CENTENNIAL HONORS COLLEGE
Western Illinois University
Undergraduate Research Day 2016
PosterPresentation
MolecularCloningandExpressionofaSecondaryAlcoholDehydrogenasefrom
NocardiaCholesterolicumNRRL5767
JoshuaDiaz
FacultyMentors:Jenq-KuenHuangandLisaWen
Chemistry
NocardiacholesterolicumNRRL5767(N.cholesterolicumNRRL5767)isawell-known-bacteriumcapable
of transforming 95% of added oleic acid to a more valuable product, 10 hydroxystearic acid (10-HSA)
(ApplMicrobiolBiotechnol32,299-304,1989).Thereactioniscatalyzedbyoleatehydratase.However,a
smallamountof10-HSAissubsequentlyconvertedto10-ketostearicacid(10-KSA)bysecondaryalcohol
dehydrogenase(2o-ADH).Since10-HSAismorevaluablethan10-KSAinindustry,10KSAisconsidereda
side product which complicates downstream separation and purification of 10-HSA. It is attractive to
hindertheproductionof10-KSAthroughbacteriumstrainimprovementbygeneticengineeringtoblock
production of genetic engineering to block 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
beensuccessfullyamplifiedbyPCRfromgenomicDNAofN.cholesterolicumNRRL5767andsub-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
monitoringtheformationofNADHspectrophotometrically.