Integrating Metabolomics and Statistical Analysis for Enhanced Serine Alkaline Protease Production in Bacillus subtilis

Sedighikamal, Hossein; Mashayekhan, Shohreh; Roostaazad, Reza

doi:10.24200/sci.2025.66954.10346

Integrating Metabolomics and Statistical Analysis for Enhanced Serine Alkaline Protease Production in Bacillus subtilis

Articles in Press

Document Type : Research Article

Authors

Hossein Sedighikamal ¹

Shohreh Mashayekhan ²

Reza Roostaazad ³

¹ Sharif University of Technology

² Chemical &Petroleum Engineering Department, Sharif University of Technology, Cell & Tissue Engineering Lab

³ Chemical & Petroleum Engineering Department, Sharif University of Technology,

10.24200/sci.2025.66954.10346

Abstract

Using quantitative analysis of intracellular metabolites is a valuable method for understanding biochemical processes in cellular systems. This study aimed to objectively evaluate various quenching–extraction techniques for intracellular metabolite profiling of Bacillus subtilis expressing serine alkaline protease (SAP) and to link metabolomics data with fermentation optimization. Among the tested methods (cold methanol, cold ethanol, and a mixture of acetonitrile and chloroform combined with fast filtration), cold methanol yielded the most efficient and reproducible extraction. Metabolomic analysis identified glucose 6-phosphate (G6P), fructose 6-phosphate (F6P), serine, and glycine as the most influential intracellular metabolites. Using the Box–Behnken design, different concentrations of the identified metabolites were systematically varied, and the model predicted optimal levels (G6P 0.52 mM, F6P 0.054 mM, serine 1.72 mM, glycine 0.07 mM) that enhanced SAP activity by nearly 300%, reaching 1530.33 U/mL compared to the control. These results establish a validated workflow that couples metabolomics with statistical methods to achieve enhanced protease production, offering both methodological and industrial relevance.

Keywords

Serine alkaline protease

Bacillus subtilis

Quenching

Extraction

Intracellular metabolomics

Subjects