Horsegram Bowman-Birk Inhibitor: Role of Intradomain Disulphide Bond on the Thermal Stability and Activity.

Abitha, P. (2013) Horsegram Bowman-Birk Inhibitor: Role of Intradomain Disulphide Bond on the Thermal Stability and Activity. [Student Project Report] (Submitted)

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Abstract

Bowman-Birk inhibitors (BBI) are a class of plant proteinase inhibitors that occur widely in legumes. They play a dominant role in natural plant defense mechanism. Their molecular masses are in the range of 6000-8000Da. BBIs have two sub domains with evolutionarily conserved seven disulphide bonds. This inhibitor interacts simultaneously and independently with trypsin and chymotrypsin. Along with protease inhibitor activity, BBI also have anticarcinogenic, radio protective, chemo protective and immune stimulating properties. Horsegram (Dolichos biflorus) is a pulse crop native to South East Asia and Tropical Africa. The major BBI of horse gram (HGI-III) is a single polypeptide with a molecular mass of 8268 Da. HGI-III occurs as dimer in solution. The role of active site residue Lys24 and C-terminal end in the dimeric state of the major inhibitor (HGI-III) was previously established by in vitro and homology modeling. The BBIs are extremely stable to extremities of pH and Heat. This has been attributed to the covalent lock formed by the evolutionarily conserved seven disulphide bridges. The role of C17-C32 disulphide bond in the trypsin domain of HGI was evaluated. Site directed mutagenesis of the two cysteine residues to serine was performed by using mutagenesis quick change method. The mutations were verified by dideoxy sequencing. The disulphide deleted HGI was expressed in E. coli B ER 2566 cells. The expression of the protein was evaluated in crude cell lysate by SDS-PAGE. The trypsin and chymotrypsin inhibitory activity were evaluated.

Item Type:	Student Project Report
Uncontrolled Keywords:	Bowman-Birk inhibitors, plant proteinase inhibitors, legumes
Subjects:	500 Natural Sciences and Mathematics > 04 Chemistry and Allied Sciences > 29 Protein Chemistry 600 Technology > 08 Food technology > 22 Legumes-Pulses
Divisions:	Protein Chemistry and Technology
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	09 May 2014 10:08
Last Modified:	09 May 2014 10:08
URI:	http://ir.cftri.res.in/id/eprint/11555

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