalysts. In this sense, the solvent tolerant knock-out mutant Pseudomonas sp. VLB120ΔC was investigated and compared with E. coli JM101 (pSPZ10). The Pseudomonas mutant strain showed a high enzyme activity (97U/g dry cells) in whole-cell assays, and a high bioconversion rate (54U/g dry cells) during continuous culture-based transformations. Most interestingly, the Pseudomonas sp. VLB120ΔC strain showed a better channeling of cell energy into the biocatalytic reaction as compared to the E. coli recombinant. Less energy was wasted for cell maintenance in the organic/aqueous environment. This resulted in a 25% higher volumetric productivity under identical conditions as compared to E. coli JM101 (pSPZ10).

As in the styrene epoxidation process described here, product toxicity often is a major limiting factor in oxygenation processes based on whole cells. Such constraints can be overcome via process optimization as well as host cell engineering, whereas an increased solvent tolerance also can improve the carbon utilization efficiency. This study contributes to the development of microorganisms as powerful catalysts in large scale organic synthesis.