Fusarium solani pisi recombinant cutinase partitioning in peg/potassium phosphate aqueous two-phase systems

Summary An aqueous two-phase system of polyethylene glycol (PEG) and potassium phosphate was developed for extraction of a cutinase from cell debris of a recombinant Escherichia coli strain. Basic studies to identify the primary factors which affect cutinase partition, namely the influence of polymer molecular weight, polymer concentration and pH were carried out using a purified preparation of the cutinase. The enzyme partition coefficient was enhanced with decreasing PEG molecular weight, increasing tie-line length and pH.     

Fusarium solani pisi cutinase

Cutinase from Fusarium solani pisi has been studied extensively with respect to its structural and functional properties. The crystal structure of the enzyme was solved to high atomic resolution (1 angstrom), while data on structural dynamics have been obtained from detailed NMR studies. Functional data were mainly derived from kinetic studies using substrate analogues that simplify the kinetic behaviour. The properties of wild-type cutinase are reviewed and discussed in relation with the effects brought about by site-directed variants of the enzyme.     

Integration of production and aqueous two-phase systems extraction of extracellular Fusarium solani pisi cutinase fusion proteins

Genetic engineering was integrated with the production and purification of Fusarium solani pisi cutinases, in order to obtain the highest amount of enzyme activity units, after purification. An aqueous two-phase system (ATPS) of polyethylene glycol 3350, dipotassium phosphate and whole broth was used for the extraction of three extracellular cutinases expressed in Saccharomyces cerevisiae. The production/extraction process was evaluated regarding cutinases secretion in the medium, partition behaviour and extraction yields in the ATPS. The proteins studied were cutinase wild type and two fusion proteins of cutinase with the tryptophane-proline (WP) fusion tags, namely (WP)(2) and (WP)(4). The (WP)(4) fusion protein enabled a 300-fold increase of the cutinase partition coefficient when comparing to the wild type. However, the secretion of the fusion proteins was lower than of the wild type cutinase secretion. A batch extraction strategy was compared with a continuous extraction in a perforated rotating disc contactor (PRDC). The batch and continuous systems were loaded with as much as 60% (w/w) whole cultivation broth. The continuous extraction strategy provided a 2.5 higher separation capacity than the batch extraction strategy. Considering the integrated process, the cutinase-(WP)(2) proved to lead to the highest product activity, enabling five and six times more product activity than the wild type and the (WP)(4) fusion proteins, respectively.     

Effect of salts and surfactants on the partitioning of Fusarium solani pisi cutinase in aqueous two-phase systems of thermoseparating ethylene oxide/propylene oxide random copolymer and hydroxypropyl starch

An aqueous two-phase system composed by a thermoseparating random copolymer of ethylene oxide/propylene oxide 50/50 (%w/w), Breox, and hydroxypropyl starch – Reppal PES 100 was evaluated for the partitioning of Fusarium solani pisi recombinant cutinase. The effect of several additives on the partitioning of pure cutinase was evaluated. Micelles of sodium dodecanoate provided a ten-fold increase of the partitioning coefficient (K=9) and recovery yields of 60-75%. The phase diagrams of the systems composed of Breox, Reppal and sodium dodecanoate were determined and it was found that in systems with high surfactant concentrations, the binodal was moved to lower polymer concentrations, enabling a two-phase system with 6% (w/w) of each polymer.     

Stability of a Fusarium solani pisi recombinant cutinase in phosphatidylcholine reversed micelles

Summary A Fusarium solani pisi recombinant cutinase solubilized in phosphatidylcholine/isooctane reversed micelles was used to catalyse the esterification reaction of butyric acid with 2-butanol at pH 10.7. The influence of temperature, Wo and substrates on lipase stability was evaluated. The enzyme displays a better stability, with a half-life over 125 days, at a temperature of 22°C and for a low water content (W_O= 6.5). Butyric acid increased the cutinase deactivation (t_1/2=0.56h), while 2-butanol led to a similar half-life (t_1/2=14h) as without substrate.     

Screening of stable cutinase from Fusarium solani pisi using plasmid display system

Although enzymes are potential candidates for industrial catalysts, their industrial applications have been limited because they are easily deactivated under harsh operational conditions. In this study, a plasmid display system was used for the screening of stable cutinase in organic solvent (20% acetonitrile) and at high temperature. The fusion proteins were expressed and bound to specific DNA sequences on the encoding plasmids. Proteolysis resistance was used as a selection tool, where well-folded proteins are more resistant to the protease digestion than poorly-folded proteins. Stable mutants, identified to be I183T, I183F, and A56V, were screened in the organic solvent and at high temperature. The I183T and I183F mutants were more stable than the A56V mutant in 20% acetonitrile, while the A56V mutant was superior to the I183T and I183F mutants at high temperature. Molecular modeling was performed in order to investigate the residual characteristics of the stable mutants; secondary structure, residual solvation energy, residual ??-carbon flexibility, number of hydrogen bonds, number of neighboring amino acids, ratio of exposed/buried residue, and surface area. This analysis provided some guidelines for increased stability.     

Direct evidence for the presence of beta-hydroxyphenylalanine and beta-hydroxytyrosine in cutinase from Fusarium solani pisi

Extracellular cutinase induced by cutin hydrolysate in glucose-grown Fusarium solani f. pisi was isolated in electrophoretically homogeneous form. This enzyme was similar to cutinase I generated by cutin-grown cells in its catalytic properties such as pH optimum, substrate specificity, and inactivation by “active serine”-directed reagents. Its molecular weight was 26,300 and this enzyme had a much larger content of serine and threonine residues than that found in cutinase from the cutin-grown cells. The hydrolysate-induced enzyme was a glycoprotein containing 6% carbohydrates. Alkaline NaB³H₄ treatment of the protein generated labeled protein and labeled carbohydrates. Analyses of the hydrolysates of these labeled products showed that alanine, α-aminobutyrate, phenylalanine, and tyrosine in the protein were labeled strongly suggesting that serine, threonine, β-hydroxyphenylalanine, and β-hydroxytyrosine were involved in O-glycosidic linkages in this protein. The protein hydrolysate also contained labeled gulonic acid, suggesting that d-glucuronic acid was attached to the protein via a base stable linkage, presumably an amide linkage at the N-terminus. The labeled reduced carbohydrates were identified by ion-exchange, thin-layer, gas-liquid, and high-performance liquid chromatographic techniques as mannitol, arabitol, gulonic acid, and 2-aminosorbitol. Thus mannose, arabinose, glucuronic acid, and glucosamine (possibly N-acetylated) were attached O-glycosidically to the hydroxyamino acids. Induction of cutinase by cutin hydrolysate in the presence of tritiated phenylalanine gave labeled cutinase. Cleavage of the O-glycosidically attached carbohydrates by anhydrous HF, followed by enzymatic hydrolysis of the labeled protein, gave rise to labeled amino acids, which upon analysis with an amino acid analyzer revealed four radioactive components. Two of them were identified as phenylalanine and tyrosine, while the other two cochromatographed with authentic β-hydroxyphenylalanine and β-hydroxytyrosine not only by the amino acid analyzer but also upon thin-layer chromatography. These results constitute the first direct evidence for the presence of the novel β-hydroxyaromatic amino acids in a protein.     

NMR studies of Fusarium solani pisi cutinase in complex with phosphonate inhibitors.

The backbone dynamics of Fusarium solani pisi cutinase in complex with a phosphonate inhibitor has been studied by a variety of nuclear magnetic resonance experiments to probe internal motions on different time scales. The results have been compared with dynamical studies performed on free cutinase. In solution, the enzyme adopts its active conformation only upon binding the inhibitor. While the active site Ser120 is rigidly attached to the stable alpha/beta core of the protein, the remainder of the binding site is very flexible in the free enzyme. The other two active site residues Asp175 and His188 as well as the oxyanion hole residues Ser42 and Gln121 are only restrained into their proper positions upon binding of the substrate-like inhibitor. The flap helix, which opens and closes the binding site in the free molecule, is also fixed in the cutinase-inhibitor complex. Our results are in contrast with the X-ray analysis results, namely that in the protein crystal, free cutinase has a well-defined active site and a preformed oxyanion hole and that it does not need any rearrangements to bind its substrate. Our solution studies show that cutinase does need conformational rearrangements to bind its substrate, which may form the rate-limiting step in catalysis.     

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.     

The interaction of Fusarium solani pisi cutinase with long chain spin label esters

We here present a study of the interaction between the Fusarium solani pisi cutinase mutant S120A and spin-labeled 4,4-dimethyloxazoline-N-oxyl-(DOXYL)-stearoyl-glycerol substrates in a micellar system. The interaction is detected by NMR measuring changes in chemical shift for 1H and 15N as well as relaxation parameters for backbone 1H (T1) and 15N (T1, T2) atoms as well as for side chain methyl groups 1H (T1). The detected interaction shows a weak binding of cutinase to the lipid micelles. Structural and mobility changes are located inside and around the active site, its flanking loops, and the oxyanion hole, respectively. Relaxation changes in the amino acid pairs Ser 92, Ala 93 and Thr 173, Gly 174 positioned at the edge of each of the active site flanking loops make these residues prime candidates for hinges, allowing for structural rearrangement during substrate binding. The cutinase mutant S120A used carries a 15 amino acid pro-peptide; the significance of this pro-peptide was so far undetermined. We show here that the pro-peptide is affected by the presence of the micellar substrate. Relaxation enhancements indicative of spatial proximity between the DOXYL group in the lipid chain and some hydrophobic residues surrounding the active site could be found.     

Crystallization and preliminary X-ray study of a recombinant cutinase from Fusarium solani pisi

Recombinant cutinase from Fusarium solani pisi is expressed and excreted with very high yields in Escherichia coli cultures. Cutinase was crystallized at 20 degrees C using the vapour diffusion technique, with polyethylene glycol 6000 as precipitant. Best crystals were obtained at pH 7.0 with polyethylene glycol 6000 as precipitant. Best crystals were obtained at pH 7.0 with polyethylene glycol at 15 to 20%. They are monoclinic, with space group P2(1) and cell dimensions a = 35.1 A, b = 67.4 A, c = 37.05 A and beta = 94.0 degrees; they diffract beyond 1.5 A resolution. The asymmetric unit contains one molecule of 22,000 Da (Vm = 1.98 A3/Da; 38% water).     

Genetic engineering of protein-peptide fusions for control of protein partitioning in thermoseparating aqueous two-phase systems

Genetic engineering has been used for the fusion of peptides, with different length and composition, on a protein to study the effect on partitioning in aqueous two-phase systems containing thermoseparating polymers. Peptides containing 2-6 tryptophan residues or tryptophan plus 1-3 lysine or aspartate residues, were fused near the C-terminus of the recombinant protein ZZT0, where Z is a synthetic IgG-binding domain derived from domain B in staphylococcal protein A. The partitioning behavior of the peptides and fusion proteins were studied in an aqueous two-phase system composed of dextran and the thermoseparating ethylene oxide-propylene oxide random copolymer, EO30PO70. The zwitterionic compound beta-alanine was used to reduce the charge-dependent salt effects on partitioning, and to evaluate the contribution to the partition coefficient from the amino acid residues, Trp, Lys, and Asp, respectively. Trp was found to direct the fusion proteins to the EO-PO copolymer phase, while Asp and Lys directed them to the dextran phase. The effect of sodium perchlorate and triethylammonium phosphate on the partitioning of the fusion proteins was also studied. Salt effects were directly proportional to the net charge of the fusion proteins. Sodium perchlorate was found to be 3.5 times more effective in directing positively charged proteins to the EO-PO copolymer phase compared to the effect of triethyl ammonium phosphate on negatively charged proteins. An empirical correlation has been tested where the fusion protein partitioning is a result of independent contributions from unmodified protein, fused peptide, and salt effects. A good agreement with experimental data was obtained which indicates the possibility, by independent measurements of partitioning of target protein and fusion peptide, to approximately predict the fusion protein partitioning.     

Scale-up of recombinant cutinase recovery by whole broth extraction with PEG-phosphate aqueous two-phase

A whole broth extraction using an aqueous two-phase system (ATPS) composed by 5% (w/w) PEG 3350 and 15% (w/w) phosphate was used for the scale-up extraction and isolation of a recombinant Fusarium solani pisi cutinase, an extracellular mutant enzyme expressed in Saccharomyces cerevisiae, containing a fusion peptide (WP)₄. The experiments were carried out at three different scales (10 ml, 1 l and 30 l). Mixing time and stirrer speed were evaluated at lab scale (1 l) with two different system compositions. Stirrer speed between 400 and 800 rpm and mixing time between 2 and 5 min led to the highest recoveries of cutinase. In all cases, inclusive of pilot scale (30 l), the equilibrium was reached after a few minutes. The performance of ATPS was reproducible within the scale range of 0.010–30 l and provided a standard deviation of the yield lower than 8%, leading to (i) a partition coefficient over 50, (ii) a yield over 95% and (iii) a concentration factor over 5. The fusion of the peptide (WP)₄ to the cutinase protein enabled a 400 increase of the partition coefficient relative to the wild-type strain.     

Production of Fusarium solani f. sp. pisi cutinase in Fusarium venenatum A3/5

Fusarium venenatum A3/5 was transformed using the Aspergillus niger expression plasmid, pIGF, in which the coding sequence for the F. solani f. sp. pisi cutinase gene had been inserted in frame, with a KEX2 cleavage site, with the truncated A. niger glucoamylase gene under control of the A. niger glucoamylase promoter. The transformant produced up to 21 U cutinase l⁻¹ in minimal medium containing glucose or starch as the primary carbon source. Glucoamylase (165 U l⁻¹ or 8 mg l⁻¹) was also produced. Both the transformant and the parent strain produced cutinase in medium containing cutin.     

Tryptophan mediated photoreduction of disulfide bond causes unusual fluorescence behaviour of Fusarium solani pisi cutinase.

The fluorescence signal of the single tryptophan residue (Trp69) of Fusarium solani pisi cutinase is highly quenched. However, prolonged irradiation of the enzyme in the tryptophan absorption band causes an increase of the tryptophan fluorescence quantum yield by an order of magnitude. By using a combination of NMR spectroscopy and chemical detection of free thiol groups with a sulfhydryl reagent we could unambiguously show that the unusual fluorescence behaviour of Trp69 in cutinase is caused by the breaking of the disulfide bond between Cys31 and Cys109 upon irradiation, while the amide-aromatic hydrogen bond between Ala32 and Trp69 remains intact. This is the first example of tryptophan mediated photoreduction of a disulfide bond in proteins.     

Ovomucoid partitioning in aqueous two-phase systems

This study evaluated the partitioning of ovomucoid from egg white, in aqueous two-phase systems (ATPS) composed of PEG 1500 and inorganic salt (lithium sulfate, sodium sulfate, magnesium sulfate, sodium carbonate or sodium citrate) at 25 °C. The results showed a great effect of the electrolyte nature on the partition coefficient. The partition coefficient value ranges from 0.02 to 6.0. The highest partition coefficients were obtained from systems composed of sodium carbonate and the lowest in systems composed of magnesium sulfate. In the system containing magnesium sulfate, a recovery percentage greater than 90% was obtained.     

Ovomucoid partitioning in aqueous two-phase systems

This study evaluated the partitioning of ovomucoid from egg white, in aqueous two-phase systems (ATPS) composed of PEG 1500 and inorganic salt (lithium sulfate, sodium sulfate, magnesium sulfate, sodium carbonate or sodium citrate) at 25 °C. The results showed a great effect of the electrolyte nature on the partition coefficient. The partition coefficient value ranges from 0.02 to 6.0. The highest partition coefficients were obtained from systems composed of sodium carbonate and the lowest in systems composed of magnesium sulfate. In the system containing magnesium sulfate, a recovery percentage greater than 90% was obtained.     

Extraction of peptide tagged cutinase in detergent-based aqueous two-phase systems

Detergent-based aqueous two-phase systems have the advantage to require only one auxiliary chemical to induce phase separation above the cloud point. In a systematic study the efficiency of tryptophan-rich peptide tags was investigated to enhance the partitioning of an enzyme to the detergent-rich phase using cutinase as an example. Up to 90% enzyme activity could be extracted in a single step from whole broth of recombinant Saccharomyces cerevisiae expressing cutinase variants carrying a (WP)₄ tag. In contrast, the extraction yield of wild type cutinase was 2–3% only. The detergent concentration and the temperature are the main parameters to optimize the extraction yield. Considering availability, extraction yields, and price the detergent Agrimul NRE 1205 served best for enzyme recovery.     

Genetic engineering of the Trichoderma reesei endoglucanase I (Cel7B) for enhanced partitioning in aqueous two-phase systems containing thermoseparating ethylene oxide--propylene oxide copolymers

Endoglucanases (endo-1,4-beta-D-glucan-4-glucanohydrolase, EC 3.2.1.4) are industrially important enzymes. In this study endoglucanase I (EGI or Cel7B) of the filamentous fungi Trichoderma reesei has been genetically engineered to investigate the influence of tryptophan rich peptide extensions (tags) on partitioning in an aqueous two-phase model system. EGI is a two-domain enzyme and is composed of a N-terminal catalytic domain and a C-terminal cellulose binding domain, separated by a linker. The aim was to find an optimal tag and fusion position, which further could be utilised for large scale extractions. Peptide tags of different length and composition were attached at various localisations of EGI. The fusion proteins were expressed from T. reesei with the use of the gpdA promoter from Aspergillus nidulans. Variations in secreted levels between the engineered proteins were obtained. The partitioning of EGI in an aqueous two-phase system composed of a thermoseparating ethylene oxide-propylene oxide random copolymer (EO(50)PO(50)) and dextran, could be significantly improved by relatively minor genetic engineering. The (Trp-Pro)(4) tag added after a short stretch of the linker, containing five proline residues, gave in the highest partition coefficient of 12.8. The yield in the top phase was 94%. The specific activity was 83% of the specific activity of unmodified EGI on soluble substrate. The efficiency of a tag fused to a protein is shown by the tag efficiency factor (TEF). A hypothetical TEF of 1.0 would indicate full tag exposure and optimal contribution to the protein partitioning by the fused tag. The location of the fusion point after the sequence of five proline residues in the linker of EGI is the most beneficial in two-phase separation. The highest TEF (0.97) was obtained with the (Trp-Pro)(2) tag at this position, indicating full exposure and intactness of the tag. However, the peptide tag composed of (Trp-Pro)(4) improved the partition properties the most but had lower TEF in comparison to (Trp-Pro)(2).