High-level fed-batch production of recombinant type II cholesterol oxidase from Brevibacterium sterolicum and its validation for clinical diagnostic applications.

Cholesterol oxidase (CO, EC 1.1.3.6) is widely used in clinical diagnostics for the enzymatic determination of cholesterol in human serum and plasma. Among bacterial COs, the type II CO from Brevibacterium sterolicum (BCO), characterized by a covalently bound FAD cofactor, is particularly attractive due to its high stability and favorable kinetic properties. In this study, we developed an integrated bioprocess for recombinant BCO production in Escherichia coli. Two His-tagged variants were constructed and compared: a C-terminally tagged form (BCO-H) and an N-terminally tagged form (H-BCO). Expression was optimized in a chemically defined modified M9 medium and scaled up using a fed-batch strategy. Both variants reached high cell densities (OD₆₀₀ ≈ 50) and showed a > 20-fold increase in volumetric productivity compared to shake-flask cultures. BCO-H outperformed H-BCO, reaching a final productivity of 102 ± 1.5 KU/L. Single-step Ni-affinity purification yielded highly pure enzymes with specific activities comparable to commercial CO. Both enzymes were successfully incorporated into a commercial cholesterol diagnostic kit formulation and evaluated according to European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) quality specifications. The in-house formulations showed excellent linearity, high precision, low bias, and total error within desirable limits, with comparable or faster kinetics than the reference formulation. Importantly, analytical performance was fully preserved after one year of storage at 4 °C. Overall, this work establishes a scalable fed-batch production process for recombinant type II CO suitable for use in real clinical diagnostic settings, achieving the highest reported volumetric productivity to date.