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

Sharmila, G. R. and Prakash, M. Halami and Venkateswaran, G. (2018) Identification and characterization of a calcium dependent bacillopeptidase from Bacillus subtilis CFR5 with novel kunitz trypsin inhibitor degradation activity. Food Research International, 103. pp. 263-272. ISSN 0963-9969

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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 2.5 mM Ca2+. BPC also showed moderate thermostability with the half-life of 4 h 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.

Item Type:	Article
Uncontrolled Keywords:	Trypsin inhibitor Protease Bacillopeptidase Kunitz trypsin inhibitor Inhibitor degrading enzyme
Subjects:	500 Natural Sciences and Mathematics > 07 Life Sciences > 04 Microbiology > 02 Bacteriology 600 Technology > 08 Food technology > 16 Nutritive value > 05 Enzymes
Divisions:	Food Microbiology
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	14 Feb 2018 05:25
Last Modified:	14 Feb 2018 05:25
URI:	http://ir.cftri.res.in/id/eprint/13408

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