Characterization of Trypsin-like Serine Proteases from Tachypleus tridentatus Amebocyte Lysate and its Potential for Endotoxin Detection

Background: Serine Proteases in the Tachypleus Amoebocyte Lysate (TAL) from horseshoe crab, Tachpleus tridentatus have many applications in biomedical industries. The sensitivity of these enzymes is a crucial factor for such usages. Until today, the catalytic behavioural and biochemical stability of these enzymes has not been properly addressed. This report aimed to characterize the physicochemical properties of serine proteases to determine their kinetic reactions and to determine the potential utilization of the enzymes for endotoxin detection. Methods: The purification process of protease from crude TAL was performed using a two-phase filtration unit by affinity and gel columns and the molecular weights of the enzyme fraction from crude, affinity and gel filtration columns were determined. The Azocasein, Aldolase and transaldolase assays were used for the determination of the enzyme Catalysis. The chromogenic assay was carried out to determine the bacterial endotoxin effects using a standard endotoxin from strain Escherichia coli 0113: H10 as a positive control. The absorbance was measured at 405 nm. The enzyme activity was determined using the constructed p-nitroanilide standard curve.
Result: A 72 kDa trypsin-like enzyme was purified at fraction-14 (F14) gel filtration and identified as transaldolase. The purified-F14 hydrolysed trypsin substrate, Ná-Benzoyl-L-Arginine-p-nitroanilide at higher activity than chymotrypsin. In conclusion, the purified enzyme showed a great potential for enhancing the TAL sensitivity.
