Recombinant Expression And Enzymatic Properties Of Crucian Carp (Carassius Auratus)Myofibril-bound Serine Proteinase

The fish myofibril-bound serine proteinase (MBSP) is similar with trypsin in substratespecificity, and it is more outstanding in thermostability than mammalian trypsins. Thus, it canbe used as a trypsin substitute. On the other hand, MBSP can degrade myofibrillar proteinsefficiently which was proposed to be responsible for the modori phenomenon during surimiproduction. Therefore, it is necessary to express a recombinant MBSP (rMBSP) and toinvestigate its characterization, which could lay the foundation for the development of a noveltool enzyme for mass spectrometric identification of proteins and the research of specific MBSPinhibitors.In this study, the freshwater crucian carp (Carassius auratus) was used as the researchobject. The crucian carp MBSP gene (MBSP) was cloned by RT-PCR with the total RNA fromskeletal muscle of crucian carp as the template. The result of sequencing showed that the cruciancarp MBSP had an open reading frame of729bp encoding a polypeptide of242amino acidresidues with a signal peptide containing20amino acid residues at its N-terminal. Thus, themature crucian carp MBSP gene (MBSP2) sequence was669bp in length encoding apolypeptide of222amino acid residues. A recombinant expression strain (GS115/pPIC9K-MBSP2) was constructed by integrating the MBSP2into the chromosome of Pichiapastoris (P. pastoris) strain GS115. The recombinant P. pastoris strain was cultured in shakeflasks with1.0%methanol used as inductor, and an approximately36kDa of recombinantprotein was obtained in the fermentation supernatant. Western blotting showed that therecombinant protein was the recombinant crucian carp MBSP (rMBSP). Periodic acid Schiff(PAS) reaction attested the rMBSP was a glycoprotein.The high-density fermentation of the recombinant expression strain (GS115/pPIC9K-MBSP2) was carried out in a7.0L fermentation vessel at the suitable fermentationconditions about temperature range of28.5-29.5°C, pH range of5.2-5.8and the amount ofdissolved oxygen maintaining above30%. The methanol used as an inductor was addedintermittently for32h to express rMBSP. At the end of the fermentation run, thefresh-cell-weight and OD600of the expression strains were123.9g/L and56.2respectively. Withthe reasonable combination of different separation and purification techniques, an optimizationalseparation and purification process for rMBSP was confirmed. After fermented in a7.0L fermentation vessel, centrifuged for supernatant, ammonium sulfate precipitation at30-60%,dialysis, Q-Sepharose anion-exchange chromatography, ultrafiltration and lyophilization orderly,the purified solid rMBSP was obtained which could be used for investigation of itscharacterization and application.Characterization of the rMBSP was investigated. Substrate specificity analysis showed thatthe rMBSP specifically cleaved substrates at the carboxyl side of lysine (Lys) residue butrevealed low activity against the substrates containing arginine (Arg) residue, which was similarto endoproteinase Lys-C that was frequently used as a tool enzyme to digest proteins inidentification of protein mass spectrometry. The rMBSP had an optimum temperature and pH of55°C and7.5with high thermostability and pH stability. Serine proteinase inhibitors and highconcentration of metal ions could significantly inhibit the activity of rMBSP. Furthermore, therMBSP could rapidly degrade myofibrillar proteins, which was similar to native crucian carpMBSP.After compared with endoproteinase Lys-C, the rMBSP had some superiority inthermostability, pH stability, substrate affinity and reaction speed. In addition, the rMBSP alsocould rapidly degrade bovine serum albumin (BSA) and arginine kinase, which was expected tobe developed as a novel tool proteinase for protein mass spectrometric identification.