Studies on the extraction and back-extraction of a recombinant cutinase in a reversed micellar extraction process

This work deals with the extraction and back-extraction of a recombinant cutinase using AOT reversed micelles in isooctane. The effect of pH, ionic strength, AOT concentration and temperature on the extraction and back-extraction of the cutinase was investigated. High extraction (97%) of the cutinase was achieved at pH 7.0 with a 50 mM Tris-HCl buffer solution containing 100 mM KCl, but a low activity was detected in the reversed micellar phase. At pH 9.0, cutinase was extracted (75%) to the reversed micelles with higher activity. Cutinase was recovered (50%) from a reversed micellar phase (100 mM AOT/isooctane) into a 50 mM Tris-HCl buffered solution at pH 9.0 with 100 mM KCl, and 20°C. Protein and cutinase activity global yields of 38 and 45%, respectively, were obtained for the global process, extraction and back-extraction steps, using low ionic strength, pH 9.0, 100 mM AOT and 20°C.Maria das Graças Carneiro da Cunha acknowledges a Ph.D. fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Centro de Pesquisas Aggeu Magalhães, Brasil. This work was partly financed by the BRIDGE Programme (Contract BIOT-CT91-0274(DTEE)).     

Liquid-liquid extraction of a recombinant cutinase from fermentation media with AOT reversed micelles

This work reports the extraction and back-extraction of an intracellular recombinant cutinase from complex biological media using AOT reversed micelles in isooctane. Cutinase was recovered from different complex media namely, fermentation broths and supernatants after cell disruption by osmotic shock and sonication. The application of the AOT reversed micellar system to the extraction of cutinase allowed activity yields and purification factors ranging from about 5% to 50% and 1.2 to 10.2, respectively, depending on the biological medium.Maria das Graças Carneiro da Cunha, from ITEP-Instituto Tecnológico do Estado de Pernambuco, acknowledges a Ph.D fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Centro de Pesquisa Aggeu Magalhães, Recife — PE — Brasil. E. P. Melo thanks Junta Nacional de Investigação Científica, Lisboa, Portugal, for providing a Ph.D. fellowship. The scientific support given by Prof. Sílvia M. B. Costa for the spectroscopic data and further discussions are particularly acknowledged.This work was partly financed by the BRIDGE and BIOTECHNOLOGY Programmes (Contracts BIOT-CT91-0274(DTEE) and BIOT 2 CT-943016).     

Recovery of a recombinant cutinase with reversed micelles in a continuous perforated rotating disc contactor

Summary A continuous perforated rotating disc contactor was used for the extraction of a recombinant cutinase from an aqueous solution to a reversed micellar phase of AOT in isooctane. Cutinase was extracted to the organic phase with protein yield of 78% after 70 minutes of operation.     

A second polycaprolactone depolymerase from Fusarium, a lipase distinct from cutinase

Polycaprolactone (PCL), a synthetic polyester with applications in biodegradable plastics, is degraded by a variety of microorganisms, including fungal phytopathogens. These pathogens secrete cutinase, which hydrolyzes cutin, the polyester structural component of plant cuticle, releasing ω-hydroxy fatty acids that induce cutinase synthesis. Our laboratory previously reported that growth of Fusarium solani on PCL requires cutinase, which is active as a PCL depolymerase and induced by the products of its action on PCL. A mutant strain of F. solani in which the cutinase gene is deleted was unable to grow on PCL and did not secrete PCL depolymerase activity in the media tested. It is now shown that this mutant produces a PCL depolymerase in media containing lipase inducers. Wild-type strains also produce this second PCL depolymerase, which is induced by Tween 80 and tributyrin, but not by PCL or cutin. The second depolymerase shows interfacial activation, indicating that it is a lipase. PCL may thus be a substrate but not an inducer of depolymerases that degrade it, and screening microorganisms on medium with PCL as the sole source of carbon and energy may fail to reveal strains with active PCL depolymerases, because of the absence of an inducer. Surprisingly, Tween 80 induces both cutinase and lipase activities in wild-type F. solani. Received: 31 March 1998 / Received revision: 27 July 1998 / Accepted: 8 August 1998     

Purification of recombinant cutinase by extraction in an aqueous two-phase system facilitated by a fatty acid substrate

Purification of recombinant wild-type cutinase from the culture supernatant of Saccharomyces cerevisiae by extraction in aqueous two-phase system was investigated. The partition of the enzyme in a polyethylene glycol (PEG)-potassium phosphate system to the top phase was increased with lower molecular weight PEG. Enzyme partition in a 20% PEG/15% phosphate two-phase system was studied in the presence of detergents, fatty acids, and alcohols, respectively. Addition of 0.5% (w/w) butyrate increased the partition coefficient from 17 to 135 and the purification factor from 10 to 23. The effect of butyrate was also confirmed by using the countercurrent mode of extraction. Recovery of cutinase from the top phase was achieved by a secondary extraction into a new salt phase at a lower pH or a lower temperature. A specific interaction of butyrate to the active site of the enzyme was demonstrated by fluorescence spectroscopy. Size exclusion chromatography showed the cutinase-butyrate complex to be over two times the size of the free enzyme.     

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.     

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.     

Cutinase: from molecular level to bioprocess development

This review analyzes the role of cutinases in nature and their potential biotechnological applications. The cloning and expression of a fungal cutinase, Fusarium solani f. pisi, in Escherichia coli and Saccharomyces cerevisiae hosts are described. The three-dimensional structure of this cutinase is also analyzed and its function as a lipase is discussed and compared with other lipases. The biocatalytic applications of cutinase are described taking into account the preparation of different cutinase forms and the media in which the different types of reactions have been performed, namely hydrolysis, esterification, transesterification, and resolution of racemic mixtures. The stability of cutinase preparations is discussed and, in particular, the cutinase stability in anionic reversed micelles is analyzed considering the role of hexanol as a substrate, a cosurfactant, and a stabilizer. Process development, based on the operation of cutinase reactors, is also reviewed.     

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.     

流加发酵对重组Bacillus subtilis发酵生产角质酶的影响

为实现基因工程菌Bacillus subtilis WSHB06-07生产角质酶的高产,在3L发酵罐中考察了不同初糖浓度对菌体生长和产酶的影响,并在选择38 g/L初始蔗糖浓度的基础上,进行碳源的分批流加和恒速流加,结果表明发酵16 h开始流加碳源,采用总补糖量60g/L,蔗糖平均流速为4g/(L·h)的恒速补料方式,角质酶酶活在31h可达到最大545.87U/ml,比分批发酵酶活提高67.8%,并获得较高的角质酶生产强度,满足工业化生产要求。     

The effect of additives and mechanical agitation in surface modification of acrylic fibres by cutinase and esterase

The surface of an acrylic fibre containing about 7% of vinyl acetate was modified using Fusarium solani pisi cutinase and a commercial esterase, Texazym PES. The effect of acrylic solvents and stabilising polyols on cutinase operational stability was studied. The half-life time of cutinase increased by 3.5-fold with the addition of 15% N,N-dimethylacetamide (DMA) and by 3-fold with 1M glycerol. The impact of additives and mechanical agitation in the protein adsorption and in the hydrolysis of vinyl acetate from acrylic fabric was investigated. The hydroxyl groups produced on the surface of the fibre were able to react specifically with Remazol Brilliant Blue R (cotton reactive dye) and to increase the colour of the acrylic-treated fabric. The best staining level was obtained with a high level of mechanical agitation and with the addition of 1% DMA. Under these conditions, the raise in the acrylic fabric colour depth was 30% for cutinase and 25% for Texazym. The crystallinity degree, determined by X-ray diffraction, was not significantly changed between control samples and samples treated with cutinase. The results showed that the outcome of the application of these enzymes depends closely on the reaction media conditions.     

Hydrolysis of plant cuticle by plant pathogens. Purification, amino acid composition, and molecular weight of two isozymes of cutinase and a nonspecific esterase from Fusarium solani f. pisi.

The extracellular fluid of the plant pathogen, Fusarium solani f. pisi, grown on the plant cuticular polymer, cutin, was shown to contain cutinase and p-nitrophenyl palmitate hydrolase activities (R.E. Purdy and P.E. Kolattukudy (1973), Arch. Biochem. Biophys. 159, 61). From this extracellular fluid two isozymes of cutinase and a nonspecific esterase (p-nitrophenyl palmitate hydrolase) were isolated using Sephedex G-100 gel filtration, QAE-Sephadex chromatography, and SE-Sephedex chromatography. Phenolics contained in the extracellular fluid were found to be associated with the cutinase but not with the nonspecific esterase, and the phenolic materials were removed from cutinase at the QAE-Sephedex step. A 34-fold purification of the nonspecific esterase and a 6.5-fold purification of cutinase were achieved by the procedure described. The two isozymes of cutinase (I and II) and the nonspecific esterase were homogeneous as judged by polyacrylamide disc gel electrophoresis and sedimentation equilibrium centrifugation. Molecular weights of cutinase I, cutinase II, and the nonspecific esterase were determined by Sephedex G-100 gel filtration, sedimentation equilibrium centrifugation, amino acid composition, and sodium dodecyl sulfate polyacrylamide disc gel electrophoresis. The values obtained with these techniques agreed with each other and were about 22,000 for both cutinases and 52,000 for the nonspecific esterase. The dodecyl sulfate gel electrophoresis indicated that a small portion of cutinase II contained proteolylic clips, near the middle of the polypeptide chain, and that the nonspecific esterase might also have undergone some proteolylic modification. The amino acid composition of cutinase I was similar to that of cutinase II except for the presence of a larger number of tryptophan residues in the latter, while the amino acid composition of the nonspecific esterase showed more differences from that of either cutinase.     

Isolation of a Fusarium solani mutant reduced in cutinase activity and virulence.

Fusarium solani isolate T-8 produces an extracellular enzyme, cutinase, which catalyzes the degradation of cutin in the plant cuticle. Cutinase activity can be measured by the hydrolysis of either the artifical substrate, p-nitrophenylbutyrate (PNB), or radioactive cutin containing [14C]palmitic acid. In the present study, the culture filtrate contained basal levels of cutinase when T-8 was grown on acetate as a sole source of carbon. After mutagenesis, a cutinase-defective mutant (PNB-1) was identified by screening acetate-grown colonies for a loss of PNBase activity. The mutant possessed an 80 to 90% reduction in cutinase activity when grown for 3 to 5 days on acetate- or cutin-containing medium. Induction of cutinase by cutin or hydrolyzed cutin after growth on glucose medium was similarly reduced. Kinetic analysis indicated that cutinase from the mutant possessed a near normal Km for PNB and a 92% reduction in Vmax. Fluorography and Western blotting of 15% sodium dodecyl sulfate-polyacrylamide gels of separated 35S-labeled proteins from cutin induction medium revealed that in the mutant the 22,000-molecular-weight band corresponding to cutinase was reduced approximately 85%. The virulence of the mutant in a pea stem bioassay was decreased by 55% and was restored to nearly the parental level by the addition of purified cutinase. The data suggest that the mutant synthesizes reduced quantities of a functional and immunoreactive cutinase enzyme and that cutinase plays a critical role in infection. The PNB1 mutation may be within a regulatory gene or a promoter for cutinase.     

Synthetic affinity ligands as a novel tool to improve protein stability

Cutinase from Fusarium solani pisi is the model-system for a new approach to assess and enhance protein stability based on the use of synthetic triazine-scaffolded affinity ligands as a novel protein-stabilizing tool. The active site of cutinase is excluded from the main surface regions postulated to be involved in early protein's thermal unfolding events. Hence, these regions are suitable targets for binding complementary affinity ligands with a potential stabilizing effect. A random solid-phase combinatorial library of triazine-bisubstituted molecules was screened for binding cutinase by a rapid fluorescence-based method and affinity chromatography. The best binding substituents were combined with those previously selected by screening a rationally designed library. A second-generation solid-phase biased library was designed and synthesized, following a semi-rational methodology. A dual screening of this library enabled the selection of ligands binding cutinase with higher affinity while retaining its functionality. These compounds were utilized for thermostability assessment with adsorbed cutinase at 60 degrees C and pH 8.0. When bound to different types of ligands, the enzyme showed markedly distinct activity retention profiles, with some synthetic affinity ligands displaying a stabilizing effect on cutinase and others a clearly destabilizing effect, when compared with the free enzyme.     

Synthesis and characterization of a new cutinase substrate, 4-nitrophenyl (16-methyl sulfone ester) hexadecanoate

Phytopathogenic fungi penetrate plants by breaking down the cuticular barrier with cutinase. Cutinases are extracellular hydrolytic enzymes that degrade cutin, a polyester composed of hydroxy and epoxy fatty acids. Until now, cutinase has been recognized by its ability to release labeled cutin monomers or by a non-specific esterase assay based on the hydrolysis of p-nitrophenyl esters of short fatty acids. In this work, an insoluble p-nitrophenyl derivative was synthesized and purified, and its structure was determined to be 4-nitrophenyl (16-methyl sulfone ester) hexadecanoate (pNMSEH) by nuclear magnetic resonance (H+ NMR) analysis. pNMSEH was tested as a new cutinase substrate with Pseudomonas mandocino cutinase and porcine liver esterase. While a linear release over time of p-nitrophenol (pNP) was recorded in the presence of cutinase, no response was obtained with the esterase. The calculated kinetic parameters of pNMSEH hydrolysis by cutinase revealed a high specificity (Km=1.8mM), albeit a low catalytic rate (Vmax=10.5 micromol min(-l)l(-1)). This new synthetic substrate may be helpful for detecting and assaying cutinase activity in mixed solutions, such as crude fungal extracellular extracts.     

Purification and characterization of a 40.8-kDa cutinase in ungerminated conidia of Botrytis cinerea Pers.: Fr

Cytoplasmic soluble proteins from ungerminated conidia of Botrytis cinerea exhibited cutinase activity. A 40.8-kDa cutinase was purified to homogeneity from this crude conidial protein extract. This cutinase does not correspond either to constitutive or to induced lytic cutin enzymes already described by other authors. The possible role of this constitutive cutinase in the induction of other cutinolytic proteins in the early stages of infection of plants by B. cinerea is discussed.     

Integration of the production and the purification processes of cutinase secreted by a recombinant Saccharomyces cerevisiae SU50 strain

By expanded bed adsorption (EBA) it was possible to simultaneously recover and purify the heterologous cutinase directly from the crude feedstock. However, it was observed that in a highly condensed and consequently economically advantageous purification process as EBA, the cultivation step highly influences the following purification step. Thus, the yeast cultivation and cutinase purification by EBA cannot be considered as independent entities, and the understanding of the interactions between them are crucial for the development of a highly cost effective overall cutinase production process. From the cultivation strategies studied, one batch, one continuous and two fed-batch cultivations, the strategy that resulted in a more economical cutinase overall production process was a fed-batch mode with a feeding in galactose. This last cultivation strategy, exhibited the highest culture cutinase activity and bioreactor productivity, being obtained 3.8-fold higher cutinase activity and 3.0-fold higher productivity that could compensate the 40% higher cultivation medium costs when compared with a fed-batch culture with a feeding on glucose and galactose. Moreover, a 3.8-fold higher effective cutinase dynamic adsorption capacity and 3.8-fold higher effective purification productivity were obtained in relation to the fed-batch culture with the feeding on glucose and galactose. The cultivation strategy with a feeding on galactose, that presented 5.6-fold higher effective purification productivity, could also compensate the 32% effective adsorption capacity obtained with a continuous cultivation broth. Furthermore, a 205-fold higher cutinase activity, 24-fold higher bioreactor productivity and 6% of the cultivation medium costs were obtained in relation to the continuous culture.     

Impaired Secretion of a Hydrophobic Cutinase by Saccharomyces cerevisiae Correlates with an Increased Association with Immunoglobulin Heavy-Chain Binding Protein (BiP)

This study focuses on the different efficiencies of secretion of two fungal cutinases by Saccharomyces cerevisiae, a wild-type cutinase (CY000) and a hydrophobic mutant cutinase (CY028). Both cutinases are placed under control of the GAL7 promoter, by which the expression levels can be regulated. Wild-type cutinase was secreted at up to 25 mg per g (dry weight), while CY028 was secreted at a level of 2 mg per g (dry weight); this difference is nearly independent of the expression level. Pulse-chase experiments revealed that whereas CY000 cutinase is secreted, CY028 is irreversibly retained in the cell. Immunogold labelling followed by electron microscopy revealed colocalization of CY028 with immunoglobulin heavy-chain binding protein (BiP) in the endoplasmic reticulum (ER). The increase of wild-type cutinase expression did not result in higher levels of the molecular chaperone BiP, but BiP levels are raised by increased induction of the hydrophobic mutant cutinase. Immunoprecipitation studies showed that in contrast to the wild-type cutinase, the hydrophobic mutant cutinase interacts with BiP. These results indicate that the introduction of two exposed hydrophobic patches in cutinase results in a higher affinity for BiP which might cause the retention of this mutant cutinase in the ER.     

Induction of a Biopolyester Hydrolase (Cutinase) by Low Levels of Cutin Monomers in Fusarium solani f. sp. pisi

Cutin hydrolysate induced the production of an extracellular cutinase by glucose-grown Fusarium solani f. sp. pisi. The rate of production depended on the amount of cutin hydrolysate added up to 80 mug/ml, and saturation was attained at this level. Glucose was found to be a repressor of cutinase production. A radial immunodiffusion assay for cutinase was developed, and the induction of cutinase by cutin hydrolysate was confirmed by this direct assay. When cutinase was induced by cutin hydrolysate, exogenous labeled phenylalanine was incorporated into cutinase, which was shown to be the major (>70%) protein in the extracellular fluid. Induction of cutinase by cutin hydrolysate was not inhibited by actinomycin D and was stimulated ( approximately 100%) by cordycepin. Addition of cycloheximide with the inducer, or up to 12 h after the addition of the inducer, resulted in a nearly immediate cessation of cutinase production. Deoxyglucose, an inhibitor of proten glycosylation, inhibited the induction of cutinase by cutin hydrolysate. omega-Hydroxy fatty acids were more effective in inducing cutinase than any of the other more polar acids of cutin. Experiments with derivatives and analogues of omega-hydroxy C(16) acid indicated that a free hydroxyl group at the omega-position was the most important factor determining the cutinase-inducing activity. n-Aliphatic primary alcohols with 14 or more carbon atoms induced cutinase, and n-C(16) was the most effective inducer. These results strongly suggest that the monomers function as the chemical signal which induces the extracellular hydrolase.     

Construction and heterologous expression of a synthetic copy of the cutinase cDNA from Fusarium solani pisi

A copy of the cutinase cDNA from Fusarium solani pisi was constructed starting from synthetic oligonucleotides. For this construction three separate cassettes were made, which were subsequently assembled to form the cutinase gene. Heterologous expression of the synthetic cutinase gene and the subsequent secretion of the recombinant enzyme was achieved in Saccharomyces cerevisiae and Aspergillus awamori.