Team:PrepaTec GarzaSadaMx/Protocols

Protocols

Naphthalene Dioxygenase This enzyme is obtained from the cells of the Pseudomonas sp. which has a melting point of 55°C. One of the early uses of the enzyme was made during the process of cloning gens which were implicated in the degradation of naphthalene. In this process the fragments that were cloned of the plasmid nah express themselves while in contact with E. coli creating blue colonies. The enzyme also works as a catalyst, transforming indole into indigo, from which we obtain the tyrian purple.

Image from: Webmaster, Department of Chemistry, University of Maine, Orono, ME 04469 What makes the indole change to indigo is an oxidation of the tryptophan that is cloned and expressed on the E. coli. The tryptophan or indole is an aromatic hydrocarbon, that when in contact with oxygen, changes to cis- dihydrodiol which makes up indigo. The oxidation is caused by combined activities of the trytophanase and napthalene diosygenase, as shown on the reaction. The optimum ph of the naphthalene dioxygenase in the Pseudomas sp. is of 7.5, meaning that the ph is almost neutral. The optimum temperature of the enzyme is 30°C. The pressure that is exerted on the enzyme is between a reduced pressure and atmospheric pressure, which is the normal pressure we feel.