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Denaturant Induced Equilibrium Unfolding and Conformational Transitional Studies of Germinated Fenugreek β-Amylase Revealed Molten Globule like State at Low pH

Author(s):

Dinesh Chand Agrawal, Anjali Yadav, Mohd. Asim Khan, Suman Kundu and Arvind M. Kayastha*   Pages 1 - 12 ( 12 )

Abstract:


Background: β-Amylase (EC 3.2.1.2) is a maltogenic enzyme, which releases β-maltose from the non-reducing end of the substrates. The enzyme plays important roles for the production of vaccine, maltiol and maltose rich syrups. Apart from these applications the enzyme protects cells from abiotic as well as oxidative damage. The enzyme is well characterized in plants and animals and crystal structures of β-amylases have been obtained from sweet potato, soybean and Bacillus cereus.

Objective: Find out correlation between structural and functional stability induced by changing in pH, temperature and chaotropes.

Methods: Activity, intrinsic fluorescence, extrinsic fluorescence, near- and far- ultraviolet circular dichroism spectroscopic measurements were performed.

Results: Peaks about 208 nm and 222 nm obtained by near- ultraviolet circular dichroism corresponds to α-helix whereas peak at 215 nm shows presence of β-sheet. At pH 2.0, absence of tertiary structures, exposed of hydrophobic regions and presence of substantial secondary structures, revealed the existence of molten globule like state. Temperature induced denaturation studies showed that the enzyme was stable up to 75 ºC and the process was found to be irreversible in nature. Chaotropes dependent equilibrium unfolding studies revealed that at low concentration of chaotropes, ellipticity and intrinsic fluorescence intensity were decreased whereas enzymatic activity remained unchanged, which revealed fenugreek β-amylase is multidomains enzyme and catalytic domain is more stable compare to non-catalytic domain. Moreover, the transition was sigmoidal and non-coincidental.

Conclusion: Results indicate the probable existence of intermediate states that might perform significant role in physiological process and biotechnological applications.

Keywords:

β-amylase, cooperative, intermediate, molten globule, fenugreek, circular dichroism.

Affiliation:

School of Biotechnology, Institute of Science, Banaras Hindu University, Varanasi 221005, School of Biotechnology, Institute of Science, Banaras Hindu University, Varanasi 221005, Department of Biochemistry, University of Delhi South Campus, New Delhi 110021, Department of Biochemistry, University of Delhi South Campus, New Delhi 110021, School of Biotechnology, Institute of Science, Banaras Hindu University, Varanasi 221005



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