Date of Award

Summer 7-15-2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

George E. Pierce

Second Advisor

Sidney A. Crow

Third Advisor

Eric S. Gilbert

Fourth Advisor

Robert B. Simmons

Abstract

Rhodococcus has diverse metabolic capabilities, such as delaying ripening of certain climacteric fruit. Nitrile hydratase (NHase), amidase, 1-aminocyclopropane-1-carboxylate deaminase (ACC deaminase), cyanidase, and β-cyanoalanine synthase-like enzyme (βCAS-like) are possibly involved in fruit ripening. The activity of these enzymes in Rhodococcus rhodochrous DAP 96253 cells were induced with selected multiple inducers (i.e. cobalt and urea).

This research showed that the supplementation of selected sugars, i.e. trehalose and maltodextrin in growth media and storage buffers of R. rhodochrous DAP 96253 affected activity and stability of the enzymes mentioned above. Thermostability and osmostability of the five enzymes in whole cells (plate grown and fermented) were evaluated in this study, i.e. βCAS-like was more stable than the other four enzymes in storage conditions.

Immobilized biocatalysts have practical advantages over the use of “free” whole cells. Immobilization of whole rhodococcal cells (plate grown and fermented) was employed, using techniques such as glutaraldehyde-polyethylenimine (GA-PEI) cross-linking, waxing and calcium-alginate entrapment. The GA-PEI immobilized catalysts were non-replicating and more stable in storage conditions than the catalysts produced by the other two methods. Wax or calcium-alginate immobilized catalysts (live catalysts) showed higher enzyme activity than the GA-PEI catalyst.

The effects of whole and immobilized catalysts were evaluated on delayed ripening of fruit. Both free whole cells and immobilized catalysts delayed the ripening of bananas and peaches. Delayed ripening experiments showed that the catalysts were effective in direct contact and not in contact with fruit. Moreover, both free whole cells and immobilized catalysts showed antifungal activity against Aspergillus niger and Penicillium spp.

Gas chromatography was performed to analyze volatile interactions between the biocatalysts and fruit. This analysis revealed that cyanide in an atmosphere with ethylene was utilized by the biocatalysts. There was also less volatile production by exposed fruit (bananas) than fruit unexposed to biocatalysts, either rhodococcal immobilized catalysts or live whole cells (plate grown and fermented).

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