Purified recombinant human β‐carotene 15–15′‐oxygenase 1 (BCO1) cleaves β‐apocarotenals and lycopene

β‐Carotene 15–15′‐oxygenase 1 (BCO1) cleaves provitamin A carotenoids at the 15–15′ bond to produce retinal (vitamin A). This study aimed to elucidate the substrate specificity using pure BCO1, rather than partially purified preparations as used in previous studies. The enzyme was cloned into an expression vector with a C‐terminal His‐tag, expressed in E.coli , and purified using Co 2+ ion affinity chromatography. The pure enzyme is soluble in detergent‐free buffer, however greater enzyme activity was observed if Triton X‐100 was present during purification and storage. BCO1 cleaved ß‐carotene, ß‐cryptoxanthin, α‐carotene, ß‐apo‐8′‐ carotenal, ß‐apo‐10′‐carotenal and ß‐apo‐12′‐carotenal to yield retinal. BCO1 cleavage of lycopene yielded acycloretinal. Lycopene and ß‐apo‐12′‐carotenal were previously reported to be unreactive with BCO1. BCO1 did not catalyze cleavage of lutein or zeaxanthin. We have calculated apparent k cat and K M values for each substrate; however, some substrates yielded kinetics that did not fit well with Michaelis‐Menten kinetics. However, among 40 carbon substrates activity was greatest with ß‐carotene, followed by ß‐cryptoxanthin, α‐carotene, and lycopene. For the ß‐apocarotenals activity decreased with decreasing length of the molecule. (NIH Grant R01‐HL49879 and a grant from the Ohio Agricultural Research Development Center (OARDC)).