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Molecular Cloning and Expression of a Secondary Alcohol Dehydrogenase From Nocardia Cholesterolicum NRRL 5767
Author(s) -
Diaz Joshua D,
Vanderway David R,
Flores Micah,
Wen Lisa,
Huang JenqKuen
Publication year - 2016
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.30.1_supplement.838.3
Subject(s) - alcohol dehydrogenase , biochemistry , chemistry , recombinant dna , enzyme , oleic acid , dehydrogenase , cloning (programming) , molecular cloning , gene , gene expression , computer science , programming language
Norcardia cholesterolicum NRRL 5767 ( N. cholesterolicum NRRL 5767) is a well‐known bacterium capable of transforming 95% of added oleic acid to a more valuable product, 10‐hydroxystearic acid (10‐HSA) (Appl Microbiol Biotechnol 32, 299–304, 1989). This reaction is catalyzed by oleate hydratase. However, a small amount of the 10‐HSA is subsequently converted to 10‐ketostearic acid (10‐KSA) by secondary alcohol dehydrogenase (2º‐ADH). Since 10‐HSA is more valuable than 10‐KSA in industry; 10‐KSA is considered a side product which complicates downstream separation and purification of 10‐HSA. It is attractive to hinder the production of 10‐KSA through bacterium strain improvement by genetic engineering to block the production of 2º‐ADH. The objective of this research is to clone, overexpress, and characterize functional recombinant 2º‐ADH. An annotated 2º‐ADH gene has been successfully amplified by PCR from genomic DNA of N. cholesterolicum NRRL 5767and sub‐cloned into pET28a expression vector. The 2º‐ADH protein has been overexpressed by IPTG induction and purified to homogeneity by Ni‐NTA affinity column. Enzyme activity of the 2º‐ADH was determined by monitoring the formation of NADH spectrophotometrically. Support or Funding Information Western Illinois University Foundation and the Department of Chemistry.