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pH-Induced Molten Globule State of Rhizopus niveus Lipase is More Resistant Against Thermal and Chemical Denaturation Than Its Native State

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Abstract

Here, we have characterized four pH-dependent states: alkaline state, “B” (pH 9.0), native state, “N” (pH 7.4), acid-induced state, “A” (pH 2.2) and molten globule state, “MG” (pH 1.8) of Rhizopus niveus lipase (RNL) by CD, tryptophanyl fluorescence, ANS binding, DLS, and enzyme activity assay. This “MG” state lacks catalytic activity and tertiary structure but it has native-like significant secondary structure. The “R h” of all the four states of RNL obtained from DLS study suggests that the molecular compactness of the protein increases as the pH of solution decreases. Kinetic analysis of RNL shows that it has maximum catalytic efficiency at state “B” which is 15-fold higher than state “N.” The CD and tryptophanyl fluorescence studies of RNL on GuHCl and temperature-induced unfolding reveal that the “MG” state is more stable than the other states. The DSC endotherms of RNL obtained at pH 9.0, 7.4, and 2.2 were with two transitions, while at pH 1.8 it showed only a single transition.

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Abbreviations

ANS:

1-Anilino-8-napthalene sulfonate

C m :

Midpoint concentration

DSC:

Differential scanning calorimetry

GuHCl:

Guanidine hydrochloride

∆H :

Enthalpy

MG:

Molten globule

T m :

Midpoint temperature

RNL:

Rhizopus niveus lipase

DLS:

Dynamic light scattering

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Acknowledgments

This study was supported by the University Grants Commission (UGC) New Delhi, funded project grant No. 32-543/2006 (SR). G. Rabbani acknowledged to Council of Scientific and Industrial Research (CSIR), New Delhi, India for financial assistance in the form of Senior Research Fellow (SRF).

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Authors and Affiliations

  1. Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, 202 002, India

    Gulam Rabbani, Ejaz Ahmad, Nida Zaidi, Sadaf Fatima & Rizwan Hasan Khan

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  1. Gulam Rabbani
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  2. Ejaz Ahmad
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Correspondence to Rizwan Hasan Khan.

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Rabbani, G., Ahmad, E., Zaidi, N. et al. pH-Induced Molten Globule State of Rhizopus niveus Lipase is More Resistant Against Thermal and Chemical Denaturation Than Its Native State. Cell Biochem Biophys 62, 487–499 (2012). https://doi.org/10.1007/s12013-011-9335-9

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