OPTIMIZATION OF THE EXTRACTION AND PURIFICATION OF A RECOMBINANT β -CAROTENE 15,15’-MONOOXYGENASE FROM INCLUSION BODIES

Abstract

Grasses used in the production of bovine livestock in the tropics contain high levels of β-carotene, which produces carcasses that contain yellow-pigmented fat, reducing their economic value. The yellow pigment is due to the low activity of the enzyme β -carotene 15,15’-monooxygenase (BCMO1) in the small intestine and liver. A biotechnological use of this enzyme could split β-carotene in two molecules of retinal, eliminating the source of coloring and optimizing the commercial value of the meat obtained from grazing cattle. The aim of this study was to obtain a recombinant BCMO1 enzyme with a similar activity to native enzymes, from bacteria modified with the gene that codifies the β-carotene 15,15’-monooxygenase in Gallus gallus (gBCMO1). The enzyme was overexpressed in an Escherichia coli XL1-Blue transformed with that gene and purified by Fast Protein Liquid Chromatography (FPLC); measuring the in vitro activity of the process by Immobilized Metal Affinity Chromatography (IMAC); and detecting the final product by High Polarity Liquid Chromatography (HPLC). A protein of approximately 63 kDa was obtained, which showed an enzyme activity of 2993 (± 108.2) pmol mg^-1 of protein h^-1 (n=3). The isolated protein can be evaluated in vitro as an additive, in order to reduce the yellow pigmentation of the livestock carcasses.