Cutinase Research Articles

Aspergillus nidulans: A Potential Resource of the Production of the Native and Heterologous Enzymes for Industrial Applications.

Aspergillus nidulans is a filamentous fungus that is a potential resource for industrial enzymes. It is a versatile fungal cell factory that can synthesize various industrial enzymes such as cellulases, β-glucosidases, hemicellulases, laccases, lipases, proteases, β-galactosidases, tannases, keratinase, cutinases, and aryl alcohol oxidase. A. nidulans has shown the potential to utilize low-cost substrates such as wheat bran, rice straw, sugarcane bagasse, rice bran, coir pith, black gram residue, and chicken feathers to produce enzymes cost-effectively. A. nidulans has also been known as a model organism for the production of heterologous enzymes. Several studies reported genetically engineered strains of A. nidulans for the production of different enzymes. Native as well as heterologous enzymes of A. nidulans have been employed for various industrial processes.

Candidate Genes for Aggressiveness in a Natural Fusarium culmorum Population Greatly Differ between Wheat and Rye Head Blight.

Fusarium culmorum is one of the species causing Fusarium head blight (FHB) in cereals in Europe. We aimed to investigate the association between the nucleotide diversity of ten F. culmorum candidate genes and field ratings of aggressiveness in winter rye. A total of 100 F. culmorum isolates collected from natural infections were phenotyped for FHB at two locations and two years. Variance components for aggressiveness showed significant isolate and isolate-by-environment variance, as expected for quantitative host-pathogen interactions. Further analysis of the isolate-by-environment interaction revealed the dominant role of the isolate-by-year over isolate-by-location interaction. One single-nucleotide polymorphism (SNP) in the cutinase (CUT) gene was found to be significantly (p < 0.001) associated with aggressiveness and explained 16.05% of the genotypic variance of this trait in rye. The SNP was located 60 base pairs before the start codon, which suggests a role in transcriptional regulation. Compared to a previous study in winter wheat with the same nucleotide sequences, a larger variation of pathogen aggressiveness on rye was found and a different candidate gene was associated with pathogen aggressiveness. This is the first report on the association of field aggressiveness and a host-specific candidate gene codifying for a protein that belongs to the secretome in F. culmorum.

A stress-responsive Escherichia coli protein, CysQ is a highly effective solubility enhancer for aggregation-prone heterologous proteins

When used as an N-terminal fusion expression partner, the Escherichia coli stress-responsive protein, CysQ dramatically increased the cytoplasmic solubility of various aggregation-prone heterologous proteins: Pseudomonas putida cutinase (CUT), human granulocyte colony-stimulating factor (hG-CSF), human ferritin light chain (hFTN-L), arginine deiminase (ADI), human interleukin-2 (IL2), human activation induced cytidine deaminase (AID), and deletion mutant of human glutamate decarboxylase (GAD448-585). As compared with well-known fusion tags such as glutathione-S-transferase (GST) and maltose-binding protein (MBP), the performance of CysQ as solubility enhancer was evidently better than GST and was similar to or better than MBP for the seven heterologous proteins above. This is likely due to the intrinsic ability of CysQ to form its native conformation, probably promoting the binding of molecular chaperones during the folding of CysQ-fusion protein. When used as a substrate, p-nitrophenyl butyrate (PNB) was successfully hydrolyzed to p-nitrophenol by CysQ–CUT fusion mutant. Even after CysQ was removed, the solubility of hFTN-L and hG-CSF, the secondary structure of hG-CSF, and self-assembly activity of hFTN-L were successfully maintained. Conclusively, it seems that CysQ is a highly effective solubility enhancer and fusion expression partner for the production of a variety of bio-active recombinant proteins.

The relationship between cutinases and the pathogenicity/virulence of Fusarium solani in potato tubers

Cutinase activity was spectrophotometrically determined in the culture supernatants of 40 isolates of Fusarium solani , obtained in Iran from various biological origins and grown in a minimal medium with 0.4% cutin as the only carbon source. Enzymatic activities, which ranged from 0 to 488 nmol min-1 mL-1, were related to the pathogenicity or virulence of the fungal isolates, determined on potato tubers using a 0–5 disease severity scale. Cutinase activity was either not detected at all or was very low in the non-pathogenic isolates, whereas it was directly correlated with the virulence of the pathogenic isolates (Radj 2 = 0.97), with an increase in cutinase activity of about 100 units corresponding to a one-point increase in the disease severity scale. SDS-PAGE analysis revealed that non-pathogenic F. solani isolates did not produce a cutinase band, while pathogenic isolates, with various degrees of virulence, produced single or double peptide bands with molecular weights of 20–23 kDa. We conclude that enzyme activity can be used as a predictive marker of the pathogenicity and virulence of F. solani isolates obtained from various hosts.

Inoculum padronization for the production of cutinase by Fusarium oxysporum

Cutinase e uma enzima versatil, que apresenta propriedades interessantes para aplicacao em processos industriais. O uso desta enzima em larga escala depende do desenvolvimento de um sistema de producao eficiente e de baixo custo. Uma das etapas mais importantes em um processo de fermentacao e a padronizacao do inoculo. Neste estudo, houve uma associacao estatisticamente significativa entre a producao de cutinase por Fusarium oxysporum e tamanho do inoculo e pH do meio PDA. As maiores atividades de cutinase foram 19,1 U/mL em PDA com pH 7,0 e 22,72 U/mL empregando um inoculo de 12,72 x 107 esporos/mL. As caracteristicas macroscopicas das colonias de Fusarium oxysporum mostraram alteracoes em funcao do pH do meio, com as maiores atividades sendo registradas em presenca de colonias brancas com aspecto cotonoso.

Transport proteins PotD and Crr of Escherichia coli, novel fusion partners for heterologous protein expression

The Escherichia coli proteome response to the stressor GdnHCl was analyzed through 2-dimensional gel electrophoresis (2-DE). We identified PotD (spermidine/putrescine-binding periplasmic protein) and Crr [glucose-specific phosphotransferase (PTS) enzyme IIA component] as a stress-responsive protein. Even under a stress situation where the total number of soluble proteins decreased by about 10%, 3.5- and 2.2-fold increase was observed in the synthesis of PotD and Crr, respectively. As fusion partners, PotD and Crr dramatically increased the solubility of many aggregation-prone heterologous proteins [e.g. human minipro-insulin (mp-INS), human epidermal growth factor (EGF), human prepro-ghrelin (ppGRN), human interleukin-2(hIL-2), human activation induced cytidine deaminase (AID), human glutamate decarboxylase (GAD 448–585), Pseudomonas putida cutinase (CUT), human ferritin light chain (hFTN-L), human granulocyte colony-stimulating factor (G-CSF), and cold autoinflammatory syndrome1 protein (NALP3) Nacht domain (NACHT)] in the E. coli cytoplasm. Presumably PotD and Crr were very effective in shielding interactive surfaces of heterologous proteins associated with non-specific protein–protein interactions leading to the formation of inclusion bodies most likely due to intrinsic high folding efficiency, chaperone-like activity, or a combination of both factors. Both the stress-induced proteins were well suited for the production of a biologically active fusion mutant of P. putida cutinase that can be expected to be of biotechnological and commercial interest.

Substrates for Cell Adhesion Prepared via Active Site-Directed Immobilization of a Protein Domain

Recent studies in basic cell biology and bioengineering call for model substrates that present active proteins, with control over protein density, pattern, and orientation, to more directly mimic the natural extracellular matrix. Herein we demonstrate a strategy for controlled, irreversible immobilization of a cell adhesion protein domain onto an otherwise bioinert substrate with well-defined protein orientation and density. Our approach uses a tri(ethylene glycol)-terminated self-assembled monolayer presenting a phosphonate ligand that is used to immobilize an engineered fusion protein. One component of the fusion protein, the 22 kDa serine esterase cutinase, reacts with the surface-bound ligand to form a site-specific covalent adduct, and the second component of the fusion protein is therefore immobilized on the surface. Here we use this approach to immobilize an engineered version of the 12 kDa 10th domain of fibronectin III (FnIII10Eng) to direct cell adhesion. Substrates presenting this protein media...

Cutinase unfolding and stabilization by trehalose and mannosylglycerate

Cutinase unfolding and stabilization by trehalose and mannosylglycerate

Surface-induced changes in the structure and activity of enzymes physically immobilized at solid/liquid interfaces.

A proteolytic enzyme, alpha-chymotrypsin, and a lipolytic enzyme, cutinase, were adsorbed from aqueous solutions on solid surfaces with different hydrophobicities and morphologies. With both enzymes the affinity of adsorption is larger for the more hydrophobic surface. Water-soluble, flexible oligomers grafted on the sorbent surface cause a decrease in enzyme adsorption. CD spectroscopy and differential scanning calorimetry (DSC) indicate severe structural perturbations in the enzymes resulting from adsorption. The CD spectra reflect an average of the structure of the whole protein population. The DSC data allow additional conclusions to be drawn on the heterogeneity in the conformational states of the adsorbed enzymes. The degree of structural perturbation, that is the fraction of the adsorbed molecules of which the structure is perturbed, is lower at a surface that (1) is less hydrophobic, (2) contains water-soluble flexible oligomers and (3) is more covered by the protein. The specific activities of the enzymes are decreased on adsorption, more or less following the extent of structural perturbation. Unlike in solution, in the adsorbed state the heat-induced inactivation process is not identical with the heat-induced unfolding process. Furthermore, when the enzymes are adsorbed their specific activities are much less sensitive to temperature variation.

Effects of Organophosphorous Pesticides on Cutinase Activity and Infection of Papayas byColletotrichum gloeosporioides

Etude de l'activite in vitro de 13 pesticides organophosphores sur la cutinase purifiee de C.g. Essai en laboratoire de prevention de l'infection des fruits. Etude de l'action du dodecyle sulfate de sodium