pH-Induced molten globule state of Rhizopus niveus lipase is more resistant against thermal and chemical denaturation than its native state.
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.
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202 002, India.
Gulam
G
Rabbani
Ejaz
E
Ahmad
Nida
N
Zaidi
Sadaf
S
Fatima
Rizwan Hasan
RH
Khan
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D019791:Guanidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012233:Rhizopus / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D000817:Anilino Naphthalenesulfonates; C027132:1-anilino-8-naphthalenesulfonate; D008049:Lipase; D019791:Guanidine