Identification and characterization of a calcium dependent bacillopeptidase from Bacillus subtilis CFR5 with novel kunitz trypsin inhibitor degradation activity

Food Res Int. 2018 Jan;103:263-272. doi: 10.1016/j.foodres.2017.10.049. Epub 2017 Oct 31.


The cereals and pulses are considered to be an important component in the food chain due to their proteinaceous nature, but the presence of anti-nutritional factors (KTI) decreases their nutrient absorption rate. Kunitz trypsin inhibitors (KTI) reduce the bioavailability of trypsin and are the primary cause for the existence of various metabolic disorders. To overcome the inhibitory effect of KTI, a KTI degrading protein (BPC) was identified and characterized from Bacillus subtilis CFR5. BPC possesses 60% identity with bacillopeptidase of B. subtilis 168. BPC cleaves at DFVLD and DFFNNY sites of KTI which results in the formation of three inactive KTI fragments. Subsequently, BPC was cloned in pHY300PLK and recombinant protein was used for the biochemical characterization, sequence alignment and mutational studies. The optimal temperature and pH of the BPC was 40°C and 8.0, respectively. BPC is a calcium dependent metalloprotease and its activity was significantly increased by 41.2-fold in the presence of 2.5mM Ca2+. BPC also showed moderate thermostability with the half-life of 4h at 55°C. Site directed mutagenesis studies in recombinant BPC revealed that mutation of Tyr49 with Phe, Tyr64 with Phe, and Pro141 to Arg affects the catalytic activity without affecting the conformation of BPC. Hence, Tyr49, Tyr64 and Pro141 were identified as the unique residues responsible for KTI cleavage. Thus, this study leads to the identification of a novel KTI degrading protease from B. subtilis CFR5 which cleaves and deactivates the kunitz trypsin inhibitor.

Keywords: Bacillopeptidase; Inhibitor degrading enzyme; Kunitz trypsin inhibitor; Protease; Trypsin inhibitor.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacillus subtilis / enzymology*
  • Bacillus subtilis / genetics
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / isolation & purification
  • Bacterial Proteins / metabolism*
  • Calcium / metabolism*
  • Cloning, Molecular
  • Enzyme Stability
  • Food Microbiology / methods*
  • Hydrogen-Ion Concentration
  • Kinetics
  • Mutation
  • Peptide Hydrolases / chemistry
  • Peptide Hydrolases / genetics
  • Peptide Hydrolases / isolation & purification
  • Peptide Hydrolases / metabolism*
  • Protein Conformation
  • Proteolysis
  • Structure-Activity Relationship
  • Substrate Specificity
  • Temperature
  • Trypsin Inhibitor, Kunitz Soybean / metabolism*


  • Bacterial Proteins
  • Trypsin Inhibitor, Kunitz Soybean
  • Peptide Hydrolases
  • Calcium