Genetic engineering of the Fusarium solani pisi lipase cutinase for enhanced partitioning in PEG-phosphate aqueous two-phase systems

The Fusarium solani pisi lipase cutinase has been genetically engineered to investigate the influence of C-terminal peptide extensions on the partitioning of the enzyme in PEG-salt based aqueous two-phase bioseparation systems. Seven different cutinase lipase variants were constructed containing various C-terminal peptide extensions including tryptophan rich peptide tags ((WP)(2) and (WP)(4)), positively ((RP)(4)) and negatively ((DP)(4)) charged tags as well as combined tags with tryptophan together with either positively ((WPR)(4)) or negatively ((WPD)(4)) charged amino acids. The modified cutinase variants were stably produced in Escherichia coli as secreted to the periplasm from which they were efficiently purified by IgG-affinity chromatography employing an introduced N-terminal IgG-binding ZZ affinity fusion partner present in all variants. Partitioning experiments performed in a PEG 4000/sodium phosphate aqueous two-phase system showed that for variants containing either (WP)(2) or (WP)(4) peptide extensions, 10- to 70-fold increases in the partitioning to the PEG rich top-phase were obtained, when compared to the wild type enzyme. An increased partitioning was also seen for cutinase variants tagged with both tryptophans and charged amino acids, whereas the effect of solely charged peptide extensions was relatively small. In addition, when performing partitioning experiments from cell disintegrates, the (WP)(4)-tagged cutinase showed a similarly high PEG-phase partitioning, indicating that the effect from the peptide tag was unaffected by the background of the host proteins. Taken together, the results show that the partitioning of the recombinantly produced cutinase model enzyme could be significantly improved by relatively minor genetic engineering and that the effects observed for purified proteins are retained also in an authentic whole cell disintegrate system. The results presented should be of general interest also for the improvement of the partitioning properties of other industrially interesting proteins including bulk enzymes.     

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.     

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.     

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.     

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).     

Predicting the partition coefficients of a recombinant cutinase in polyethylene glycol/phosphate aqueous two-phase systems

A model for the prediction of protein partition coefficients in aqueous two-phase systems has been developed. This model accounts for both charge-independent and electrostatic effects. The determination of nonelectrostatic effects was based on the model of Eiteman and Gainer for uncharged solutes while the electrostatic contribution was computed using TITRA, a program that uses a continuum electrostatic model to treat charge interactions in proteins and considers the effect of pH and ionic strength. The partition coefficients of Fusarium solani pisi recombinant cutinase have been satisfactorily predicted in polyethylene glycol (PEG) 1000 and phosphate aqueous two-phase systems at a pH range of 6.0-9.0. The model failed to predict the enzyme partitioning behavior at pH 4.5. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 248-257, 1997.     

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.     

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.     

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.     

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.     

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.     

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.     

Purification of recombinant phenylalanine dehydrogenase by partitioning in aqueous two-phase systems

This study presents the partitioning and purification of recombinant Bacillus badius phenylalanine dehydrogenase (PheDH) in aqueous two-phase systems (ATPS) composed of polyethylene glycol 6000 (PEG-6000) and ammonium sulfate. A single-step operation of ATPS was developed for extraction and purification of recombinant PheDH from E. coli BL21 (DE3). The influence of system parameters including; PEG molecular weight and concentration, pH, (NH(4))(2)SO(4) concentration and NaCl salt addition on enzyme partitioning were investigated. The best optimal system for the partitioning and purification of PheDH was 8.5% (w/w) PEG-6000, 17.5% (w/w) (NH(4))(2)SO(4) and 13% (w/w) NaCl at pH 8.0. The partition coefficient, recovery, yield, purification factor and specific activity values were of 92.57, 141%, 95.85%, 474.3 and 10424.97 U/mg, respectively. Also the K(m) values for L-phenylalanine and NAD(+) in oxidative deamination were 0.020 and 0.13 mM, respectively. Our data suggested that this ATPS could be an economical and attractive technology for large-scale purification of recombinant PheDH.     

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.     

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.     

Partitioning of beta-galactosidase fusion proteins in PEG/potassium phosphate aqueous two-phase systems.

Four different beta-galactosidase fusion proteins have been partitioned in poly(ethylene glycol) (PEG) 4000/potassium phosphate aqueous two-phase systems. The partition coefficients (K) of staphylococcal protein A-beta-galactosidase (SpA beta gal) (K = 3.5) and staphylococcal protein A-streptococcal protein G-beta-galactosidase (AG beta gal) (K = 2.8) were compared with the partition coefficients of their constituent molecules, beta-galactosidase, SpA, and protein AG. It was found that by fusing beta-galactosidase to the smaller proteins SpA and protein AG, their partition coefficients were increased four to five times. Experimental data were fitted into, and found to agree with, the Albertsson partition model of interacting molecules. The compatibility with PEG and potassium phosphate of beta-galactosidase, SpA, and two different versions of the SpA beta gal protein, displayed as precipitation curves, showed a relationship to the protein partition coefficients in PEG/potassium phosphate systems. High solubility in one phase component was accompanied by preferential partitioning to the phase rich in the same component in the PEG/potassium phosphate system. Also, a changed linker region in SpA beta gal resulted in a more soluble protein. This, together with the improved K values of the target proteins by fusion, shows that it is possible to use beta-galactosidase as an affinity handle.     

Protein partitioning in two-phase aqueous polymer systems

Theories of protein partitioning in two-phase polymer systems which account for the effects of different aspects of system composition-such as the choice of materials, protein size, polymer molecular weight, polymer concentration, salt concentration, and affinity ligands-are reviewed. Although the present models provide some information about specific aspects of partitioning, a comprehensive and fundamental theory which can be used to predict protein partitioning behavior has not yet been developed. Some recommendations for future work are given.