Differential Scanning Calorimetry of the Effects of Ca<sup>2+</sup>on the Thermal Unfolding of<i>Pseudomonas cepacia</i>Lipase

Tanaka, Akiyoshi; Sugimoto, Hayuki; Muta, Yuko; Mizuno, Takafumi; Senoo, Keishi; Obata, Hitoshi; Inouye, Kuniyo

doi:10.1271/bbb.67.207

Cited by 13 publications

(4 citation statements)

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“…In such cases, addition of a cofactor molecule shifts the equilibrium to favor the native‐state side and leads to apparent stabilization. The unfolding temperature of these proteins, as observed by DSC, continued to increase even in the presence of the cofactor molecule concentrations at which the proteins are fully saturated with cofactor molecules . However, no dependency of the unfolding peak temperature on the protein concentration and the added Cu 2+ concentration (Figure ) was observed for holoCBP21.…”

Section: Resultsmentioning

confidence: 89%

“…Cofactor binding is one factor that affects protein stability. In many cases, protein‐cofactor complexes undergo unfolding with simultaneous dissociation of the cofactor molecule, as N∙L ⇌ U + L, where L is the cofactor . In such cases, addition of a cofactor molecule shifts the equilibrium to favor the native‐state side and leads to apparent stabilization.…”

Section: Resultsmentioning

confidence: 99%

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Unfolding of CBP21, a lytic polysaccharide monooxygenase, without dissociation of its copper ion cofactor

et al. 2019

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Chitin-binding protein 21 (CBP21) from Serratia marcescens is a lytic polysaccharide monooxygenase that contains a copper ion as a cofactor. We aimed to elucidate the unfolding mechanism of CBP21 and the effects of Cu 2+ on its structural stability at pH 5.0. Thermal unfolding of both apo-and holoCBP21 was reversible. ApoCBP21 unfolded in a simple two-state transition manner. The peak temperature of the DSC curve, t p , for holoCBP21 (74.4 C) was about nine degrees higher than that for apoCBP21 (65.6 C). The value of t p in the presence of excess Cu 2+ was around 75 C, indicating that Cu 2+ does not dissociate from the protein molecule during unfolding.The unfolding mechanism of holoCBP21 was considered to be as follows: NÁCu 2+ Ð UÁCu 2+ , where N and U represent the native and unfolded states, respectively. Ureainduced equilibrium unfolding analysis showed that holoCBP21 was stabilized by 35 kJ mol −1 in terms of the Gibbs energy change for unfolding (pH 5.0, 25 C), compared with apoCBP21. The increased stability of holoCBP21 was considered to result from the structural stabilization of the protein-Cu 2+ complex itself.

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Section: Resultsmentioning

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Unfolding of CBP21, a lytic polysaccharide monooxygenase, without dissociation of its copper ion cofactor

et al. 2019

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“…The lid consists of a helix-loop-helix motif with helix α4 (residues 118–127) and helix α5 (residues 134–150) which are linked to helix α6 (residues 160–166) [ 14 ]. BCL contains an essential Ca 2+ -ion site [ 15 ] which stabilizes a β-hairpin (residues 214–228) [ 16 ]. While no experimental structure of BCL in the closed conformation is available, a homologous lipase from Burkholderia glumae (BGL) [PDB: 1QGE ] [ 17 ] with a sequence identity of 84% has been crystallized in the closed conformation.…”

Section: Introductionmentioning

confidence: 99%

Modeling of solvent-dependent conformational transitions in Burkholderia cepacia lipase

2009

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BackgroundThe characteristic of most lipases is the interfacial activation at a lipid interface or in non-polar solvents. Interfacial activation is linked to a large conformational change of a lid, from a closed to an open conformation which makes the active site accessible for substrates. While for many lipases crystal structures of the closed and open conformation have been determined, the pathway of the conformational transition and possible bottlenecks are unknown. Therefore, molecular dynamics simulations of a closed homology model and an open crystal structure of Burkholderia cepacia lipase in water and toluene were performed to investigate the influence of solvents on structure, dynamics, and the conformational transition of the lid.ResultsThe conformational transition of B. cepacia lipase was dependent on the solvent. In simulations of closed B. cepacia lipase in water no conformational transition was observed, while in three independent simulations of the closed lipase in toluene the lid gradually opened during the first 10–15 ns. The pathway of conformational transition was accessible and a barrier was identified, where a helix prevented the lid from opening to the completely open conformation. The open structure in toluene was stabilized by the formation of hydrogen bonds.In simulations of open lipase in water, the lid closed slowly during 30 ns nearly reaching its position in the closed crystal structure, while a further lid opening compared to the crystal structure was observed in toluene. While the helical structure of the lid was intact during opening in toluene, it partially unfolded upon closing in water. The closing of the lid in water was also observed, when with eight intermediate structures between the closed and the open conformation as derived from the simulations in toluene were taken as starting structures. A hydrophobic β-hairpin was moving away from the lid in all simulations in water, which was not observed in simulations in toluene. The conformational transition of the lid was not correlated to the motions of the β-hairpin structure.ConclusionConformational transitions between the experimentally observed closed and open conformation of the lid were observed by multiple molecular dynamics simulations of B. cepacia lipase. Transitions in both directions occurred without applying restraints or external forces. The opening and closing were driven by the solvent and independent of a bound substrate molecule.

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“…One further point of attention is represented by the presence of a calcium ion in the structure of PcL (Figure 1b), which strongly affects the protein stability and this might be lost upon recycling [33]. …”

Section: Resultsmentioning

confidence: 99%

Lipases Immobilization for Effective Synthesis of Biodiesel Starting from Coffee Waste Oils

Ferrario

Veny

Angelis³

et al. 2013

Biomolecules

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Immobilized lipases were applied to the enzymatic conversion of oils from spent coffee ground into biodiesel. Two lipases were selected for the study because of their conformational behavior analysed by Molecular Dynamics (MD) simulations taking into account that immobilization conditions affect conformational behavior of the lipases and ultimately, their efficiency upon immobilization. The enzymatic synthesis of biodiesel was initially carried out on a model substrate (triolein) in order to select the most promising immobilized biocatalysts. The results indicate that oils can be converted quantitatively within hours. The role of the nature of the immobilization support emerged as a key factor affecting reaction rate, most probably because of partition and mass transfer barriers occurring with hydrophilic solid supports. Finally, oil from spent coffee ground was transformed into biodiesel with yields ranging from 55% to 72%. The synthesis is of particular interest in the perspective of developing sustainable processes for the production of bio-fuels from food wastes and renewable materials. The enzymatic synthesis of biodiesel is carried out under mild conditions, with stoichiometric amounts of substrates (oil and methanol) and the removal of free fatty acids is not required.

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Differential Scanning Calorimetry of the Effects of Ca²⁺on the Thermal Unfolding ofPseudomonas cepaciaLipase

Cited by 13 publications

References 16 publications

Unfolding of CBP21, a lytic polysaccharide monooxygenase, without dissociation of its copper ion cofactor

Unfolding of CBP21, a lytic polysaccharide monooxygenase, without dissociation of its copper ion cofactor

Modeling of solvent-dependent conformational transitions in Burkholderia cepacia lipase

Lipases Immobilization for Effective Synthesis of Biodiesel Starting from Coffee Waste Oils

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Differential Scanning Calorimetry of the Effects of Ca2+on the Thermal Unfolding ofPseudomonas cepaciaLipase

Cited by 13 publications

References 16 publications

Unfolding of CBP21, a lytic polysaccharide monooxygenase, without dissociation of its copper ion cofactor

Unfolding of CBP21, a lytic polysaccharide monooxygenase, without dissociation of its copper ion cofactor

Modeling of solvent-dependent conformational transitions in Burkholderia cepacia lipase

Lipases Immobilization for Effective Synthesis of Biodiesel Starting from Coffee Waste Oils

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Product

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Differential Scanning Calorimetry of the Effects of Ca²⁺on the Thermal Unfolding ofPseudomonas cepaciaLipase