Optimization of Catalase Production from CRISPR-Cas9-Mediated Editing of the LacZ Gene in Escherichia coli Using Various Carbon Sources (Manihot esculenta Crantz, Oryza sativa L., Solanum tuberosum L., and Glycin max (L.) Merr.)

Abstract

Catalase is an important antioxidant enzyme widely applied in food processing, textiles, and biomedical industries, yet it is largely imported into Nigeria at considerable cost. This study investigated the use of CRISPR-Cas9–edited Escherichia coli HB101-pBRKan and locally available agricultural by-products as low-cost carbon sources for catalase production. Cassava peel, potato peel, rice bran, and soybean meal were processed into powdered substrates and incorporated into a basal medium containing KH₂PO₄ (2.13 g/L), MgSO₄ (0.5 g/L), and NaH₂PO₄ (1.5 g/L). Fermentations were carried out at varying temperatures (4, 37, and 50 °C) and pH values (4.5, 6.5, and 9.0). Growth was monitored for seven days using OD₅₄₀ nm, and the spectrophotometer was used to assess the catalase activity through hydrogen peroxide decomposition. The lacZ gene was targeted with CRISPR-Cas9 to examine its influence on catalase production. Colony colour on X-gal plates reflected editing outcomes: blue colonies appeared where lacZ remained intact, white colonies indicated successful editing, and plates lacking arabinose showed no growth due to insufficient Cas9 induction. Multiplex PCR confirmed gene disruption, producing an approximately 650 bp amplicon in edited strains and 1100 bp fragment in unedited controls. Temperature influenced enzyme output markedly. The edited strain showed improved tolerance to higher temperatures, achieving its highest catalase activity at 50 °C (1.6 U/mL), whereas the unedited strain reached maximal activity at 37 °C (1.0 U/mL). Clear differences were observed in the substrates: cassava peel supported the highest catalase yield (OD₅₄₀ = 1.591 at pH 6.5 and 50 °C), while potato peel substrates demonstrated optimal activity (OD₅₄₀ = 0.595 at pH 6.5 and 37 °C). In contrast, soybean meal and rice bran favoured the unedited strain in both growth and enzyme production. Overall, this study shows that CRISPR-Cas9–Edited E. coli can produce higher levels of catalase under the right environmental conditions. It also identifies cassava peel waste as a strong, practical option for local enzyme production. Together, these findings point to a realistic path for turning agricultural waste into value and using gene-edited microbes to cut Nigeria’s reliance on imported catalase.