Masters Theses

Date of Award

8-2013

Degree Type

Thesis

Degree Name

Master of Science

Major

Animal Science

Major Professor

Jun Lin

Committee Members

John C. Waller, Michael O. Smith, Niki Labbe

Abstract

Currently there is a global trend to eliminate the use of antibiotic growth promoters (AGP) in animal production due to the risk of creating reservoirs of antibiotic resistant bacteria. Previous studies have shown that the ability of AGP to promote growth is highly correlated with a decrease in the activity of bile salt hydrolase (BSH), an enzyme produced by commensal bacteria and whose actions may compromise host lipid metabolism; thus, BSH inhibitors could be a novel AGP alternative. In this study, a recombinant bile salt hydrolase (rBSH) from Lactobacillus salivarius was produced in an E. coli expression system, purified, and enzymatically characterized. Purification by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography consistently yielded approximately 15 mg of rBSH per liter of induced culture. A standard 2-step BSH activity assay which estimates the amount of amino acids liberated from conjugated bile acids was used to determine substrate specificity of the BSH and the effect of pH, temperature, and dietary compounds on BSH activity. The rBSH displayed its highest hydrolysis activity for glycochenodeoxycholic acid although there was no preference among other substrates tested. Optimal activity was observed between pH of 5.0 and 6.0 and between temperatures of 35°C to 55°C. Preliminary assays identified different dietary compounds that were potent inhibitors, including copper and zinc compounds which have previously been shown to boost feed efficiency and promote growth of poultry and swine. Furthermore, a high-throughput screening system (HTS) was developed for fast and efficient identification of potent BSH inhibitors. This HTS system was utilized to screen a compound library comprised of 2,240 biologically active and structurally diverse compounds. The pilot screen led to 107 hits and a preliminary review of biochemical information of the corresponding compounds further narrowed down those of interest. Several lead compounds have been validated by the standard 2-step BSH activity assay and will be subjected to future in vivo analysis in a large-scale animal study. Overall, this study characterized a BSH with broad substrate specificity and developed and validated different strategies for identification of BSH inhibitors, the promising alternatives to AGP for enhancing the productivity and sustainability of food animals.

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