The novel non-glycosylated invertase from Candida utilis (the properties and the conditions of production and purification).

The Candida utilis yeast, which is cultivated in liquid media enriched with saccharose, synthesizes the well-known invertase of 300 kDa (EC 3.2.1.26). This enzyme is present both intracellularly in the periplasmic space and extracellularly in the culture broth. However, it was determined that the same C. utilis strain cultured in certain conditions is simultaneously capable of producing another, still unknown form of invertase with a molecular mass of 60 kDa. The presence of the latter enzymatic form was detected in cells as well as in the liquid culture medium. Both invertase forms were purified using a three-step process (ion-exchange chromatography, affinity chromatography, and preparative column electrophoresis) and named, due to their different migration ratio in polyacrylamide gel electrophoresis, F-form (Fast; 60 kDa) and S-form (Slow; 300 kDa). The F-form of invertase was found to be nonglycosylated as opposed to the well-known S-form of invertase from the same source. The physicochemical properties of the F-form of invertase (isoelectric point, substrate specificity, pH, and temperature optima) were determined and compared with those of the S-form of the enzyme.

Screening the keratinolytic activity of dermatophytes in vitro.

Sixteen strains out of 12 species dermatophytes were examined in respect to their ability of utilizing keratin substrates as the only sources of C and N. The employed keratin substrates included a solubilized preparation of feather keratin (KS) and native keratin, guinea pig hair and chicken feathers. It has been shown that the preparation KS constitutes a convenient model for a preliminary estimation of fungal keratinolytic activity and it can be a source of information about the localization of these enzymes. It has been found that, among the 16 fungal strains, 13 strains synthesize mainly intracellular keratinases while 3 strains of T. verrucosum release enzymes mainly to the medium. Native keratin from hair and feathers was degraded only by some of the examined strains which, under the experimental conditions, developed characteristic spore forms. Keratin of guinea pigs hair was attacked only by the T. mentagrophytes strains, T. verrucosum and K. ajelloi, and only T. gallinae grew on native keratin from chicken feathers.

Feather keratin as a ligand in an affinity chromatographic technique for isolation of protease from Trichophyton verrucosum.

A technique of affinity chromatography was developed and optimized for proteases from the postculture fluid of Trichophyton verrucosum. The technique employs porous glass with adsorbed feather keratin or keratin covalently bound to the glass. Modifications of the amounts of proteases introduced into the columns and the manner of elution (pH gradient, buffer concentration, EDTA) made it possible to achieve yields of the isolated enzymes of the order of 80%. The degree of purification of four fractions isolated by the proposed technique was about 6-fold and allowed electrophoretically almost homogeneous enzymatic forms to be isolated. Substrate and inhibition tests on the four chromatographically purified proteolytic enzymes indicated a specifically keratinolytic nature of the enzymes studied.

Immobilization of enzymes to porous-bead polymers and silica gels activated by graft polymerization of 2,3-epoxypropyl methacrylate.

Three types of organic polymers and bead-shape silica gels were activated by graft polymerization of 2,3-epoxypropyl methacrylate; in some cases, epoxide groups on the support surface were modified to NH2 groups. Eight active matrices so obtained were assessed as supports for immobilized enzymes using peroxidase, glucoamylase and urease. The immobilization yield of protein and specific activities of enzymes were better with supports containing NH2 groups than with those containing epoxide spacer arms. Maximum enzyme immobilization and storage stabilities were obtained with silica-gel beads activated by graft polymerization of 2,3-epoxypropyl methacrylate. With all eight matrices tested, the immobilized enzymes showed good stability with not less than 82% of the original activity persisting after 28 days. The developed matrices have potential for use in process-scale biotechnological operations.

Comparative characterization of proteolytic enzymes from Trichophyton gallinae and Trichophyton verrucosum.

The proteolytic activities of culture filtrates and cell homogenates of Trichophyton gallinae and Trichophyton verrucosum were compared when the fungi were grown in the presence of a readily assimilable source of C and N (Sabouraud's broth) and a poorly assimilable source (mineral medium containing soluble keratin (KS) protein prepared from either chicken feathers or guinea pig hair). The proteolytic activity of T. gallinae was found to be located predominantly in the cell homogenate although this depended somewhat on the nature of the C and N source in the medium. Enzyme activity in T. verrucosum on the other hand was located in the culture filtrate. The use of KS as a substrate revealed only one peak of enzymatic activity which occurred at pH 7.0 in both fungi. In the case of T. gallinae the total proteolytic activity was not related to the fungal biomass but did depend on the nature of the substrate; poorly assimilable substrates (native feathers and hair) stimulated a higher enzymatic activity per unit biomass than readily assimilable Sabouraud's broth. In T. verrucosum, proteolytic activity was related to the fungal biomass and was highest in Sabouraud's broth which proved most suitable for growth of the fungus. On the basis of the findings with six different inhibitors of protease activity, it is concluded that the proteolytic enzymes of T. gallinae differ from those of T. verrucosum in terms of their metal-dependence and contained serine as part of the active centre.

Intracellular keratinase of Trichophyton gallinae.

Trichophyton gallinae could grow on agar medium containing chicken feathers as sole sources of carbon and nitrogen, but it could not utilize human or guinea pig hair. The fungus grows rapidly when feather keratin solubilized in dimethyl sulphoxide was included in the growth medium. Keratinase activity could be detected by release of amino acids from solubilized keratin and by creation of zones of lysis in gel diffusion assay. However, the activity was observed only in homogenates of T. gallinae mycelia: it was not possible to detect it in culture filtrates.

New matrices for the purification of pectinases by affinity chromatography.

Polygalacturonic acid was used as a ligand in the affinity technique for pectinases purification from the filtrate of Aspergillus niger 71 culture. For this purpose four matrices were examined, namely, alkylamine controlled porous glass (CPG), alkylamine silica gel as well as keratin or polyamide coated silica gel. Good results of pectinase purification was obtained on silanized CPG or keratin coated silica gel supports.

Keratin and polyamide-coated inorganic matrices as supports for glucoamylase immobilization.

Previously solubilized feather keratin and polyamide were used for coating sand, glass beads and silica gel. These new seven supports were employed for comparative studies on pure glucoamylase / EC 3.2.1.3 / immobilization. The immobilization yield of glucoamylase on keratin and polyamide coated supports was comparable with conventional matrices used earlier. The highest activity per 1 g of support was shown by the enzyme bound to polyamide-coated CPG, and the bests operational stability by the enzyme immobilized on polyamide-coated CPG with keratin subsequently deposited on it.

Induction by ferulic acid of multiple forms of peroxidase in the fungus Trametes versicolor (Basidiomycetes).

Two forms of peroxidase (EC 1.11.1.7) were induced in mycelium of Trametes versicolor by ferulic acid, with an about 2,5-fold increase of their specific activity. Both inducible forms of peroxidase were isolated and partially purified by ion-exchange chromatography on CM-Sephadex C-50 and DEAE-Sepharose Cl 6B; some of their properties were also characterized.

Hemoproteid peroxidase from Inonotus radiatus.

The exoperoxidase from Inonotus radiatus was first identified as hemoproteid enzyme. The purified peroxidase Ia and IIa contained 1.09% 1.15% of hematin, respectively. It is established that oxidation products of phenolic compounds form stable complex with these two fungal peroxidases.