Control of enzyme ionization state in supercritical ethane by sodium/proton solid-state acid–base buffers

Fontes, Nuno; Halling, Peter J.; Barreiros, Susana

doi:10.1016/j.enzmictec.2003.07.003

Cited by 10 publications

(10 citation statements)

References 15 publications

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“…No products were detected in experiments with CAPSO buffer. Cutinase thus behaves very differently from subtilisin Carlsberg (Fontes et al, 2002(Fontes et al, , 2003bHarper and Barreiros, 2002). Although the two enzymes share a similar active site architecture and catalytic mechanism, they have substantially different structures, and this must account for the observed differences in behavior.…”

Section: Resultsmentioning

confidence: 91%

“…One question that we sought to answer was whether rates of transesterification could be improved by providing acid -base control in the medium. In a recent study with subtilisin Carlsberg, we observed that the transesteriflcation activity of the enzyme increased substantially with increasing a w in the presence of a relatively basic solid-state acid -base buffer (Fontes et al, 2003b). Another issue that we sought to investigate was whether buffers that set a more basic protonation state of cutinase affected the competition between 2-phenyl-1-propanol and water for the acyl-enzyme.…”

Section: Resultsmentioning

confidence: 98%

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Effect of immobilization support, water activity, and enzyme ionization state on cutinase activity and enantioselectivity in organic media

Vidinha

Harper

Micaêlo

et al. 2004

Biotech & Bioengineering

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We studied the reaction between vinyl butyrate and 2-phenyl-1-propanol in acetonitrile catalyzed by Fusarium solani pisi cutinase immobilized on zeolites NaA and NaY and on Accurel PA-6. The choice of 2-phenyl-1-propanol was based on modeling studies that suggested moderate cutinase enantioselectivity towards this substrate. With all the supports, initial rates of transesterification were higher at a water activity (a(w)) of 0.2 than at a(w) = 0.7, and the reverse was true for initial rates of hydrolysis. By providing acid-base control in the medium through the use of solid-state buffers that control the parameter pH-pNa, which we monitored using an organo-soluble chromoionophoric indicator, we were able, in some cases, to completely eliminate dissolved butyric acid. However, none of the buffers used were able to improve the rates of transesterification relative to the blanks (no added buffer) when the enzyme was immobilized at an optimum pH of 8.5. When the enzyme was immobilized at pH 5 and exhibited only marginal activity, however, even a relatively acidic buffer with a pK(a) of 4.3 was able to restore catalytic activity to about 20% of that displayed for a pH of immobilization of 8.5, at otherwise identical conditions. As a(w) was increased from 0.2 to 0.7, rates of transesterification first increased slightly and then decreased. Rates of hydrolysis showed a steady increase in that a(w) range, and so did total initial reaction rates. The presence or absence of the buffers did not impact on the competition between transesterification and hydrolysis, regardless of whether the butyric acid formed remained as such in the reaction medium or was eliminated from the microenvironment of the enzyme through conversion into an insoluble salt. Cutinase enantioselectivity towards 2-phenyl-1-propanol was indeed low and was not affected by differences in immobilization support, enzyme protonation state, or a(w).

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Section: Resultsmentioning

confidence: 91%

Section: Resultsmentioning

confidence: 98%

Effect of immobilization support, water activity, and enzyme ionization state on cutinase activity and enantioselectivity in organic media

Vidinha

Harper

Micaêlo

et al. 2004

Biotech & Bioengineering

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“…However, changes in pressure do not affect the stability of most enzymes. [38] To improve the activities and stabilities of enzymes, many techniques have been developed that use various SCFs: immobilised enzymes, [39,40] lipid-coated enzymes, [41,42] cross-linked enzyme crystals (CLECs), [43][44][45] cross-linked enzyme aggregate (CLEAs) [46,47] or reverse micelles. [48] The last category of green solvents is composed those organic solvents obtained from renewable sources, also called "bio-solvents".…”

Section: Green Solventsmentioning

confidence: 99%

Applied Biotransformations in Green Solvents

Hernáiz

Alcántara

Garcı́a

et al. 2010

Chemistry A European J

104

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The definite interest in implementing sustainable industrial technologies has impelled the use of biocatalysts (enzymes or cells), leading to high chemo-, regio- and stereoselectivities under mild conditions. As usual substrates are not soluble in water, the employ of organic solvents is mandatory. We will focus on different attempts to combine the valuable properties of green solvents with the advantages of using biocatalysts for developing cleaner synthetic processes.

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“…However, if the enzyme is exposed to acidic or basic species (substrates, byproducts, impurities) in the organic solvent, as is frequently the case, the protonation-state of the enzyme is altered, resulting in a large change to the catalytic activity and stability. Organo-soluble and also insoluble (solid-state) conjugate acid -base pairs have been introduced to control the ionisation-state of enzymes in nonaqueous media (Blackwood et al, 1994;Fontes et al, 2003;Harper et al, 2000Harper et al, , 2001Partridge et al, 2000;Quirós et al, 2001;Theppakorn et al, 2003;Zacharis et al, 1997).…”

Section: Introductionmentioning

confidence: 99%

High‐activity biocatalysts in organic media: solid‐state buffers as the immobilisation matrix for protein‐coated microcrystals

Kreiner

Parker²

2004

Biotech & Bioengineering

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Recently, we reported a new high-activity biocatalyst for use in organic media termed protein-coated microcrystals (PCMC) (Kreiner et al. [2001] Chem Commun 12:1096-1097). These novel particles consist of water-soluble micron-sized crystalline particles coated with the given biocatalyst(s) and are prepared in a one-step rapid dehydration process. In this study we extended the choice of immobilisation matrix from a simple inorganic salt, K(2)SO(4), to other compounds, both inorganic and zwitterionic, that act as solid-state buffers for biocatalysis in organic media. The catalytic activity of serine proteases subtilisin Carlsberg (SC) and alpha-chymotrypsin (CT) were significantly increased when coated onto the surface of solid-state buffers, as measured in acetonitrile/1wt% H(2)O. SC-PCMC with both organic and inorganic buffer carriers (Na-AMPSO, Na(2)CO(3), and NaHCO(3)) showed a 3-fold greater activity than that observed when using the unbuffered system (PCMC-SC/K(2)SO(4)). In comparison with freeze-dried preparations, this represents an approximately 3,000-fold increase in catalytic activity. Importantly, there is no improvement in catalytic activity upon external addition of any of the solid-state buffers to the reaction mixture. When acting in a solid-state buffer capacity, good buffering capacity was observed with SC-PCMC (3 wt% protein loading) prepared from a 1:1 mixture of AMPSO and AMPSO-Na. Alternatively, increasing the amount of solid-state buffer in the system allows improvement of the buffering. This can be achieved either by decreasing the protein loading of the SC/Na-AMPSO-PCMC or by addition of further external solid-state buffer to the reaction mixture. The catalytic activity of lipase-PCMC prepared from solid-state buffers was found less responsive to immobilisation.

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Control of enzyme ionization state in supercritical ethane by sodium/proton solid-state acid–base buffers

Cited by 10 publications

References 15 publications

Effect of immobilization support, water activity, and enzyme ionization state on cutinase activity and enantioselectivity in organic media

Effect of immobilization support, water activity, and enzyme ionization state on cutinase activity and enantioselectivity in organic media

Applied Biotransformations in Green Solvents

High‐activity biocatalysts in organic media: solid‐state buffers as the immobilisation matrix for protein‐coated microcrystals

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