Meedium engineering of haloalkane dehalogenase catalysis: Effect of water-miscible organic solvents

Bc. Veronika Štěpánková, Ph.D.

Master's thesis

Abstract:

Enzymes represent a potential alternative to chemical synthesis due to their high selectivities and rapid catalysis under ambient reaction conditions. Nevertheless, synthetic chemists have been reluctant to employ enzymes as catalysts, because it was believed that enzymes are only active in aqueous media. Unfortunately, most organic compounds are water insoluble. The realization that enzymes can work and sometimes even improve their substrate specificity, enantioselectivity or stability in the organic media, has changed the prospects of employing enzymes in organic synthesis and industrial bioconversions. This approach, called „medium engineering“, can be also exploited as an alternative to protein engineering. Recently, the use of enzymes in all sorts of non-conventional media, such as organic solvents, supercritical fluids or ionic liquids has been one of the most exciting facets of enzymology. Little information is available on the mechanism of action of organic solvents on enzyme activity. Therefore, the influence of organic solvents on given enzyme can not be predicted and their effect has to be tested empirically. This Thesis is focused on medium engineering of hydrolytic enzymes. The attention was devoted to the study of three haloalkane dehalogenases (HLDs) known to catalyse the variety of reactions of great environmental and industrial significance. The HLDs LinB from Sphingobium japonicum UT26 and DhaA from Rhodococcus rhodochrous NCIMB 13064 were selected for their wide substrate specificities and ability to detoxify 2-chloroethyl-ethylsulfide (yperite). To some extent, the biological detoxification is limited by low solubility of yperite, therefore the presence of organic solvents in decontamination mixture is advantageous. Another HLD selected for our experiment was DbjA from Bradyrhizobium japonicum USDA110. The DbjA enzyme exhibits new substrate specificity and high enantioselectivity. Thanks to this property DbjA belongs to the enzymes suitable for industrial scale synthesis of optically pure compounds.

Keywords

circular dichroism, enzyme activity, enzyme structure, fluorescence, haloalkane dehalogenase, LogP, medium engineering, water-miscible organic solvents

Language used: English

Date on which the thesis was submitted / produced: 29. 4. 2008

Thesis defence

• Date of defence: 10. 6. 2008
• Supervisor: prof. RNDr. Zbyněk Prokop, Ph.D.
• Reader: RNDr. Lubomír Janda, Ph.D.

Masaryk University

Faculty of Science

Master programme / field:
Biochemistry / Biochemistry