Stability Of Cutinase Research Articles

Immobilization of Cutinase from Fussarium oxysporum into Sea Sand Matrix for Catalytic Degradation of Polyethylene Terephthalate (PET)

Cutinase is an enzyme that can be used as a catalyst in the hydrolysis reaction of polyethylene terephthalate (PET) waste into terephthalic acid and ethylene glycol. Generally, cutinase has low stability in its free form. Therefore, to increase cutinase stability, cutinase needs to be immobilized. In this study, cutinase was immobilized into a sea sand matrix by adsorption technique. The purpose of this study was to determine the optimum conditions of PET hydrolysis (pH, temperature, and incubation time), and to study the ability of immobilized cutinase for catalytic degradation of PET. The results show that immobilized cutinase has optimum conditions at pH 8, temperature of 50 °C, and incubation time of 24 hrs. Moreover, the immobilized cutinase can be used for up to three cycles with a residual activity percentage of 66.2% and an activity value of 23.224 nmol. g-1. min-1. The total enzyme activity of immobilized cutinase after being used in three cycles was 89.99 nmol. g-1. min-1 or 28.6% of its free enzyme activity.

Recycling by-design of plastic through formulation with thermally protected enzymes in layered double hydroxide structures

The use of polymer-degrading enzymes in polymer formulation is a very attractive strategy for the life-cycle of plastics. The design of a material able to degrade on-demand could help reach the ambitious targets pursued by recent European policies. Only 32% of the resulting waste from the 50 million tonnes of plastics consumed each year in Europe is actually recycled. Since packaging accounts for more than 40% of all plastics produced every year, the improvement of its circularity from origin to subsequent life cycles is now a priority. The present research suggests a solution to improve the recyclability of plastics via a life cycle approach employing thermally stable enzymes as innovative materials providing a new potential for plastic and its end life. More specifically, cutinase, selected as a highly degrading polyester hydrolytic enzyme, was thermally protected by immobilization in Mg/Al layered double hydroxide structures. The cutinase immobilization efficiency was found to be high, as well as its release ability in an appropriate medium. The thermal stability of cutinase was strongly improved after immobilization, as highlighted by a 6-times increase of its half-life at 90 °C, compared to the free enzyme, and by a high activity retention (>60%) after short exposure to temperatures up to 200 °C. Moreover, it was demonstrated that a film of poly (butylene succinate-co-adipate) formulated with 5 wt% of immobilized cutinase, completely degraded within 24 h.

Depolymerization of post-consumer PET bottles with engineered cutinase 1 fromThermobifida cellulosilytica

The enhanced enzymatic activity and thermal stability of cutinase 1 fromThermobifida cellulosilyticaby enzyme engineering were utilized to achieve efficient degradation of post-consumer polyethylene terephthalate (PET) bottle particles.

Granulomes hépatiques pseudo-sarcoidosiques et carcinome hépato-cellulaire : à propos d’un cas et revue de la littérature

Fusarium solani pisi cutinase microencapsulated in reversed micelles of AOT (bis(2-ethylhexyl) sodium sulfosuccinate) was used to catalyse the alcoholysis of butyl acetate (acyl donor) with hexanol. In the present study the stability of cutinase is evaluated comparatively with the initial activity in order to obtain a set of conditions to fully optimise the system. The start conditions were selected based on a synergy of the results assigned in previous work, where relevant parameters for the cutinase activity, such as temperature, buffer molarity, pH, amount of water, surfactant, hexanol and butyl acetate concentrations were studied. Using a 2(5−1) factorial design expanded further to a central composite design (CCD), the effects of each factor mentioned above (with exception of temperature and acetate concentration) on the stability were calculated and the interactions were determined. The surface methodology applied to the results showed that cutinase, incubated at 40°C retained a high activity level (90%) after 3 days when microencapsulated at W0 (molar ratio of water to surfactant) 2.7. The W0 values in the range 5–8 led to optimal-specific activities with lower stability. A hexanol concentration of 500–600 mm was essential to prevent deactivation and at the same time allowed relatively high initial rates. A pH value of 8.0–8.55 and a buffer molarity of 200 mm were suitable for both activity and stability. Cutinase in AOT reversed micelles has been usually reported to be very sensitive to temperature, however, in this work, at an incubation temperature of 40°C, cutinase displayed half-lives significantly greater than previously stated. The main reasons for this are the presence of hexanol and the reduced water content that promote a stabilisation of cutinase structure inside the micelles. The thermostability experiments performed with the selected conditions led to a half-life of 53 days, confirming the model predictions.

Operational stability of cutinase in organic solvent system: model esterification of alkyl esters

AbstractBACKGROUND: The present work aims to gain further insight on the use of Fusarium solani pisi cutinase from a Saccharomyces cerevisiae strain for the synthesis of short chain ethyl esters, a group of important fruit flavor compounds, in a non‐conventional environment. Synthesis is promoted by cutinase in organic media, in particular, in iso‐octane, an organic solvent recognized as a safe ingredient in food and beverage industrial processes.RESULTS: The effect of solvent and substrate components of the reaction mixture on the enzyme stability was measured separately. Focus was given to the effect of reaction medium on the operational stability of cutinase and on the esterification yield after 24h. The feasibility of the operation in fed batch mode was successfully evaluated. The effect of the addition of substrate in consecutive pulses in the activity and stability of the biocatalyst was again assessed.CONCLUSIONS: The bioconversion system used in this work allowed for the sustained production of short chain alkyl esters for more than 45 days, which is suggestive of the stability of cutinase in the organic environment evaluated. Copyright © 2010 Society of Chemical Industry

Stability of cutinase, wild type and mutants, in AOT reversed micellar system—effect of mixture components of alkyl esters production

AbstractBACKGROUND: Fusarium solani pisi cutinase was microencapsulated in reversed micelles of bis(2‐ethylhexyl) sodium sulfosuccinate (AOT) in isooctane for the production of alkyl esters, known as biodiesel; its stability in this system was also evaluated. Results using cutinase wild type and three mutants, L153Q, T179C and S54D, were compared.RESULTS: There is a loss of 45% of cutinase wild type activity when incubated in the micellar system for 3 h, indicating a denaturing effect of AOT towards the enzyme, and furthermore a loss of 90% of the activity is observed in the presence of methanol with only 10 min of incubation. In contrast, an improvement of cutinase performance was achieved when incubated with ethanol or butanol, indicating that these alcohols have a protective role regarding the enzyme. Mutant T179C displayed high stability in the presence of methanol with an activity loss of only 16%.CONCLUSIONS: Mutant T179C presented not only high resistance to the denaturing effect of AOT but also high stability in the presence of methanol. This is a promising biocatalyst for a continuous process. Copyright © 2010 Society of Chemical Industry

Improving activity and stability of cutinase towards the anionic detergent AOT by complete saturation mutagenesis

Cutinase is an enzyme suitable for detergent applications as well as for organic synthesis in non-aqueous solvents. However, its inactivation in the presence of anionic surfactants is a problem which we have addressed by creating a complete saturation library. For this, the cutinase gene from Fusarium solani pisi was mutated to incorporate all 19 possible amino acid exchanges at each of the 214 amino acid positions. The resulting library was screened for active variants with improved stability in the presence of the anionic surfactant dioctyl sulfosuccinate sodium salt (AOT). Twenty-four sites in cutinase were discovered where amino acid replacements resulted in a 2-11-fold stability increase as compared to the wild-type enzyme.

Modulation of cutinase stability and structure by phospholipid detergents

Fusarium solani pisi cutinase hydrolyses triglycerides of different lengths. Here we show that micelle-forming short-chain (C6–C9) phospholipids significantly reduce cutinase stability (both below and above the critical micelle concentration cmc) and rates of folding (only above cmc), trapping cutinase in an inactive state which only regains activity over hours to days, rather than the few seconds required for refolding in the absence of detergent. Destabilization decreases with increasing chain length, and increases with cmc, indicating that monomers and micelles cooperate in destabilizing cutinase. Detergents have little effect on enzymatic activity and confer no changes in secondary structure. Some changes in chemical shift occur around the enzyme active site, although distant regions are also affected. To our knowledge, this is the first example of marked destabilization of a water-soluble protein by zwitterionic detergents, highlighting the multitude of different detergent interactions with enzymes that target amphiphilic substrates and providing means of trapping a protein in a metastable state. We propose a model for destabilization where monomers via various binding sites on the native state prime it for interacting with micelles in a destabilizing fashion, whereas only micelles halt refolding due to the absence of these monomer-binding sites in the denatured state.

Influence of organic solvents on cutinase stability and accessibility to polyamide fibers

AbstractThe stability of cutinase was measured in the presence of dimethylacetamide (DMA) and benzyl alcohol (BA). These solvents are known swelling agents of polyamide fibers. We found that cutinase retains some stability (half‐life of 14 h), enough for an enzymatic treatment with a solvent composition of 1.5% DMA + 10% BA. Enzymatic treatment in the presence of these solvent compositions yields more soluble amino groups as well as more amino end groups at the fiber level. We have shown evidence that the presence of minor quantities of solvent can increase the accessibility of the enzyme towards the polyamide fiber. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 2749‐2753, 2005

Improving cutinase stability in aqueous solution and in reverse micelles by media engineering

The stability of a recombinant cutinase from the fungus Fusarium solani was evaluated in aqueous media and in reverse micelles. Thermal unfolding in aqueous solution is a two-state process at the pH values tested and trehalose increased the temperature at the mid-point of the unfolding transitions. Irreversible inactivation is a first-order process at pH 9.2, but two inactivation phases were resolved at pH 4.5. Trehalose did not change the irreversible inactivation pathway but increased the kinetics of the irreversible inactivation step. Unfolding of cutinase induced by guanidine hydrochloride was more complex, showing a stable intermediate, molten globule in character, within the transition region. Trehalose did not change the three-state nature of the unfolding process. Encapsulation of cutinase in AOT reverse micelles induced unfolding at room temperature due to an enzyme location at the micellar interface. The presence of 1-hexanol as co-surfactant delayed or even prevented the unfolding of cutinase by promoting the establishment of a new equilibrium in the system. Cutinase is encapsulated in a 10-fold larger AOT/hexanol reverse micelle built up by the fusion of empty reverse micelles. When tested in a membrane reactor in the presence of 1-hexanol, an operational half-life of 674 days was achieved.

DSC studies of Fusarium solani pisi cutinase: consequences for stability in the presence of surfactants

The application of cutinase from Fusarium solani pisi as a fat-stain removing ingredient in laundry washing is hampered by its lack of stability in the presence of anionic surfactants. We postulate that the stability of cutinase towards anionics can be improved by mutations increasing its temperature stability. Thermal unfolding as measured with DSC, appears to be irreversible, though the thermograms are more symmetric than predicted by a simple irreversible model. In the presence of taurodeoxycholate (TDOC), the unfolding temperature is lower and the unfolding is reversible. We conclude that an early reversible unfolding intermediate exists in which a number of additional hydrophobic patches are exposed to the solvent, or preferentially are covered with TDOC. Improvement of the stability of cutinase with respect to both surfactants and thermal denaturation, should thus be directed toward the prevention of exposure of hydrophobic patches in the early intermediate.

Effects of ionic surfactants used in reversed micelles on cutinase activity and stability

The effects of aqueous surfactant solutions on the kinetics and stability of cutinase from Fusarium solani pisi were studied. The surfactant sodium bis[2-ethylhexyl]ester sulfosuccinic acid (AOT) acts as a pseudo-competitive inhibitor within a limited concentration range relative to the hydrolysis of short-chain p-nitrophenyl esters. For higher concentrations a hyperbolic mixed inhibition takes place. A pseudo-activation of hydrolysis in presence of AOT and hexadecyltrimethyl-ammonium bromide (CTAB) was observed. CTAB has similar effects on kinetics of cutinase. Cutinase revealed to be stable in CTAB solutions, with activity retention as high as 80%. AOT has a deleterious effect on the enzyme in the time course, resulting in acute loss of activity possibly related with unfolding of the protein structure. A relation between deactivation rate constants and AOT/cutinase concentration ratios is suggested. The presence of the linear alcohol, 1-hexanol, was included in these solutions, in the attempt to interpret the deactivation of cutinase when encapsulated in reversed micelle systems in the absence of this co-surfactant.

Trehalose delays the reversible but not the irreversible thermal denaturation of cutinase.

The effect of trehalose (0.5 M) on the thermal stability of cutinase in the alkaline pH range was studied. The thermal unfolding induced by increasing temperature was analyzed in the absence and in the presence of trehalose according to a two-state model (which assumes that only the folded and unfolded states of cutinase were present). Trehalose delays the reversible unfolding. The midpoint temperature of the unfolding transition (Tm) increases by 4.0 degrees C and 2. 6 degrees C at pH 9.2 and 10.5, respectively, in the presence of trehalose. At pH 9.2 the thermal unfolding occurs at higher temperatures (Tm is 52.6 degrees C compared to 42.0 degrees C at pH 10.5) and a refolding yield of around 80% was obtained upon cooling. This pH value was chosen to study the irreversible inactivation (long-term stability) of cutinase. Temperatures in the transition range from folded to unfolded state were selected and the rate constants of irreversible inactivation determined. Inactivation followed first-order kinetics and trehalose reduced the observed rate constants of inactivation, pointing to a stabilizing effect on the irreversible inactivation step of thermal denaturation. However, if the contribution of reversible unfolding on the irreversible inactivation of cutinase was taken into account, i.e., considering the fraction of cutinase molecules in the reversible unfolded conformation, the intrinsic rate constants can be calculated. Based on the intrinsic rate constants it was concluded that trehalose does not delay the irreversible inactivation. This conclusion was further supported by comparing the activation energy of the irreversible inactivation in the absence and in the presence of trehalose. The apparent activation energy in the absence and in the presence of trehalose were 67 and 99 Kcal/mol, respectively. The activation energy calculated from intrinsic rate constants was higher in the absence (30 Kcal/mol) than in the presence of trehalose (16 Kcal/mol), showing that kinetics of the irreversible inactivation step increased in the presence of trehalose. In fact, trehalose stabilized only the reversible step of thermal denaturation of cutinase.

Cutinase stability in AOT reversed micelles: system optimization using the factorial design methodology

Fusarium solani pisi cutinase microencapsulated in reversed micelles of AOT (bis(2-ethylhexyl) sodium sulfosuccinate) was used to catalyse the alcoholysis of butyl acetate (acyl donor) with hexanol. In the present study the stability of cutinase is evaluated comparatively with the initial activity in order to obtain a set of conditions to fully optimise the system. The start conditions were selected based on a synergy of the results assigned in previous work, where relevant parameters for the cutinase activity, such as temperature, buffer molarity, pH, amount of water, surfactant, hexanol and butyl acetate concentrations were studied. Using a 2(5−1) factorial design expanded further to a central composite design (CCD), the effects of each factor mentioned above (with exception of temperature and acetate concentration) on the stability were calculated and the interactions were determined. The surface methodology applied to the results showed that cutinase, incubated at 40°C retained a high activity level (90%) after 3 days when microencapsulated at W0 (molar ratio of water to surfactant) 2.7. The W0 values in the range 5–8 led to optimal-specific activities with lower stability. A hexanol concentration of 500–600 mm was essential to prevent deactivation and at the same time allowed relatively high initial rates. A pH value of 8.0–8.55 and a buffer molarity of 200 mm were suitable for both activity and stability. Cutinase in AOT reversed micelles has been usually reported to be very sensitive to temperature, however, in this work, at an incubation temperature of 40°C, cutinase displayed half-lives significantly greater than previously stated. The main reasons for this are the presence of hexanol and the reduced water content that promote a stabilisation of cutinase structure inside the micelles. The thermostability experiments performed with the selected conditions led to a half-life of 53 days, confirming the model predictions.

Deactivation and conformational changes of cutinase in reverse micelles

Deactivation data and fluorescence intensity changes were used to probe functional and structural stability of cutinase in reverse micelles. A fast deactivation of cutinase in anionic (AOT) reverse micelles occurs due to a reversible denaturation process. The deactivation and denaturation of cutinase is slower in small cationic (CTAB/1-hexanol) reverse micelles and does not occur when the size of the cationic reverse micellar water-pool is larger than cutinase. In both systems, activity loss and denaturation are coupled processes showing the same trend with time. Denaturation is probably caused by the interaction between the enzyme and the surfactant interface of the reversed micelle. When the size of the empty reversed micelle water-pool is smaller than cutinase (at W0 5, with W0 being the water:surfactant concentration ratio) a three-state model describes denaturation and deactivation with an intermediate conformational state existing on the path from native to denaturated cutinase. This intermediate was clearly detected by an increase in activity and shows only minor conformational changes relative to the native state. At W0 20, the size of the empty water-pool was larger than cutinase and the data was well described by a two-state model for both anionic and cationic reverse micelles. For AOT reverse micelles at W0 20, the intermediate state became a transient state and the deactivation and denaturation were described by a two-state model in which only native and denaturated cutinase were present. For CTAB/1-hexanol reverse micelles at W0 20, the native cutinase was in equilibrium with an intermediate state, which did not suffer denaturation. 1-Hexanol showed a stabilizing effect on cutinase in reverse micelles, contributing to the higher stabilities observed in the cationic CTAB/1-hexanol reverse micelles. Copyright 1998 John Wiley & Sons, Inc.

Deactivation and conformational changes of cutinase in reverse micelles

Deactivation data and fluorescence intensity changes were used to probe functional and structural stability of cutinase in reverse micelles. A fast deactivation of cutinase in anionic (AOT) reverse micelles occurs due to a reversible denaturation process. The deactivation and denaturation of cutinase is slower in small cationic (CTAB/1-hexanol) reverse micelles and does not occur when the size of the cationic reverse micellar water-pool is larger than cutinase. In both systems, activity loss and denaturation are coupled processes showing the same trend with time. Denaturation is probably caused by the interaction between the enzyme and the surfactant interface of the reversed micelle. When the size of the empty reversed micelle water-pool is smaller than cutinase (at W0 5, with W0 being the water:surfactant concentration ratio) a three-state model describes denaturation and deactivation with an intermediate conformational state existing on the path from native to denaturated cutinase. This intermediate was clearly detected by an increase in activity and shows only minor conformational changes relative to the native state. At W0 20, the size of the empty water-pool was larger than cutinase and the data was well described by a two-state model for both anionic and cationic reverse micelles. For AOT reverse micelles at W0 20, the intermediate state became a transient state and the deactivation and denaturation were described by a two-state model in which only native and denaturated cutinase were present. For CTAB/1-hexanol reverse micelles at W0 20, the native cutinase was in equilibrium with an intermediate state, which did not suffer denaturation. 1-Hexanol showed a stabilizing effect on cutinase in reverse micelles, contributing to the higher stabilities observed in the cationic CTAB/1-hexanol reverse micelles. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:380–386, 1998.

Triglyceride hydrolysis and stability of a recombinant cutinase from Fusarium solani in AOT-iso-octane reversed micelles.

A recombinant cutinase from Fusarium solani was encapsulated in AOT reversed micelles. Physicochemical parameters of the system were optimized relative to triolein hydrolysis. Kinetic studies of triglyceride hydrolysis showed a decrease in specificity with increase of the acyl chain length. Stability of cutinase in the system under study is lower than in aqueous solution and decreases with increase in the water content in the system (W0 = [H2O]/[AOT]). The products of triolein hydrolysis had little effect on the cutinase stability. Although glycerol did not alter the stability, oleic acid decreases the enzyme stability. The increase in log P of solvent (from iso-octane to n-dodecane) decreased the stability. Deactivation profiles were fitted with the Henley and Sadana model (1).

Synthesis of fatty acid esters by a recombinant cutinase in reversed micelles

Fusarium solani pisi recombinant cutinase, solubilized in AOT/isooctane-reversed micelles, was used to catalyze the esterification of fatty acids with aliphatic alcohols. Some relevant parameters for the enzyme activity such as pH, W(o) (water/surfactant molar ratio), temperature, and substrate concentration were optimized. Maximal specific activity was obtained for hexanol. The cutinase showed selectivity for short-chain fatty acids. The stability of the microencapsulated cutinase was investigated at various concentrations of water and different values of pH. Oleic acid had a negative effect on the cutinase stability, while hexanol proved to be a strong stabilizer increasing the half-life of the enzyme about 45 times.