J. Stano

Decarboxylation of l-tyrosine and l-dopa by immobilized cells of Papaver somniferum

Abstract Papaver somniferum (opium poppy) cells, after permeabilization in Tween 80, were immobilized by glutaraldehyde without any insoluble carrier. Cells immobilized by cross-linking decarboxylated l -dopa and l -tyrosine for three months, without significant loss of either activity.

Localization and measurement of extracellular plant galactosidases

A simple, rapid and sensitive procedure for the identification and determination of plant extracellular alpha-galactosidase and beta-galactosidase is described using callus cultures and seedlings from tomato. Synthetic substrates (1-naphthyl- and p-nitrophenyl-alpha-D- and beta-D-galactopyranosides) were used for the identification and determination of intracellular and extracellular activity of alpha-galactosidase and beta-galactosidase, respectively. Many iminosugars or azasugars are strong glycosidase inhibitors and some of them show promising chemotherapeutic effects against viral diseases, and are potentially antidiabetic agents, as well as antitumor agents. These facts initiated our interest in a rapid and sensitive assay to determine activity of alpha-galactosidase and beta-galactosidase in plant tissues. The results presented here show the potential of the assay of the activity of intracellular and extracellular galactosidases of plant origin in inhibitory and/or biotechnological studies.

A Simple and Rapid Method of Identification of Extracellular Plant Galactosidases

A simple, rapid, and sensitive procedure for the identification of plant extracellular α- and β-galactosidase is described using callus cultures of seedlings from the tested plant, roots of 4-6 day old seedlings of Californian poppy germinating on agar plates, and cell suspension cultures cultivated from callus cultures. Synthetic substrates were used for the determination of the intra- and extracellular activities of α- and β-galactosidase. Many imino- or azasugars are strong glycosidase inhibitors, and some of them show promising chemotherapeutic effects against viral disease and are potential antidiabetic as well as antitumor agents. This fact aroused our interest for a rapid and sensitive assay method of the activity of α- and β-galactosidases of plant origin. The results presented here show the perspective usefulness of extracellular galactosidases of plant origin in inhibitory and/or biotechnological studies.

A simple procedure for the detection of plant extracellular proteolytic enzymes

A simple procedure for the detection of extracellular plant proteolytic enzymes using insoluble dye stained gelatin substrates incorporated into an appropriate culture medium is described. Extracellular proteinases produced by the tested plant cells (callus culture and cell suspension) hydrolyzed the substrates and dyed peptide fragments were released. Dyed zones around and under the proteinase-producing callus cultures were formed on the agar medium. Similarly, coloration of the culture media using proteinase-producing cell suspensions was observed.

Melibiase in Immobilized Cells of Watermelon

Cells of suspension culture Citrullus vulgaris cv. “Samara” were permeabilized by Tween 80 and immobilized by glutaraldehyde. The highest melibiase activity was at pH 5.4 and 60°C. The hydrolysis of substrate was linear for 3.5 h, reaching 65–70% conversion of the substrate. The cells, characterized by high enzyme activity and stability in long-term storage, showed convenient physico-mechanical properties (physical protection from shear forces and easy separation of product from biocatalysts).

Invertase in Immobilized Cells of Eschscholtzia Californica

Cell suspension culture of Eschscholtzia californica Cham. were permeabilized by Tween 20 or 80, and immobilized by glutaraldehyde. The highest invertase activity was at pH 4.5 and temperature 50 °C. The hydrolysis of the substrate was linear for 5 h reaching 60 % conversion. The cells had high invertase activity and a good stability, and in long-term storage they showed convenient physico-mechanical properties.

Demonstration of Activity of α-Galactosidase Secreted by Cucumis sativus L. Cells

A simple, rapid and reproducible procedure for the identification of extracellular cucumber (Cucumis sativus L.) α-galactosidase is described using callus cultures of seedlings from the tested plant, hairy roots of 2-day-old seedlings of cucumber germinating on agar plates as well as cell suspension cultures derived from callus cultures. For the determination of the intracellular and extracellular activities of α-galactosidase, 6-bromo-2-naphthyl--α--D-galactopyranoside and /? -nitrophenyl- «-/ ->-galactopyranoside, respectively, were used as synthetic substrates. The extracellular α-galactosidase activity was identified by evaluating the dye-zones in agar medium. The enzyme from cucumber callus cultures and seedling roots, cultivated on agar plates supplemented with 6-bromo-2-naphthyl-α-D-galactopyranoside, hydrolyzed this substrate releasing 6-bromo-2-naphthol. By simultaneous coupling with hexazonium p-rosaniline the corresponding azodye was formed. Thus, the extracellular enzyme was detected by the presence of reddish-brown zones on the agar plates around the plant material. The parallel extracellular and intracellular activities were determined in cell suspension cultures derived from callus cultures. The results show a 44.6% intracellular and 55.4% extracellular distribution of α-galactosidase activity. The described agar plate method enables a rapid, simple and specific detection of plant producers of extracellular α-galactosidase.

Β-Galactosidase in immobilized cells ofPapaver somniferum

Cell suspension culture of poppy was permeabilized by Tween 80 and immobilized by glutaraldehyde. Β-Galactosidase showed optimum pH 4.8 and temperature of 50 ‡C. The enzyme hydrolysis was linear during 3 h reaching 68 % conversion. The cells characterized by high Β-galactosidase activity and stability in long-term storage showed convenient physico-mechanical properties.

Study of gherkin lactase in cell culture and in seedlings.

A synthetic substrate replacing lactose has facilitated application of a simple, rapid and sensitive method for the identification and determination of extracellular and intracellular gherkin lactase. The intracellular enzyme activity was estimated from the cell suspension, while the extracellular enzyme activity was established within the cell free cultivation medium. A suspension of gherkin cells was permeabilized by Tween 20, or Tween 80, or hexadecyltrimethyl ammonium bromide, or hexadecylpyridinium chloride or ethanol added one at a time and then immobilized by glutaraldehyde. The highest lactase activity was at pH 4.8 at a temperature of 55°C. The hydrolysis of substrate was linear for 4.5h and reached 60% conversion. The cells had high lactase activity and good stability. During long-term storage they demonstrated convenient physico-mechanical properties.

Identification and determination of aminopeptidase activities secreted by lemon balm

Using synthetic substrates, an uncomplicated and sensitive procedure for the identification and determination of extracellular aminopeptidase was developed. The β-naphthylamides of the amino acids were applied for the identification of extracellular aminopeptidase, whereas the 4-(phenylazo) phenylamides of the amino acids were used for the determination of intra-and extracellular aminopeptidase activity. The results show a 81.8–88.9% intracellular and 11.1–18.2% extracellular distribution of the studied enzyme activity.