Title

Investigation of Coenzyme Q10 Production in Sporidiobolus johnsonii

Date of Award

2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Advisor(s)

Robert P. Doyle

Keywords

Affinity purification, Coenzyme q10, Fermentation, Purification, Saposin b, Sporidiobolus johnsonii

Subject Categories

Chemistry

Abstract

There is considerable current interest in CoenzymeQ10 (CoQ10) from a medical perspective. Coenzyme Q10 (CoQ10) plays a critical role in energy production in humans. Low levels of CoQ10 have been linked to diseases such as Parkinson's and Huntingtons's disease. CoQ10 levels decrease as we age, and the use of statin drugs also lowers CoQ10 levels. Oral supplementation increases CoQ10 levels. However, a lack of industrial sources of CoQ10 and the difficulty in CoQ10 purification has resulted in a need to solve both of these problems. Sporidiobolus johnsonii ( S. johnsonii ) has been reported to produce CoQ10 in papers that used only standards on thin-layer chromatography (TLC) and also suggested the production of Coenzyme Q9 (CoQ9). This work set out to verify CoQ9/CoQ10 production in S. johnsonii and quantify as appropriate. We show that S. johnsonii produces CoQ10, but find no evidence for CoQ9 biosynthesis. The specific production of CoQ10 was noted at 10 mg / g dry cell weight (DCW) in media supplemented with para -hydroxy benzoic acid (HBA). This makes S. johnsonii a naturally high CoQ10 producer. Identification of a closely eluting side product under normal phase chromatography is reported. Current purification methods for CoQ10 are difficult and expensive. A protein based purification method may alleviate the current problems associated with typical LC purification. Saposin B (sapB) has been shown to bind with CoQ10 at selective pH's. We hypothesized that utilizing a sapB coated support resin that an affinity purification method for CoQ10 could be produced. To this end, sapB was recombinantly expressed by fermentation producing 105 mg sapB per liter. The recombinant sapB was characterized beyond what is currently in literature. This includes pH dependent circular dichroism demonstrating a pH dependent alpha-helical structure, and investigation into the presence and effect of the disulfide bonds and their effect upon thermal stability and resin binding. The affinity resin was made by binding sapB via a his-tag to a sepharose IMAC bead. This work demonstrates that CoQ10 can be bound by a sapB based affinity resin with pH controlled binding and release.

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