Cells in suspension culture of celandine (Chelidonium maius L.) were permeabilized with Tween 80 and immobilized with glutaraldehyde, alginate or pectinate. Glutaraldehyde-immobilized celandine cells lost their viability. Celandine cells immobilized with pectinate or alginate have retained the high activity of dipeptidylpeptidase (DP) IV/CD 26 with a pH optimum at 7.8. The immobilized cells showed a good activity, fair stability and convenient physicomechanical properties. The extracellular activity of DP IV/CD 26 estimated in cell suspension accounts for 82.2 % of the total activity the rest being due to the intracellular activity. The latter activity is 1.87 times higher than the extracellular one. Secretion of DP IV/CD 26 by root tips, root hairs, callus and suspension cells was detected histologically. The method permits a rapid, simple and specific identification of DP IV/CD 26.

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.

Cells in suspension culture of mouse-ear cress (Arabidopsis thaliana (L), Heynh cultivar, Columbia were permeabilized with Tween 20, Tween 80, ethanol, and hexadecyltrimethylammonium bromide or hexadecylpyridinium chloride, and immobilized using glutaraldehyde Lactase showed a pH optimum at 4 7 and the optimal temperatures for immobilized cells and cell cultures was 60 degrees C and 56 degrees C, respectively Four-hour enzyme hydrolysis of the tested substrate proceeded with a conversion of 60-70% The immobilized cells showed a high lactase activity, fair stability in long-term storage and convenient physicomechanical properties The culture medium after removing cells was used for identification and determination of enzyme activity The extracellular activity of lactase estimated in cell suspension accounts for 61% of the total activity, the rest being due to the intracellular activity The specific extratracellular activity is 4 15 times higher than the intracellular one Using a histochemical method, secretion of lactase by root tips, root hairs, callus and suspension cells was detected The described method permits a rapid, simple and specific identification of lactase

A simple, rapid and straightforward procedure for identification and determination of intracellular and extracellular activity of aminopeptidases employing synthetic substrates beta-naphtylamides of L-Ala, L-Phe, and L-Tyr was used. Poppy cells (Papaver somniferum L.) permeabilized by Tween 80 were immobilized via crosslinking by glutaraldehyde. Glutaraldehyde immobilized poppy cells lost their viability and demonstrated significantly lower aminopeptidase activities than untreated control cells probably due to a damage to the enzyme active centre. Poppy cells immobilized by pectate and alginate have retained high activity of studied aminopeptidases. The culture medium (without cells) used for the identification and determination of extracellular enzyme activities retained 20-21%, whereas intracellular activities were estimated to be 79-80% of total enzyme activity. Thus the intracellular specific activity was 1.00-1.07 higher.

Cell suspensions of lemon balm (Melissa officinalis L.) were permeabilized by Tween 20, Tween 80, ethanol, hexadecyltrimethylammonium bromide, and hexadecylpyridinium chloride, and immobilized by glutaraldehyde. The invertase pH optimum was 4.5 at temperature 50°C. The hydrolysis of substrate was linear for 4 h, reaching 60% conversion. The cells had high invertase activity and good stability, and in longterm storage they showed good physicomechanical properties. The culture medium (without cells) was used for the identification and determination of extracellular enzyme activity. Intracellular activity was estimated from the cell suspension. For the lemon balm cell suspension, the intracellular activity accounted for 83.7% of the total activity, and the extracellular one for 12.7%. The intracellular specific activity is 4.2 times higher. Our method permits the rapid, simple, and specific identification and determination of plant invertase.

Cells in suspension Culture of foxglove (Dilgitalis lanata Ehrh.) were permeabilized with Tween 20, Tween 80, ethanol, hexadecyltrimethylammonium bromide or hexadecylpyridinium chloride and immobilized using glutaraldehyde. Saccharase showed a pH optimum at 4.6 and 5.4; the optimum, temperature for immobilized cells and for cell suspension Culture were 50 degrees C and 45 degrees C, respectively. Four-hour hydrolysis of the substrate proceeded with a conversion of 63%. The immobilized cells showed a high saccharase activity, good stability of plant saccharase in long-term storage and convenient physico-mechanical properties. The Culture medium (without cells) was used for identification and determination of extracellular enzyme activity. The intracellular activity of saccharase estimated in cell Suspension accounts for 88.4% of the total activity the rest is due to the extracellular activity. The specific intracellular activity is 4.6 times higher than the extracellular one. The described method permits a rapid, simple and specific identification.

Celandine cells, after permeabilization in Tween 80, were immobilized by crosslinking with glutaraldehyde without any carrier. The cells showed significantly lower aminopeptidase activities than untreated cells. Pectate and alginate hydrogels were successfully used for immobilization of greater celadine cells while retaining the activity of some aminopeptidases. A simple and rapid procedure for determination of extracellular aminopeptidases was developed using synthetic substrates. 4-Nitroanilides of amino acids were used as substrates for the determination of extracellular and intracellular enzymatic activities. The former were determined in culture media (without cells) whereas the latter in a cell suspension culture.

Celandine cells, after permeabilization in Tween 80, were immobilized by crosslinking with glutaraldehyde without any carrier. The cells showed significantly lower aminopeptidase activities than untreated cells. Pectate and alginate hydrogels were successfully used for immobilization of greater celadine cells while retaining the activity of some aminopeptidases. A simple and rapid procedure for determination of extracellular aminopeptidases was developed using synthetic substrates. 4-Nitroanilides of amino acids were used as substrates for the determination of extracellular and intracellular enzymatic activities. The former were determined in culture media (without cells) whereas the latter in a cell suspension culture.

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.

A simple, rapid and reproducible procedure for the identification and determination of extracellular saccharase from culture medium of watermelon cell suspension cultures is described. The culture medium (without cells) was used for the identification and determination of extracellular enzyme activity. Intracellular activity was estimated from the cell suspension. Watermelon cell suspension was permeabilized by Tween 80 and immobilized by glutaraldehyde. The highest saccharase activity was at pH 4.6 at a temperature of 50°C. The hydrolysis of substrate was linear 5h after reaching 60% conversion. The cells had high saccharase activity and good stability, and in long-term storage they showed convenient physico-mechanical properties.

Permeabilized tomato cells were cross‐linked with glutaraldehyde in the absence of a carrier. The immobilized cells demonstrated significantly lower aminopeptidase (AP) activities than untreated control cells. However, when immobilized with pectate and alginate gels, the tomato cells retained their AP activities. A new method for the determination of the activity of both extra‐ and intracellular AP was developed, based on enzyme‐catalyzed hydrolysis of a series of synthetic β‐naphthylamides (βNA) of the L‐amino acids Ala, Arg, Leu, Pro, Tyr, or of the synthetic β‐methoxynaphthylamides (βMNA) of Ala and Arg. Extracellular AP – produced by calli, cell‐suspension culture, or seedlings of tomato cells grown on agar – hydrolyzed these peptidic substrates to the free naphthalene amines and amino acids. Staining with Fast Garnet GBC salt under formation of bright reddish azo dyes readily allowed the determination of AP activities. For the tomato‐cell suspension, the intracellular activity accounted for 91.3–93.9% of the total activity, and the extracellular one for 6.1–8.7%, respectively. Our method permits the rapid, simple, and specific determination of plant aminopeptidases.

A simple, rapid and reproducible procedure for the identification of extracellular a-glucosidase is described using californian poppy (Eschscholtzia californica CHAM.) callus cultures, roots of 3-6 days-old seedlings of californian poppy, cucumber, melon, pea and tomato seedlings germinated on agar plates. 1-Naphthyl-alpha-D-glucopyranoside and p-nitrophenyl-alpha-D-glucopyranoside were used as substrates for the determination of alpha-glucosidase intracellular and extracellular activities in californian poppy cell suspensions. The extracellular a-glucosidase activity was identified by evaluating the dye-zones in the agar medium. The enzyme from californian poppy callus cultures or from seedling roots cultivated on agar plates supplemented with 1-naphthyl glucopyranoside hydrolysed this substrate releasing 1-naphthol. By simultaneous coupling with hexazonium p-rosaniline the corresponding (reddish-brown) azo-dye was formed.

The activity of (soluble acid) sucrase was detected in a culture medium of the cell suspension culture of watermelon (Citrullus vulgaris L.). A simple and rapid procedure for the identification and determination of extracellular sucrase from a culture medium of watermelon cell suspension cultures is described. Sucrose was used as a substrate for the determination of extracellular and intracellular activities of the enzyme. Intracellular activity was estimated from the cell suspension. The results show a 91.5-92.0% intracellular and 8.0-8.5% extracellular distribution of sucrase activity. The described method enables to carry out a rapid, simple and specific detection of extracellular sucrase in plants.

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).

Using synthetic substrates, an uncomplicated and sensitive procedure for the determination of extracellular aminopeptidase was developed. The studied enzyme produced by the tested plant material (calli, cell suspension culture and roots of Amsonia tabernaemontana Walt. seedlings) hydrolyzed the substrates beta-naphthylamides (beta NA) and 4-(phenylazo) phenylamides (PAP-amide) of the amino acids to beta-naphthylamine and 4-(phenylazo) aniline, respectively, and amino acid. The beta-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. By simultaneous azocoupling of beta-naphthol with Fast Garnet GBC salt on agar plates a corresponding brown-red hardly water-soluble azo-dye was produced. The evaluation of dyed zones allowed the extracellular aminopeptidase activity to be assessed. No coloration of the agar medium was observed without inoculum, with heat-inactivated cells (10 min at 100 degrees C) or in medium inoculated without substrate. On the agar plates with substrate and sterile Amsonia seedlings, changes in coloration were observed indicating a release of aminopeptidase from the roots during germination. The results show a 91.0 % intracellular and 9.0 % extracellular distribution of aminopeptidase activity, when a cell suspension culture of A. tabernaemontana Walt. as the plant material was used. The agar plate method described permits the rapid, uncomplicated and specific detection of plant producers of extracellular aminopeptidase, which could be particularly useful in future inhibitory and/or biotechnological studies.

Lactase activity was detected in a culture medium of the cell suspension culture of watermelon (Citrullus vulgaris L.). A simple, rapid and reproducible procedure for identification of extracellular lactase is described using callus cultures of seedlings from the tested plant, hairy roots of 2.5 days old seedlings of watermelon germinating on agar plates as well as cell suspension cultures derived from callus cultures. For the determination of intracellular activities of lactase, 6-bromo-2-naphthyl-β-D-galactopyranoside and p-nitrophenyl-β-D-galactopyranoside were used as synthetic substrates. The extracellular lactase activity was determined by evaluating the day-zone in agar medium. The enzyme from watermelon callus cultures and seedling roots, cultivated on agar plates supplemented with 6-bromo-2-naphthyl-2-bromo-β-D-galactopyranoside, hydrolyzed this substrate releasing 6-bromo-naphthyl. By simultaneous coupling with hexazonium p-rosaniline or Fast Blue BB the corresponding azo dye was formed. The parallel extracellular and intracellular activities were determined in cell suspension cultures derived from callus cultures. The results show a 43.8% intracellular and 54.2% extracellular distribution of lactase activity. The described agar plate method enables a rapid, simple and specific detection of plant processes of extracellular lactase.

A simple and rapid procedure for the identification and determination of extracellular invertase from a culture medium of tomato cell suspension cultures is described. Sucrose was used as substrate for the determination of the extracellular and intracellular activities of the enzyme. The culture medium (without cells) was used for identification and determination of extracellular enzyme activity. Intracellular activity was estimated from the cell suspension.

Using a synthetic substrate, a simple and sensitive procedure for the determination of extracellular lactase was developed. The enzyme studied produced by the tested plant material hydrolyzed the substrate (1‐naphtyl‐α‐D‐galactopyranoside) to α‐D‐galactose and 1‐naphthol. By simultaneous azocoupling of 1‐naphthol hardly water‐soluble azo‐dyes were produced. The evaluation of the intensity of dyed zones allowed the extracellular lactase activity to be assessed. The agar plate method described permitted rapid, simple, and specific detection of plant producers of extracellular lactase and proved to be perspectively useful in inhibitory and/or biotechnological studies.