Development of soluble human catechol-O-methyltransferase using P. pastoris as a host and stabilizing it by immobilization in agar-agar matrix

Human soluble catechol-O-methyltransferase (hSCOMT) plays a crucial role in the O-methylation of catecholamines and active catechols, influencing neurotransmitter metabolism and the bioavailability of drugs. This research focused on the development of an improved biocatalyst through immobilization techniques. hSCOMT was firstly produced in Pichia pastoris X-33 using the pPICZαA vector, regulated by the methanol-inducible AOX1 promoter, which facilitated efficient secretion into the culture medium and simplified purification through ultrafiltration, yielding a protein recovery of 2.8 mg/mL from the crude extract, concentrated to 0.6 mg/mL. The free hSCOMT exhibited a specific activity of 168.63 ± 2.66 nmol/h/mg and was highly sensitive to entacapone inhibition (IC50 of 3.716 nM), confirming its authenticity. Then to enhance the catalytic performance beyond that of free enzyme systems, we proceeded with an immobilization method using agar-agar as a natural, biocompatible support matrix. Entrapment under gentle conditions maintained the enzyme’s structure and function, achieving 89% immobilization efficiency with minimal protein leakage. The immobilized hSCOMT demonstrated significantly enhanced specific activity at 231.73 ± 1.98 nmol/h/mg in an optimized colorimetric assay. The agar-agar matrix enhanced thermal and pH stability, maintaining strong activity in a pH range of 8–9 and temperatures up to 50 °C, while demonstrating excellent reusability over multiple cycles. These findings establish agar-agar immobilized hSCOMT as an advanced biocatalyst platform for COMT inhibitor screening, drug metabolism research, biosensor applications or continuous-flow processes.