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The anionic biosurfactant rhamnolipid does not denature industrial enzymes.

Madsen JK, Pihl R, Møller AH, Madsen AT, Otzen DE, Andersen KK - Front Microbiol (2015)

Bottom Line: It efficiently unfolds both LT and CZ, but LT is unfolded by SDS through formation of SDS clusters on the enzyme well below the cmc, while CZ is only unfolded by bulk micelles and on average binds significantly less SDS than LT.In contrast, RL does not affect the tertiary or secondary structure of any enzyme at room temperature, has little impact on thermal stability and only binds detectably (but at low stoichiometries) to SZ.Furthermore, all enzymes maintain activity at both monomeric and micellar concentrations of RL.

View Article: PubMed Central - PubMed

Affiliation: Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology and Genetics, Aarhus University Aarhus, Denmark.

ABSTRACT
Biosurfactants (BS) are surface-active molecules produced by microorganisms. Their combination of useful properties and sustainable production make them promising industrial alternatives to petrochemical and oleochemical surfactants. Here we compare the impact of the anionic BS rhamnolipid (RL) and the conventional/synthetic anionic surfactant sodium dodecyl sulfate (SDS) on the structure and stability of three different commercially used enzymes, namely the cellulase Carezyme® (CZ), the phospholipase Lecitase Ultra® (LT) and the α-amylase Stainzyme® (SZ). Our data reveal a fundamental difference in their mode of interaction. SDS shows great diversity of interaction toward the different enzymes. It efficiently unfolds both LT and CZ, but LT is unfolded by SDS through formation of SDS clusters on the enzyme well below the cmc, while CZ is only unfolded by bulk micelles and on average binds significantly less SDS than LT. SDS binds with even lower stoichiometry to SZ and leads to an increase in thermal stability. In contrast, RL does not affect the tertiary or secondary structure of any enzyme at room temperature, has little impact on thermal stability and only binds detectably (but at low stoichiometries) to SZ. Furthermore, all enzymes maintain activity at both monomeric and micellar concentrations of RL. We conclude that RL, despite its anionic charge, is a surfactant that does not compromise the structural integrity of industrially relevant enzymes. This makes RL a promising alternative to current synthetic anionic surfactants in a wide range of commercial applications.

No MeSH data available.


Thermal stability of enzymes with surfactants monitored by far-UV CD thermal scans at 222 nm. (A)tm of LT is reduced from 60°C in buffer to ~54°C around the cmc of RL. Higher concentrations of RL do not reduce tm any further, while SDS progressively lowered the tm and no thermal transition was observed above ~1 mM SDS. (B)tm of CZ is lowered by RL by a few degrees while SDS lowers the tm from 74°C to ~58°C at 2.5 mM SDS. A thermal transition was not observed >2.5 mM SDS. (C)tm of SZ was increased by both RL and SDS. (D) Change in enzyme thermal stability with increasing concentrations of surfactants.
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Figure 5: Thermal stability of enzymes with surfactants monitored by far-UV CD thermal scans at 222 nm. (A)tm of LT is reduced from 60°C in buffer to ~54°C around the cmc of RL. Higher concentrations of RL do not reduce tm any further, while SDS progressively lowered the tm and no thermal transition was observed above ~1 mM SDS. (B)tm of CZ is lowered by RL by a few degrees while SDS lowers the tm from 74°C to ~58°C at 2.5 mM SDS. A thermal transition was not observed >2.5 mM SDS. (C)tm of SZ was increased by both RL and SDS. (D) Change in enzyme thermal stability with increasing concentrations of surfactants.

Mentions: Pyrene fluorescence and CD indicate that at room temperature, none of the enzymes are denatured by RL, while LT is denatured by SDS monomers and CZ by SDS micelles. Pyrene investigations indicate that SZ interact with both RL and SDS monomers, but the interactions do not lead to enzyme denaturation. To determine how SDS and RL influenced enzyme stability at elevated temperatures, we subjected all 3 enzymes to thermal scans monitored by far-UV CD at 222 nm. In 50 mM Tris pH 8 and in the absence of surfactant, melting temperatures were 86, 74, and 60°C for SZ, CZ, and LT, respectively (Figures 5A–C). As summarized in Figure 5D, both SDS and RL shifted SZ's unfolding curve to higher temperatures, indicating that the surfactants bind to the native state of SZ and actually stabilize it against denaturation. This is in excellent agreement with the observation that SZ interact with both monomeric SDS and RL but is not denatured by either surfactant. SZ's high intrinsic thermal stability made it difficult to determine melting temperatures (tm) in the presence of surfactants, since unfolding was incomplete at 95°C (the temperature limit in the experiment). Therefore it is not possible to determine the exact tm at concentrations above 1 mM surfactant.


The anionic biosurfactant rhamnolipid does not denature industrial enzymes.

Madsen JK, Pihl R, Møller AH, Madsen AT, Otzen DE, Andersen KK - Front Microbiol (2015)

Thermal stability of enzymes with surfactants monitored by far-UV CD thermal scans at 222 nm. (A)tm of LT is reduced from 60°C in buffer to ~54°C around the cmc of RL. Higher concentrations of RL do not reduce tm any further, while SDS progressively lowered the tm and no thermal transition was observed above ~1 mM SDS. (B)tm of CZ is lowered by RL by a few degrees while SDS lowers the tm from 74°C to ~58°C at 2.5 mM SDS. A thermal transition was not observed >2.5 mM SDS. (C)tm of SZ was increased by both RL and SDS. (D) Change in enzyme thermal stability with increasing concentrations of surfactants.
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Related In: Results  -  Collection

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Figure 5: Thermal stability of enzymes with surfactants monitored by far-UV CD thermal scans at 222 nm. (A)tm of LT is reduced from 60°C in buffer to ~54°C around the cmc of RL. Higher concentrations of RL do not reduce tm any further, while SDS progressively lowered the tm and no thermal transition was observed above ~1 mM SDS. (B)tm of CZ is lowered by RL by a few degrees while SDS lowers the tm from 74°C to ~58°C at 2.5 mM SDS. A thermal transition was not observed >2.5 mM SDS. (C)tm of SZ was increased by both RL and SDS. (D) Change in enzyme thermal stability with increasing concentrations of surfactants.
Mentions: Pyrene fluorescence and CD indicate that at room temperature, none of the enzymes are denatured by RL, while LT is denatured by SDS monomers and CZ by SDS micelles. Pyrene investigations indicate that SZ interact with both RL and SDS monomers, but the interactions do not lead to enzyme denaturation. To determine how SDS and RL influenced enzyme stability at elevated temperatures, we subjected all 3 enzymes to thermal scans monitored by far-UV CD at 222 nm. In 50 mM Tris pH 8 and in the absence of surfactant, melting temperatures were 86, 74, and 60°C for SZ, CZ, and LT, respectively (Figures 5A–C). As summarized in Figure 5D, both SDS and RL shifted SZ's unfolding curve to higher temperatures, indicating that the surfactants bind to the native state of SZ and actually stabilize it against denaturation. This is in excellent agreement with the observation that SZ interact with both monomeric SDS and RL but is not denatured by either surfactant. SZ's high intrinsic thermal stability made it difficult to determine melting temperatures (tm) in the presence of surfactants, since unfolding was incomplete at 95°C (the temperature limit in the experiment). Therefore it is not possible to determine the exact tm at concentrations above 1 mM surfactant.

Bottom Line: It efficiently unfolds both LT and CZ, but LT is unfolded by SDS through formation of SDS clusters on the enzyme well below the cmc, while CZ is only unfolded by bulk micelles and on average binds significantly less SDS than LT.In contrast, RL does not affect the tertiary or secondary structure of any enzyme at room temperature, has little impact on thermal stability and only binds detectably (but at low stoichiometries) to SZ.Furthermore, all enzymes maintain activity at both monomeric and micellar concentrations of RL.

View Article: PubMed Central - PubMed

Affiliation: Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology and Genetics, Aarhus University Aarhus, Denmark.

ABSTRACT
Biosurfactants (BS) are surface-active molecules produced by microorganisms. Their combination of useful properties and sustainable production make them promising industrial alternatives to petrochemical and oleochemical surfactants. Here we compare the impact of the anionic BS rhamnolipid (RL) and the conventional/synthetic anionic surfactant sodium dodecyl sulfate (SDS) on the structure and stability of three different commercially used enzymes, namely the cellulase Carezyme® (CZ), the phospholipase Lecitase Ultra® (LT) and the α-amylase Stainzyme® (SZ). Our data reveal a fundamental difference in their mode of interaction. SDS shows great diversity of interaction toward the different enzymes. It efficiently unfolds both LT and CZ, but LT is unfolded by SDS through formation of SDS clusters on the enzyme well below the cmc, while CZ is only unfolded by bulk micelles and on average binds significantly less SDS than LT. SDS binds with even lower stoichiometry to SZ and leads to an increase in thermal stability. In contrast, RL does not affect the tertiary or secondary structure of any enzyme at room temperature, has little impact on thermal stability and only binds detectably (but at low stoichiometries) to SZ. Furthermore, all enzymes maintain activity at both monomeric and micellar concentrations of RL. We conclude that RL, despite its anionic charge, is a surfactant that does not compromise the structural integrity of industrially relevant enzymes. This makes RL a promising alternative to current synthetic anionic surfactants in a wide range of commercial applications.

No MeSH data available.