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Purification and CNBr cleavage of secondary alcohol dehydrogenase from Micrococcus luteus WIU/JH20
Author(s) -
Weber Scott A.,
Huang JenqKuen,
Wen Lisa
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a471-a
Subject(s) - oleic acid , chemistry , biochemistry , alcohol dehydrogenase , fatty acid , yield (engineering) , micrococcus luteus , amino acid , linoleic acid , enzyme , organic chemistry , gene , escherichia coli , materials science , metallurgy
The conversion of oleic acid and linoleic acid to their corresponding hydroxyl and keto‐ fatty acid derivatives has industrial applications. Both of these fatty acids are found in soapstock, a waste product from the production of edible oils. The conversion of oleic acid to 10‐ hydroxysteric acid (10‐HSA) is catalyzed by oleate hydratase, and the conversion of 10‐HSA to 10‐ketosteric acid (10‐KSA) is catalyzed by secondary alcohol dehydrogenase (2°‐ADH). Of the two fatty acid derivatives, 10‐ HSA is more valuable for industrial applications then 10‐ KSA. Eliminating the production of 10‐KSA would make downstream processing of the 10‐HSA easier. Nocardia cholesterolicum NRRL 5767 is known to convert oleic acid to 10‐ HSA with high yields, but a small amount of 10‐KSA is also produced. The strain improvement of this microorganism to posses the oleate hydratase and lack the 2°‐ADH would allow high production of 10‐HSA and eliminate 10‐KSA making down stream processing more economical. Therefore, it is our interest to study the gene encoding 2°‐ADH. The 2°‐ADH has been purified to homogeneity and its N‐terminal amino acid sequence determined. Preliminary data shows that CNBr treatment of the purified enzyme yield five peptide fragments. We are in the process of trying to yield enough peptide fragments for internal sequencing. The deduced N‐ and internal sequences will be used to synthesize a probe by Polymerase Chain Reaction which will be used to clone the gene. This work is supported in part by grants from USDA CSREES 04‐35504‐14712, 02‐015470, and the University Research Council at Western Illinois University.