Biocatalysts are important for the synthesis of fine chemicals and steroidal drugs in the biopharmaceutical industry. Cytochromes P450 (P450) monooxygenases are significant biocatalysts due to their high selectivity for oxidation reactions. CYP119 is the first characterized thermoacidophilic P450. CYP119 was isolated from Sulfolobus acidocaldirius. CYP119 enzyme shows high stability at low pH and high temperature. CYP119 can utilize the peroxidase shunt pathway in the catalytic cycle of P450. These abilities make CYP119 attractive biocatalyst for production of fine chemicals and drugs. In this study, Leu69Gly mutant CYP119 enzyme was cloned by site-directed mutagenesis. L69G and WT CYP119 was expressed successfully in Escherichia coli BL21 (DE3) cells with isopropyl β-D-1- thiogalactopyranoside (IPTG). This study shows that L69G mutation is important for binding to progesterone. This was predicted by in silico mutagenesis in a previous computational study. Isolation and purification of the WT and L69G CYP119 were carried out. Activity assays and substrate binding studies of the enzymes were performed and compared each other. L69G mutation did not cause significant effect on Amplex Red® oxidation and styrene epoxidation activities. L69G CYP119 (KS: 34.55 ± 7.4 μM) showed higher affinity for progesterone compared to WT CYP119 (KS: 69.8 ± 48.9 mM). A new product, thought to be hydroxylated progesterone, was formed as result of hydroxylation of progesterone by L69G CYP119 using peroxidase shunt pathway.