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Identification and characterization of a calcium dependent bacillopeptidase from Bacillus subtilis CFR5 with novel kunitz trypsin inhibitor degradation activity. [Mass Spectrometry Facility - Proteomics]

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TitleIdentification and characterization of a calcium dependent bacillopeptidase from Bacillus subtilis CFR5 with novel kunitz trypsin inhibitor degradation activity. [Mass Spectrometry Facility - Proteomics]
Publication TypeJournal Article
Year of Publication2018
AuthorsSharmila GRamachandr, Halami PMotiram, Venkateswaran G
JournalFood Res Int
Volume103
Pagination263-272
Date Published2018 Jan
ISSN1873-7145
Abstract

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 Ca. 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 Tyr with Phe, Tyr with Phe, and Pro to Arg affects the catalytic activity without affecting the conformation of BPC. Hence, Tyr, Tyr and Pro 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.

DOI10.1016/j.foodres.2017.10.049
Alternate JournalFood Res. Int.
PubMed ID29389614