Characterization of the Altai Maral Chymosin Gene, Production of a Chymosin Recombinant Analog in the Prokaryotic Expression System, and Analysis of Its Several Biochemical Properties

S. V. Belenkaya, A. A. Bondar, T. A. Kurgina, V. V. Elchaninov, A. Yu Bakulina, E. A. Rukhlova, O. I. Lavrik, A. A. Ilyichev, D. N. Shcherbakov

Research output: Contribution to journalArticlepeer-review

Abstract

For the first time, the chymosin gene (CYM) of a maral was characterized. Its exon/intron organization was established using comparative analysis of the nucleotide sequence. The CYM mRNA sequence encoding a maral preprochymosin was reconstructed. Nucleotide sequence of the CYM maral mRNA allowed developing an expression vector to ensure production of a recombinant enzyme. Recombinant maral prochymosin was obtained in the expression system of Escherichiacoli [strain BL21 (DE3)]. Total milk-coagulation activity (MCA) of the recombinant maral chymosin was 2330 AU/ml. The recombinant maral prochymosin relative activity was 52955 AU/mg. The recombinant maral chymosin showed 100-81% MCA in the temperature range 30-50°C, thermal stability (TS) threshold was 50°C, and the enzyme was completely inactivated at 70°C. Preparations of the recombinant chymosin of a single-humped camel and recombinant bovine chymosin were used as reference samples. Michaelis–Menten constant (Km), turnover number (kcat), and catalytic efficiency (kcat/Km) of the recombinant maral chymosin, were 1.18 ± 0.1 µM, 2.68 ± 0.08 s−1 and 2.27± 0.10 µm M−1·s−1, respectively.

Original languageEnglish
Pages (from-to)781-791
Number of pages11
JournalBiochemistry (Moscow)
Volume85
Issue number7
DOIs
Publication statusPublished - 1 Jul 2020

Keywords

  • Michaelis–Menten kinetics parameters
  • milk-clotting activity
  • recombinant chymosin
  • thermal stability
  • CAMEL CHYMOSIN
  • PASTORIS
  • BOVINE CHYMOSIN
  • Michaelis-Menten kinetics parameters
  • ENZYME
  • CLONING
  • PURIFICATION
  • PREPROCHYMOSIN
  • ACTIVE CHYMOSIN

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