Enzovera Recombinant Porcine Trypsin (EV-PRO-001) is an animal-origin-free (AOF) recombinant serine protease supplied as a lyophilized powder with a specific activity of >10,000 U/mg, available in 500 mg and 5 g bottles. Produced by recombinant expression without any animal-derived raw materials in the manufacturing process, EV-PRO-001 delivers the biochemical performance of native porcine trypsin — precise, specific proteolysis at lysine and arginine residues — while fully satisfying animal-origin-free (AOF) and animal-derived component-free (ADCF) requirements for cell culture in pharmaceutical and biopharmaceutical production environments.

Mechanism and substrate specificity. Recombinant Porcine Trypsin (EC 3.4.21.4) is a serine endopeptidase that cleaves peptide bonds specifically at the carboxyl side of lysine and arginine residues, with a preference for Arg > Lys when proline does not occupy the P1′ position immediately C-terminal to the cleavage site. The catalytic mechanism proceeds through the serine protease triad (Asp-102, His-57, Ser-195) via an acyl-enzyme tetrahedral intermediate. Activity is maximal at pH 7.5–8.5 and 37°C, is Ca²⁺-stimulated (but not Ca²⁺-dependent), and is rapidly inhibited by serine protease inhibitors including PMSF, AEBSF, and soybean trypsin inhibitor (SBTI) — commonly used to terminate cell dissociation reactions and prevent over-digestion of cell-surface proteins.

Animal-origin-free cell culture and proteomics. The primary use of trypsin in biopharmaceutical cell culture is enzymatic dissociation of adherent mammalian cell monolayers for passaging. Trypsin cleaves cell-surface adhesion proteins — including fibronectin, vitronectin, and E-cadherin — at exposed Lys/Arg residues, releasing cells from the matrix. The AOF designation of EV-PRO-001 is critical for GMP biomanufacturing, where regulatory guidelines for biologic drugs, vaccines, gene therapy vectors, and cell therapies require minimization of adventitious agent risk from conventional animal-pancreatic trypsin. In proteomics, EV-PRO-001 provides consistent batch-to-batch digestion performance for LC-MS/MS peptide mapping and mass fingerprinting.

Recommended applications:

• Adherent mammalian cell dissociation for GMP-compliant passaging in biopharmaceutical cell culture
• Upstream bioprocessing for biologics, cell therapies, and gene therapy vector production
• Proteomics: in-solution and in-gel trypsin digestion for LC-MS/MS peptide mass fingerprinting
• Recombinant protein propeptide removal and activation cleavage
• Vaccine manufacturing: cell substrate preparation and cell banking operations
• Protein sequence analysis and comprehensive peptide mapping
• Replacement for conventional porcine pancreatic trypsin in all AOF-required workflows