Overproduction of a recombinant heat-stable catalase for use in an extremophile enzyme lab exercise

ABSTRACT The manganese-dependent catalase of Pyrobaculum calidifontis , a facultative aerobe that grows optimally at 90 ˚C, has been previously used in a teaching lab to highlight salient characteristics of hyperthermophilic extremophiles. However, accruing enough enzyme from the native organism, which would allow each student in a typical classroom to carry out the experiment individually, is difficult for several reasons, ranging from the fastidious growth requirements of P. calidifontis to the need of specialized equipment for preparing cell extracts. Here, E. coli was used in the overproduction of recombinant P. calidifontis catalase (kat Pc ). The recombinant catalase enzyme was activated with manganese and purified by heat treatment. Experiments discussed here indicate that the use of the recombinant enzyme is a viable alternative for instructors who may or may not have access to P. calidifontis or mechanical means of cell lysis.