Kinetics of Detergent-Induced Activation and Inhibition of a Minimal Lipase

Kübler, Daniel; Bergmann, Anna; Weger, Lukas; Ingenbosch, Kim N.; Hoffmann-Jacobsen, Kerstin

doi:10.1021/acs.jpcb.6b11037

Cited by 14 publications

(32 citation statements)

References 45 publications

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“…Enhanced activity at 28°C, coupled with no significant loss in activity at 40°C, could be explained by the concentration of SDS. Below the Critical Micelle Concentration CMC (8.2mM or 0.24% w/v at 25°C) SDS binds to the lid of lipase and activates it by conformational changes and the enzyme requires less interfacial activation [44]. Also, detergents may also alter the hydrophobicity of the enzyme and, therefore, the availability of substrate to the enzyme [45].…”

Section: Influence Of Metal Ions and Chemical Reagentsmentioning

confidence: 99%

Isolation, purification and characterization of a novel solvent stable lipase from Pseudomonas reinekei

Priyanka¹,

Kinsella²,

Henehan³

et al. 2019

Protein Expression and Purification

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Section: Influence Of Metal Ions and Chemical Reagentsmentioning

confidence: 99%

Isolation, purification and characterization of a novel solvent stable lipase from Pseudomonas reinekei

Priyanka¹,

Kinsella²,

Henehan³

et al. 2019

Protein Expression and Purification

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“…The FP spectroscopy was previously used to inspect: (i) the interactions of mild 14 and harsh 15 detergents with water-soluble proteins, (ii) the harsh detergent-induced unfolding 16 and resistance of soluble proteins to denaturation, 17 (iii) the detergent-mediated oligomerization of hydrophobic proteins into proteomicelles, 18 and (iv) the impact of detergent on conformational changes 14 and enzymatic activity 19 of soluble proteins.…”

Section: Introductionmentioning

confidence: 99%

Quantification of Membrane Protein-Detergent Complex Interactions

Wolfe

Zhang

et al. 2017

J. Phys. Chem. B

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Although fundamentally significant in structural, chemical, and membrane biology, the interfacial protein-detergent complex (PDC) interactions have been modestly examined because of the complicated behavior of both detergents and membrane proteins in aqueous phase. Membrane proteins are prone to unproductive aggregation resulting from poor detergent solvation, but the participating forces in this phenomenon remain ambiguous. Here, we show that using rational membrane protein design, targeted chemical modification, and steady-state fluorescence polarization spectroscopy, the detergent desolvation of membrane proteins can be quantitatively evaluated. We demonstrate that depleting the detergent in the sample well produced a two-state transition of membrane proteins between a fully detergent-solvated state and a detergent-desolvated state, the nature of which depended on the interfacial PDC interactions. Using a panel of six membrane proteins of varying hydrophobic topography, structural fingerprint, and charge distribution on the solvent-accessible surface, we provide direct experimental evidence for the contributions of the electrostatic and hydrophobic interactions to the protein solvation properties. Moreover, all-atom molecular dynamics simulations report the major contribution of the hydrophobic forces exerted at the PDC interface. This semi-quantitative approach might be extended in the future to include studies of the interfacial PDC interactions of other challenging membrane protein systems of unknown structure. This would have practical importance in protein extraction, solubilization, stabilization, and crystallization.

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“…This experiment allowed to highlight the main difference in the hydrolytic performance between the variants, where P5F3 maintains its performance throughout the assessed substrates, potentially allowing the use of BSLA backbones for the catalysis of longer fatty acid ester substrates. From the substrate saturation curves of the more hydrophobic substrates (Figure A2) for P5F3 variant, we can observe a possible substrate availability (effective concentration) decrease in concentrations higher than 0.5 mM, due to the low water solubility of the substrates and the complex kinetics of substrate availability for lipases [46]. For wildtype BSLA, we can see a significantly lower hydrolytic rate even before 0.5 mM substrate and a saturation beyond that point.…”

Section: Characterization Of Variant P5f3mentioning

confidence: 87%

Deletion and Randomization of Structurally Variable Regions in B. subtilis Lipase A (BSLA) Alter Its Stability and Hydrolytic Performance Against Long Chain Fatty Acid Esters

Martínez

Bernal

Álvarez

et al. 2020

IJMS

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The continuous search for novel enzyme backbones and the engineering of already well studied enzymes for biotechnological applications has become an increasing challenge, especially by the increasing potential diversity space provided by directed enzyme evolution approaches and the demands of experimental data generated by rational design of enzymes. In this work, we propose a semi-rational mutational strategy focused on introducing diversity in structurally variable regions in enzymes. The identified sequences are subjected to a progressive deletion of two amino acids and the joining residues are subjected to saturation mutagenesis using NNK degenerate codons. This strategy offers a novel library diversity approach while simultaneously decreasing enzyme size in the variable regions. In this way, we intend to identify and reduce variable regions found in enzymes, probably resulting from neutral drift evolution, and simultaneously studying the functional effect of said regions. This strategy was applied to Bacillus. subtilis lipase A (BSLA), by selecting and deleting six variable enzyme regions (named regions 1 to 6) by the deletion of two amino acids and additionally randomizing the joining amino acid residues. After screening, no active variants were found in libraries 1% and 4%, 15% active variants were found in libraries 2% and 3%, and 25% for libraries 5 and 6 (n = 3000 per library, activity detected using tributyrin agar plates). Active variants were assessed for activity in microtiter plate assay (pNP-butyrate), thermal stability, substrate preference (pNP-butyrate, -palmitate), and compared to wildtype BSLA. From these analyses, variant P5F3 (F41L-ΔW42-ΔD43-K44P), from library 3 was identified, showing increased activity towards longer chain p-nitrophenyl fatty acid esters, when compared to BSLA. This study allowed to propose the targeted region 3 (positions 40–46) as a potential modulator for substrate specificity (fatty acid chain length) in BSLA, which can be further studied to increase its substrate spectrum and selectivity. Additionally, this variant showed a decreased thermal resistance but interestingly, higher isopropanol and Triton X-100 resistance. This deletion-randomization strategy could help to expand and explore sequence diversity, even in already well studied and characterized enzyme backbones such as BSLA. In addition, this strategy can contribute to investigate and identify important non-conserved regions in classic and novel enzymes, as well as generating novel biocatalysts with increased performance in specific processes, such as enzyme immobilization.

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Kinetics of Detergent-Induced Activation and Inhibition of a Minimal Lipase

Cited by 14 publications

References 45 publications

Isolation, purification and characterization of a novel solvent stable lipase from Pseudomonas reinekei

Isolation, purification and characterization of a novel solvent stable lipase from Pseudomonas reinekei

Quantification of Membrane Protein-Detergent Complex Interactions

Deletion and Randomization of Structurally Variable Regions in B. subtilis Lipase A (BSLA) Alter Its Stability and Hydrolytic Performance Against Long Chain Fatty Acid Esters

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