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 Schematic illustration of the strategy for OTA detection.

Fig. 2 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. 3 Results of pET-29a(+)-SUMO-MCPA. a. The plasmid map of pET-29a(+)-SUMO-MCPA. b. 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. c. SDS-PAGE analysis of the purified protein SUMO-M-CPA (48.5KDa) in SHuffle T7 E. coli cultured in 2xYT medium express protein for 12 hours at 20°C. Lane M: protein marker. Lanes 1-9: flow through and elution containing 10, 10, 20, 20, 50, 50, 100, 100, 250 mM imidazole, respectively.

Fig. 4 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.

Fig. 5 High performance liquid chromatography (HPLC) chromatogram retention time of OTA and OTα. (a) 10 μg/mL OTA after incubation with methanol solution(control). (b) HPLC chromatogram of degradation products of OTA after incubation with 5 U/mL M-CPA for 24 h. (c) 50 μg/mL OTA after incubation with methanol solution(control). (d) HPLC chromatogram of degradation products of OTA after incubation with 5 U/mL ADH3 for 30 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]