Part:BBa_K4613001

Mature Carboxypeptidase A（M-CPA）

Carboxypeptidase A (CPA), derived from bovine pancreas, is a 47 kDa zinc-dependent metal carboxypeptidase. Mature Carboxypeptidase A (M-CPA) removed the signal peptide composed of 16 amino acid residues and the leading peptide composed of 94 amino acid residues and contains a mature peptide of composed of 307 amino acid residues (35 kDa). Mature Carboxypeptidase A has an efficient capacity to hydrolyze ochratoxin α (OTA) into the non-toxic product ochratoxin α and L-α-phenylalanine (Phe). The degradation rate was up to 93.36%. Compared with commercial CPA (S-CPA), M-CPA obtained better thermal tolerance and stability at a wider range of pH.

Fig.1 Enzymatic properties of Mature Carboxypeptidase A(M-CPA) and commercial Carboxypeptidase A (S-CPA) (a) The optimum temperature of M-CPA and S-CPA; (b) The thermal tolerance of M-CPA and S-CPA; (c) The optimum pH of M-CPA and S-CPA; (d) enzymatic dynamic response curve of M-CPA and S-CPA.

Fig.2 SDS-PAGE analysis of protein expression trials in SHuffle T7 E. coli cultured in 2xYT medium for 12 hours using pET29a(+)-SUMO-MCPA. The temperature was 20 °C. Lane M：protein marker. Lane 1: induced total protein. Lane 2: precipitate. Lane 3: supernatant.

Fig.3 Assay of ADH3 activity. A reaction mixture containing 290 μl of 25 mM Tris buffer, 500 mM NaCl (pH 7.5), 3.26 mg/mL Hippuryl-L-phenylalanine (HLP), and 10 μl of ADH3 dissolved in 20 mM Tris-HCl (pH 8.0) in eppendorf tube was incubated at 25℃ for 5 min.

Due to its high efficiency and stability, M-CPA can be an ideal enzyme for our project.

Reference

1. Xiong L, Peng M, Zhao M, et al. Truncated expression of a carboxypeptidase a from bovine improves its enzymatic properties and detoxification efficiency of ochratoxin A[J]. Toxins, 2020, 12(11): 680.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 88
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 198
  
• 1000
  COMPATIBLE WITH RFC[1000]