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

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.

Calorimetric and spectroscopic binding studies of amoxicillin with human serum albumin

“…The value of k q obtained from Eq. (2) is of the order of 10 12 , which is 100 times higher than the maximum scatter collision quenching constant of various quenchers with biopolymers (2 9 10 M -1 s -1 ) [29]. This shows that quenching is not initiated by dynamic diffusion but occurs by formation of a strong complex between HSA and AX.…”

“…There are only three aromatic fluorophores amino acids, tryptophan (Trp), tyrosine (Try) and phenylalanine (Phe) in HSA which are used for studying conformational changes in HSA on drug binding. However, among them contribution of tryptophan is maximum [28,29]. The intrinsic fluorescence of HSA excited at 295 nm is mainly contributed by the Trp residue alone, because the Phe residue has a very low quantum yield and the fluorescence of Tyr is almost totally quenched when it is ionized or nearby to an amino group, a carboxyl group or a Trp [30].…”

Facile preparation of magnetically functionalized graphite nanosheets for porcine pancreatic lipase immobilization

“…In addition, the IPPL kept the residual activity at about 50 % when the concentration of urea was about 6 mol L -1 . One of the possible reasons for the effect of denaturant on the hydrolysis activity of the IPPL is the lipase which was covalently attached with the amino groups, and it is possibly conducted a higher activation energy for the molecules to reorganize and prevented the ternary structure of lipase from denaturation (Rabbani et al 2012). …”

A differential behavior of α-amylase, in terms of catalytic activity and thermal stability, in response to higher concentration CaCl2

“…The structural stability of ␣-amylases depends on various intrinsic (e.g., amino acid sequence) and extrinsic (e.g., solution conditions, such as pH, presence of cofactors, metal ions, etc.) factors [10][11][12][13][14][15][16]. Calcium is an essential metal required for catalytic activity and structural stability of most of the ␣-amylases [14].…”