Baqiyatallah University of Medical Sciences Journal of Applied Biotechnology Reports 2322-1186 13 1 2026 03 01 Expression of Mesophilic-Alkali-Stable Catalase Hydroperoxidase II from Staphylococcus equorum in Escherichia coli BL21(DE3) 1928 1939 242577 10.30491/jabr.2025.488165.1806 EN Rahma Ziska Pharmaceutical Biotechnology Laboratory, Department of Pharmaceutics, School of Pharmacy, Institut Teknologi Bandung, Bandung 40132, Indonesia Faculty of Pharmacy, Bhakti Kencana University, Bandung 40614, Indonesia 0000-0001-6939-0535 Catur Riani Pharmaceutical Biotechnology Laboratory, Department of Pharmaceutics, School of Pharmacy, Institut Teknologi Bandung, Bandung 40132, Indonesia 0000-0001-7082-0264 Ratna Annisa Utami Pharmaceutical Biotechnology Laboratory, Department of Pharmaceutics, School of Pharmacy, Institut Teknologi Bandung, Bandung 40132, Indonesia 0000-0002-5660-9961 Diky Mudhakir Department of Pharmaceutics, School of Pharmacy, Institut Teknologi Bandung, Bandung 40132, Indonesia 0000-0002-1081-5241 Journal Article 2024 11 11 Introduction: Catalase is a widely used enzyme with numerous advantages in industrial, diagnostic, and therapeutic applications. This study elucidates the characteristics of recombinant catalase hydroperoxidase II (rHPIISeq) from Staphylococcus equorum. Materials and Methods: A synthetic gene of catalase (hpII) was expressed in Escherichia coli BL21(DE3). The gene was codon-optimized and cloned commercially into vector pET-15b. Gene expression was performed under 0.5 mM of isopropyl-β-D-1-thiogalactopyranoside (IPTG) induction for 24 hours at 25 °C. The soluble recombinant catalase, rHPIISeq, was partially purified using ammonium sulfate precipitation followed by dialysis. Its activity was measured at 440 nm using a UV-visible spectrophotometer. Additionally, the effects of pH and temperature on the enzyme activity and stability were evaluated by incubating the enzyme across various pH levels and temperatures. Results: The pET-15b_HPIISeq recombinant plasmid was successfully constructed. The optimized gene of hpII consisted of 1,989 bp and encoded the rHPIISeq protein with a size of 75.22 kDa. The yield of soluble rHPIISeq was 9.73 mg in 1 L culture with 1.5–1.6 g of wet weight bacterial mass. Notably, approximately 90% of the produced protein formed inclusion bodies (IBs). Following partial purification, a 7-fold increase in the purification of soluble rHPIISeq was achieved using 40% ammonium sulfate precipitation, resulting in a purity level of about 60% with a yield of 93.7%. The enzyme exhibits optimal activity at a pH of 7 and a temperature of 40 °C, and it remains stable within a pH range of 6 to 10 at temperatures between 20 °C and 50 °C. Conclusions: This recombinant catalase is proposed as a mesophilic-alkali-stable enzyme, which is potentially beneficial for industrial applications, particularly in processes under alkaline conditions and a wide range of temperatures.

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