Abstract
Serine proteases are thought to play a key role in the muscle softening of gazami crab (Portunus trituberculatus) during storage. A serine protease, Pt-sp2, was purified from the hepatopancreas of gazami crab using ammonium sulfate precipitation, anion-exchange and gel filtration chromatography, and was analyzed by mass spectrometry, transcriptome and bioinformatics. It revealed that Pt-sp2 was trypsin-like, with no 100% identical proteins in the NCBI database. The molecular weight of Pt-sp2 was approximately 37.2 kDa. Its optimum pH and temperature were 9.0 and 50 °C, respectively, using t-Butyloxy‑carbonyl-Phe-Ser-Arg-4-methyl-coumaryl-7-amide as a substrate. Pt-sp2 was activated in the presence of Ca2+. Both soybean trypsin inhibitor and Nα-Tosyl-l-lysine chloromethyl ketone hydrochloride completely suppressed Pt-sp2 activity, while it was only partially inhibited by phenylmethylsulfonyl fluoride and EDTA. However, PMSF, Pepstatin A and cystatin inhibitor E-64 showed no inhibition on Pt-sp2 protease activity. The Km value of Pt-sp2 was 0.82 μM, and Pt-sp2 effectively hydrolyzed myofibrillar protein at 37 °C.
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