Stability and Activity of Porcine Lipase Against Temperature and Chemical Denaturants

Chaitanya, P. Krishna; Prabhu, N. Prakash

doi:10.1007/s12010-014-1220-8

Cited by 14 publications

(4 citation statements)

References 37 publications

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“…Although our knowledge on the structure and cellular function of PTPase is still increasing, little is known how the activity of PTPase from thermus thermophiles HB27 is determined by its conformation. Denaturant is usually used to characterize the activity and conformation of an enzyme [13][14][15][16][17][18] . Urea is known to break non-covalent interactions.…”

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confidence: 99%

Effects of SDS on the activity and conformation of protein tyrosine phosphatase from thermus thermophilus HB27

Hou

Wang

2020

Sci Rep

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Deciphering the activity-conformation relationship of PTPase is of great interest to understand howPTPase activity is determined by its conformation. Here we studied the activity and conformational transitions of PTPase from thermus thermophilus HB27 in the presence of sodium dodecyl sulfate (SDS). Activity assays showed the inactivation of PTPase induced by SDS was in a concentration-dependent manner. Fluorescence and circular dichroism spectra suggested SDS induced significant conformational transitions of PTPase, which resulted in the inactivation of PTPase, and the changes of α-helical structure and tertiary structure of PTPase. Structural analysis revealed a number of hydrophobic and charged residues around the active sites of PTPase may be involved in the hydrophobic and ionic bonds interactions of PTPase and SDS, which are suggested to be the major driving force to result in PTPase inactivation and conformational transitions induced by SDS. Our results suggested the hydrophobic and charged residues around the active sites were essential for the activity and conformation of PTPase. Our study promotes a better understanding of the activity and conformation of PTPase.Although we have discussed the conformational transitions and the inactivation of PTPase induced by SDS in detail, more experimental evidences such as the complex structure of PTPase-SDS and the dynamics of PTPase-SDS interactions are still required to clarify the details of molecular interaction. Our study is valuable toward the long-term goal to better understand the activity and conformation of PTPase. Materials and MethodsReagents and materials. All chemicals used in this research such as para-nitrophenyl phosphate (pNPP), Isopropyl-β-D-1-thiogalactopyranoside (IPTG), Dithiothreitol (DTT), SDS and 1-anilinonaphtalene-8-sulfonate (ANS) were of the highest purity commercially available. PTPase from thermus thermophilus HB27 was cloned into pET-28a (+) vector (Novagen, Germany) and overexpressed in E. coli BL21 (DE3). PTPase was purified as described 12 . The concentration of recombinant PTPase was determined by BCA protein assay kit (Pierce, USA).PTPase activity assay. PTPase activity was assayed as described previously 25,44 . Briefly, pNPP (10 mM) and PTPase (2.4 μM) were added into 200 µL, 50 mM acetic acid-sodium acetate buffer (pH 3.8) plus 5 mM DTT. After incubation at 30 °C for 10 min, NaOH (1 M, 1 mL) was added into the mixture to terminate the reaction. The absorption change at 405 nm was recorded on a Helios-γ UV-VIS spectrophotometer (Thermo Scientific, USA).

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confidence: 99%

Effects of SDS on the activity and conformation of protein tyrosine phosphatase from thermus thermophilus HB27

Hou

Wang

2020

Sci Rep

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“…Similar results have been reported by other researchers for porcine and rice brown lipase at similar concentration [ 38 ]. Thus, quenching binding constants of over 10 5 M -1 are taken to be significantly strong [ 39 , 40 ]. Prominent red shift in on reset of lipase indicates major changes in tertiary structure and disruption of native structure.…”

Section: Discussionmentioning

confidence: 99%

Interaction of fungal lipase with potential phytotherapeutics

et al. 2022

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Interaction of thymol, carvacrol and linalool with fungal lipase and Human Serum Albumin (HSA) have been investigated employing UV-Vis spectroscopy Fluorescence and Circular dichroism spectroscopy (CD) along with docking studies. Thymol, carvacrol and linalool displayed approximately 50% inhibition at 1.5 mmol/litre concentrations using para-nitrophenyl palmitate (pNPP). UV-Vis spectroscopy give evidence of the formation of lipase-linalool, lipase-carvacrol and lipase—thymol complex at the ground state. Three molecules also showed complex formation with HSA at the ground state. Fluorescence spectroscopy shows strong binding of lipase to thymol (Ka of 2.6 x 109 M-1) as compared to carvacrol (4.66 x 107 M-1) and linalool (5.3 x 103 M-1). Number of binding sites showing stoichiometry of association process on lipase is found to be 2.52 (thymol) compared to 2.04 (carvacrol) and 1.12 (linalool). Secondary structure analysis by CD spectroscopy results, following 24 hours incubation at 25°C, with thymol, carvacrol and linalool revealed decrease in negative ellipticity for lipase indicating loss in helical structure as compared with the native protein. The lowering in negative ellipticity was in the order of thymol > carvacrol > linalool. Fluorescence spectra following binding of all three molecules with HSA caused blue shift which suggests the compaction of the HSA structure. Association constant of thymol and HSA is 9.6 x 108 M-1 which along with ‘n’ value of 2.41 suggests strong association and stable complex formation, association constant for carvacrol and linalool was in range of 107 and 103 respectively. Docking results give further insight into strong binding of thymol, carvacrol and linalool with lipase having free energy of binding as -7.1 kcal/mol, -5.0 kcal/mol and -5.2 kcal/mol respectively. To conclude, fungal lipases can be attractive target for controlling their growth and pathogenicity. Employing UV-Vis, Fluorescence and Circular dichroism spectroscopy we have shown that thymol, carvacrol and linalool strongly bind and disrupt structure of fungal lipase, these three phytochemicals also bind well with HSA. Based on disruption of lipase structure and its binding nature with HSA, we concluded thymol as a best anti-lipase molecule among three molecules tested. Results of Fluorescence and CD spectroscopy taken together suggests that thymol and carvacrol are profound disrupter of lipase structure.

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“…Spectroscopic analysis has shown lipase structural unfolding in the presence of urea [13]. Urea-induced unfolding in the lipase native structure can result in amyloid fibril formation, in which proteins aggregate in a continuous β-sheet structure [14,15].…”

Section: Introductionmentioning

confidence: 99%

Unfolded Lipase at Interfaces Studied via Interfacial Dilational Rheology: The Impact of Urea

Dowlati

Javadi

Miller

et al. 2022

Colloids and Interfaces

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Unfolding can interrupt the activity of enzymes. Lipase, the enzyme responsible for triglyceride catalysis, can be deactivated by unfolding, which can significantly affect the yield of enzymatic processes in biochemical engineering. Different agents can induce lipase unfolding, among which we study the impact of urea as a strong denaturant. Unfolding weakens the rigidity and stability of globular proteins, thereby changing the viscoelastic properties of the protein adsorbed layers. These changes can be detected and quantified using interfacial dilational rheology. The urea-induced unfolding of lipase destructs its globular structure, making it more flexible. The interfacial tension and viscoelastic moduli of lipase adsorbed layers reduce upon the addition of urea in the range of studied concentrations. The results agree with the theory that, upon unfolding, a distal region of the loop and tail domain forms adjacent to the proximal region of the interface. The exchange of matter between these regions reduces the viscoelasticity of the unfolded lipase adsorbed layers. Additionally, unfolding reduces the rigidity and brittleness of the lipase adsorbed layers: the aged adsorbed layer of native lipase can break upon high-amplitude perturbations of the interfacial area, unlike the case for urea-induced unfolded lipase.

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Stability and Activity of Porcine Lipase Against Temperature and Chemical Denaturants

Cited by 14 publications

References 37 publications

Effects of SDS on the activity and conformation of protein tyrosine phosphatase from thermus thermophilus HB27

Effects of SDS on the activity and conformation of protein tyrosine phosphatase from thermus thermophilus HB27

Interaction of fungal lipase with potential phytotherapeutics

Unfolded Lipase at Interfaces Studied via Interfacial Dilational Rheology: The Impact of Urea

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