Flexibility of Enzymes Suspended in Organic Solvents Probed by Time-Resolved Fluorescence Anisotropy. Evidence That Enzyme Activity and Enantioselectivity Are Directly Related to Enzyme Flexibility

Broos, Jaap; Visser, Antonie J. W. G.; Engbersen, Johan F.J.; Verboom, Willem; Hoek, A. van; Reinhoudt, David N.

doi:10.1021/ja00156a001

Cited by 159 publications

(119 citation statements)

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“…Enantioselectivity was higher at 21°C for all crown ethers, particularly for 18-crown-6, than at 5°C or 45°C. However, crown ethers did not improve enantioselectivity substantially in contrast to the macrocyclic compound MβCD Santos et al, 1999), a fact that has also been reported for α-chymotrypsin colyophilized with 18-crown-6 (Broos et al, 1995b).…”

Section: Effect Of Crown Ethers On Catalytic Properties Of Subtilisinmentioning

confidence: 67%

“…Enzyme denaturation is unlikely in this instance because of the first aforementioned fact and thus the changes in dynamic properties of the catalyst might be responsible for the decrease in enantioselectivity. Somewhat differing results have also been presented (Broos et al, 1995b), showing increased enantioselectivity to be associated with increased enzyme flexibility.…”

Section: Introductionmentioning

confidence: 91%

“…Thus, again this effect cannot be responsible for the crown ether activation of subtilisin. It is of interest to note that 18-crown-6 does not influence the microscopic protein structural mobility as measured by timeresolved fluorescence anisotropy (Broos et al, 1995b), further indicating that dynamic changes are not responsible for the crown ether activation.…”

Section: Subtilisin Structure and Dynamics In Organic Solventsmentioning

confidence: 99%

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Effect of crown ethers on structure, stability, activity, and enantioselectivity of subtilisin Carlsberg in organic solvents

Santos

Vidal

Pacheco

et al. 2001

Biotech & Bioengineering

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Colyophilization or codrying of subtilisin Carlsberg with the crown ethers 18-crown-6, 15-crown-5, and 12-crown-4 substantially improved enzyme activity in THF, acetonitrile, and 1,4-dioxane in the transesterification reactions of N-acetyl-L-phenylalanine ethylester and 1-propanol and that of (±)-1-phenylethanol and vinylbutyrate. The acceleration of the initial rate, V 0 , ranged from less than 10-fold to more than 100-fold. All crown ethers activated subtilisin substantially, which excludes a specific macrocyclic effect from being responsible. The secondary structure of subtilisin was studied by Fourier-transform infrared (FTIR) spectroscopy. 18-Crown-6 and 15-crown-5 led to a more nativelike structure of subtilisin in the organic solvents employed when compared with that of the dehydrated enzyme obtained from buffer alone. However, the high level of activation with 12-crown-4 where this effect was not observed excluded overall structural preservation from being the primary cause of the observed enzyme activation. The conformational mobility of subtilisin was investigated by performing thermal denaturation experiments in 1,4-dioxane. Although only a small effect of temperature on subtilisin structure was observed for the samples prepared with or without 12-crown-4, both 18-crown-6 and 15-crown-5 caused the enzyme to denature at quite low temperatures (38°C and 56°C, respectively). No relationship between this property and V 0 was evident, but increased conformational mobility of the protein decreased its storage stability. The possibility of a "molecular imprinting" effect was also tested by removing 18-crown-6 from the subtilisin-18-crown-6 colyophilizate by washing. V 0 was only halved as a result of this procedure, an effect insignificant compared with the ca. 80-fold rate enhancement observed prior to washing in THF. This suggests that molecular imprinting is likely the primary cause of sub-tilisin activation by crown ethers, as recently suggested.

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Section: Effect Of Crown Ethers On Catalytic Properties Of Subtilisinmentioning

confidence: 67%

Section: Introductionmentioning

confidence: 91%

Section: Subtilisin Structure and Dynamics In Organic Solventsmentioning

confidence: 99%

See 1 more Smart Citation

Effect of crown ethers on structure, stability, activity, and enantioselectivity of subtilisin Carlsberg in organic solvents

Santos

Vidal

Pacheco

et al. 2001

Biotech & Bioengineering

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“…It has been shown experimentally that enzyme flexibility is correlated with enzyme activity and enantioselectivity in organic solvents. [13] To investigate the effect of organic solvent on CRL on a molecular level, we performed molecular dynamics simulations of four models (the open and closed conformation of CRL solvated in two solvents each, water and carbon tetrachloride) and compared the effect of the two solvents on the average protein structure and flexibility of backbone and side chains.…”

Section: Introductionmentioning

confidence: 99%

Structure and dynamics of Candida rugosa lipase: the role of organic solvent

2004

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The effect of organic solvent to structure and dynamics of proteins was investigated by multiple molecular dynamics simulations (1 ns each) of Candida rugosa lipase in water and in carbon tetrachloride. The choice of solvent had only a minor structural effect. For both solvents the open and the closed conformation of the lipase were near to their experimental X-ray structures (C α rms deviation 1-1.3 Å). However, the solvents had a highly specific effect on the flexibility of solvent-exposed side chains: polar side chains were more flexible in water, but less flexible in organic solvent. In contrast, hydrophobic residues were more flexible in organic solvent, but less flexible in water. As a major effect solvent changed the dynamics of the lid, a mobile element involved in activation of the lipase, which fluctuated as rigid body about its average position. While in water the deviations were about 1.6 Å, organic solvent reduced flexibility to 0.9 Å. This increase rigidity was caused by two salt bridges (Lys85-Asp284, Lys75-Asp79) and a stable hydrogen bond (Lys75-Asn 292) in organic solvent. Thus organic solvents stabilize the lid but render the side chains in the hydrophobic substrate binding site more mobile.

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“…4 On the other hand, reactions carried out in organic solvents, in general, result in enzymes with greater rigidity, since in aqueous medium the breaking and formation of hydrogen bonds involved in the formation of the enzymesubstrate complex are assisted by water, which does not occur in hydrophobic solvents. 56 Another factor that may influence the selectivity of biocatalysts is immobilization. 4 Nascimento et al 57 reported that the use of immobilized lipase increased the enantioselectivity and the conversion percentage in the resolution of α-methylene-β-hydroxy esters, when compared to the reaction with the free enzyme.…”

Section: Synthetic Screeningmentioning

confidence: 99%

Potential application in biocatalysis of mycelium-bound lipases from Amazonian fungi

Zanotto¹,

Romano

Duvoisin

et al. 2009

J. Braz. Chem. Soc.

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Neste trabalho foram avaliados 212 fungos isolados de plantas da Região Amazônica quanto ao potencial de aplicação em biocatálise como fornecedores de lipases ligadas ao micélio. Estes isolados foram submetidos a ensaios de atividade hidrolítica e sintética. Quando submetidos ao ensaio com o substrato tributirina, 87% dos isolados apresentaram atividade hidrolítica. Destes, 30% mostraram bom crescimento em meio líquido indutor de lipase, e foram submetidos à avaliação sintética em reações de esterificação e transesterificação em solvente orgânico. Os nove fungos que apresentaram os melhores desempenhos sintéticos foram avaliados quanto a sua enantiosseletividade na reação de resolução do (R,S)-2-octanol. O isolado UEA_115 foi o biocatalisador mais versátil, apresentando bom desempenho nas reações de esterificação (conversão > 90%) e boa capacidade para a resolução do (R,S)-2-octanol (ee s 29%; ee p 99%; c 22%; E > 200). Com este estudo foi possível demonstrar o grande potencial dos fungos amazônicos como fornecedores de lipases para biocatálise.In this study, 212 fungi were isolated from Amazon region plants, aiming to obtain mycelium bound-lipase-producing biocatalysts. These isolates were submitted to hydrolytic and synthetic activity assays. When submitted to the tributyrine substrate test, 87% of the isolates showed hydrolytic activity. Of these, 30% showed good growth in lipase inducing liquid media and were submitted to evaluation of synthetic activity in esterification and transesterification reactions in organic solvents. The nine fungi which had the best synthetic activity were evaluated in the (R,S)-2-octanol resolution reaction, in order to verify the enantioselectivity of mycelium-bound lipases. The isolate UEA_115 was the most versatile biocatalyst, showing good performance in esterification reactions (conversion > 90%) and good ability for the resolution of (R,S)-2-octanol (ee s 29%; ee p 99%; c 22%; E > 200). Thus, this study has demonstrated the great potential of the Amazonian fungi as lipase suppliers for biocatalysts. Keywords: fungi, Amazon region, mycelium-bound lipase, biocatalyst screening, biocatalysis IntroductionIn recent years, global concern regarding the impact of human actions on the environment has been growing. The implementation of the concept of sustainable development in several productive activities has become an urgent issue. Fortunately, biotechnology also offers an increasing potential to meet the global demands for processes and products in greater harmony with the sustainability concept. 1,2 In this regard, one of the most promising tools in biotechnology is the field of applied biocatalysis, also referred to by some authors as biotransformation or enzymatic technology. 2,3 The advantages of using enzymes as biocatalysts in industrial processes have been shown to be ever more evident. Great versatility of catalyzed reactions, mild reaction conditions, and regio, chemo and enantioselective natures are those most frequently mentioned in the literature. 4 Such characteristic...

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Flexibility of Enzymes Suspended in Organic Solvents Probed by Time-Resolved Fluorescence Anisotropy. Evidence That Enzyme Activity and Enantioselectivity Are Directly Related to Enzyme Flexibility

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Cited by 159 publications

References 2 publications

Effect of crown ethers on structure, stability, activity, and enantioselectivity of subtilisin Carlsberg in organic solvents

Effect of crown ethers on structure, stability, activity, and enantioselectivity of subtilisin Carlsberg in organic solvents

Structure and dynamics of Candida rugosa lipase: the role of organic solvent

Potential application in biocatalysis of mycelium-bound lipases from Amazonian fungi

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