Date of Award

2018

Degree Type

Thesis

Degree Name

Master of Science (MS)

Author's Department

Biological Sciences

First Advisor

Steven L. Daniel

Abstract

Bile acids are one group of steroids that can be metabolically transformed by human gut anaerobes. One of these transformations is the conversion of primary bile acids to secondary bile acids by 7α-dehydroxylating gut bacteria such as Clostridium scindens, Clostridium hylemonae, and Clostridium hiranonis. These anaerobes metabolize bile acid derivatives via 3α- and 7α-hydroxysteroid dehydrogenases (HSDH). Studies to date have not reported if these organisms are capable of converting 12-oxolithocholic acid (12-oxoLCA) to deoxycholic acid (DCA). The emphasis of this study was to determine the ability of C. scindens ATCC 35704, C. hiranonis DSM 13275, and C. hylemonae DSM 15053 to reduce 12-oxoLCA to DCA during growth, to identify 12α-HSDH genes in, C. scindens and C. hiranonis, to express, purify, and characterize the 12α-HSDH gene product (protein) of C. scindens. These organisms were grown anaerobically at 37°C in brain heart infusion (BHI) broth. Bile acids, prepared in methanol, were added to BHI broth to a final concentration of 0.1 mM. Following growth, cultures were extracted with ethyl acetate; extracts were dried, re-suspended in methanol, and subjected to thin-layer chromatography (TLC) for the detection of bile acids. In addition, 12α-HSDH recombinant enzymes were characterized and purified by affinity chromatography using Strep-Tactin® resin. Kinetic constants (Km, Kcat, Vmax, and Kcat/Km) were determined for the purified recombinant proteins for the forward and reverse directions. Catalytic efficiencies Kcat/Km toward the reductive direction were between 2.8-3.5-fold greater than oxidative direction. However, Vmax and Kcat in the oxidative direction were between 1.6-2.9-fold higher than the reductive direction. The 12α-HSDH recombinant enzyme from each strain was shown to reduce 12-oxoLCA to DCA. Moreover, these organisms were able to transform other oxo-bile acids during growth.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

Included in

Microbiology Commons

Share

COinS