Date of Graduation

5-2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Poultry Science (PhD)

Degree Level

Graduate

Department

Poultry Science

Advisor/Mentor

Ricke, Steven C.

Committee Member

Slavik, Michael F.

Second Committee Member

Kwon, Young Min

Third Committee Member

Du, Yuchun

Keywords

Biological sciences; Bacillus; Linoleic acid; Methionine; Recombinant protein

Abstract

Linoleic acid isomerase (LAI) is the enzyme that converts linoleic acid to conjugated linoleic acids (CLAs) which appear to be capable of lowering cancer risk and enhancing immunity. There is an interest in developing commercial processes for the production of single isomers of CLA by biotransformation of LA using microbial cells and enzymes. However, the evaluation of these strains suggest that growth and LA isomerase production levels by these anaerobes are insufficient to support economic commercial production of single CLA isomers. A better alternative is to clone the isomerase gene and to generate new production strains using recombinant technology. Bifidobacterium LAI genes were cloned into pET24a(+) as an expression vector and transferred into E. coli BL21 (DE3) as the expression host while Propionibacterium acnes LAI were cloned into Bacillus species as the expression hosts. Unfortunately, the expressed Bifodobacterium LAI formed inclusion bodies and did not exhibit detectable enzymatic activities. Propeptide Staphylococcus hyicus lipase fused with P. acnes LAI expressed in B. megaterium YYBm1 was secreted into the surrounding medium. Based on MALDI-TOF MS results showed that this propeptide S. hyicus lipase still attached with the secreted LAI and might inhibit activity. However, other propeptides (B. subtilis nprE, B. subtilis amyE, B. megaterium nprM) did not protect LAI from proteolytic degradation.

Methionine is the first limiting amino acid in poultry feed and must be supplemented to the poultry feed mixture. Many studies attempted to isolate methionine producing microoragnisms from environments and genetically modified by using chemical mutagens. However, genetically-modified organisms are considered unacceptable for use in organic food production. Therefore, wild type strains with methionine-producing ability are necessary for the organic poultry industry. In this study, numerous bacteria were isolated from natural environments that appeared to produce high methionine; however, all of them were identified as pathogens. In addition, bacteria excreted methionine into the surrounding medium and it was concluded that this could be impractical for large scale recovery of methionine. Conversely, isolated yeast strain K1 yielded high methionine content, compared to other yeasts. Based on large subunit rRNA sequences, these isolated strains were identified as Pichia kudriavzevii/Issatchenkia orientalis. P. kudriavzevii/I. orentalis is recognized as a "generally recognized as safe " (GRAS) organism. Yeast strain K1 may be suitable as a source of methionine for dietary supplements in organic poultry feed.

Download

Share

COinS