Targeted Assay Engineering Enhances Bile Salt Hydrolase Activity in Heyndrickxia Coagulans ATCC 7050 and Lactiplantibacillus Plantarum ATCC 10012

The recent emergence of bile salt hydrolase (BSH) enzyme as a therapeutic target reflects its unbound potential in mitigating hypercholesterolemia, obesity, and gastrointestinal issues. However, to bolster its industrial application, optimization of BSH assay lays the cornerstone for enhancing sensitivity, specificity, and reproducibility. The current study delved into optimizing the BSH assay parameters utilizing response surface methodology (RSM) and one-factor-at-a-time (OFAT) method for two novel, natural BSH producers, Heyndrickxia coagulans ATCC 7050 and Lactiplantibacillus plantarum ATCC 10012. Factors such as pH, temperature, cell concentration, and substrate concentration were optimized using RSM and numerical optimization. The analysis of responses unveiled significant insights into the biochemical characteristics of BSH from both organisms. The optimal pH for BSH activity from H. coagulans ATCC 7050 and L. plantarum ATCC 10012 was determined to be 6.1 and 6.0, with their corresponding optimal temperatures being 60 °C and 40 °C, respectively. Subsequent to RSM, optimization of the remaining parameters such as buffer type, buffer molarity, cells-to-substrate ratio, etc., performed using the classical OFAT approach further enhanced BSH activity, with H. coagulans ATCC 7050 and L. plantarum ATCC 10012 exhibiting a 1.45 and 0.87-fold increase, respectively. Conventionally, even though BSH has been optimized using the OFAT approach, this is the first instance in which a hybrid model using RSM has been applied to achieve a greater performance. These findings offer valuable insights in augmenting the specificity, efficiency, and stability of BSH and broaching new avenues for industrial and therapeutic applications.