Lactase in Immobilized Cells of Watermelon

Tilemann, I.; Tokhtaeva, Elmíra; Sedlárová, Eva; Barth, A; Valent, Aladár; Siekel, P.; Ďuríček, Martin

doi:10.1023/b:conc.0000003424.33324.e4

Chemistry of Natural Compounds

2003

DOI: 10.1023/b:conc.0000003424.33324.e4

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Lactase in Immobilized Cells of Watermelon

I. Tilemann¹,

Elmíra Tokhtaeva

Eva Sedlárová

et al.

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Cited by 6 publications

(2 citation statements)

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“…It is very interesting that the activity of extracellular α-galactosidase and β-galactosidase [15] is 3-to 4-fold higher than the activity of invertase [14] and aminopeptidase studied. The production of extracellular aminopeptidases as well as other hydrolases, which are released from plant cells, might be of some importance for biotechnological applications in food and pharmaceutical research as well as in industry [16,17]. Biotransformations using free or immobilized biocatalysts not only provide an alternative and efficient solution to the synthesis of many compounds, but also offer environmentally clean technologies that profit from very mild reaction conditions [18,19].…”

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confidence: 99%

Identification and determination of aminopeptidase activities secreted by lemon balm

et al. 2007

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UDC 615.322Using 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.Plant proteolytic enzymes play many roles in metabolism such as peptide and protein degradation, posttranslation protein modification, and other processes as well [1,2]. Proteolysis is ubiquitous in biological systems, providing a means for cells to change their protein content during development and adaptation to altered environmental conditions. Proteinases participate in the mobilization of storage proteins by transforming stored polypeptides through oligopeptides to free amino acids, which are indispensable for the development and primary and secondary metabolism of cells [3,4]. Germination and ripening of seeds and pollen is associated with the expression of various hydrolytic enzymes [5,6].In the last years, several methods for the identification and determination of the activity of aminopeptidases have been developed [5,7]. Naturally occurring or synthetic substrates may be used for these purposes [8,9].Aminopeptidases (aminoacylpeptide hydrolase EC3.4.11) catalyze the release of N-terminal amino acids from peptides or synthetic substrates. The determination of aminopeptidase activities plays an important role in many fields of basic and applied research [1, 10].The availability of a simple and rapid screening method for the detection of aminopeptidase activity is of great importance for both scientific and production purposes. One advantage is the use of synthetic substrates such as 4-(phenylazo) phenylamides (PAP-amides) and β-naphthyl amides (βNA) of the amino acids [5,11]. The aim of this work was to show that the synthetic substrates L-Arg-βNA, L-Phe-βNA, and L-Leu-βNA could be employed for the identification of the activity of extracellular plant aminopeptidases in an uncomplicated and rapid procedure, whereas L-Arg-PAP amide, L-Phe-PAP-amide, and L-Leu-PAP-amide could be employed for its determination.The synthetic substrates L-Arg-PAP amide, L-Phe-PAP amide, and L-Leu-PAP amide were used in this study to determine the intracellular and extracellular activities of aminopeptidase. The results presented are mean values ± SD of five experiments. Culture media (agar plates) with and without the substrates L-Arg-βNA, L-Phe-βNA, L-Leu-βNA, and GBC salt [12,13] were inoculated with cells from growing callus cultures and then incubated for 30-90 min. The corresponding azo dye was formed by simultaneous azocoupling of the β-naphthylamine released by the enzyme activity with Fast Garnet GBC salt (GBC salt).The activities of extracellular aminopeptidase were detected by the presence ...

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Identification and determination of aminopeptidase activities secreted by lemon balm

et al. 2007

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“…105 lus vulgaris cv. Samara as previously described by TILEMANN et al (2003).Identification and determination of intra-and extracellular sucraseUsing sucrose as a substrate the intra-and extracellular activity of soluble acid sucrase was identified. Eight days old cell suspension cultures were used to determine the intracellular enzyme activity.…”

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Identification of sucrase activity in cell suspension and culture medium of melon

Stano¹,

Mičieta²,

Barth³

et al. 2005

Hort. Sci. (Prague)

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ABSTRACT: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. 105 lus vulgaris cv. Samara as previously described by TILEMANN et al. (2003).Identification and determination of intra-and extracellular sucraseUsing sucrose as a substrate the intra-and extracellular activity of soluble acid sucrase was identified. Eight days old cell suspension cultures were used to determine the intracellular enzyme activity. The cells (5 g) were filtered and washed twice with 1,000 ml of distilled water. Soluble proteins were extracted by grinding the cells in a pre-cooled mortar using a ratio 1:2 (g/ml) of cells and Mc Ilvaine buffer pH 4.5 at 4°C. The homogenate was filtered through two layers of nylon cloth and centrifuged at 15,000 g at 4°C. For determinations of extracellular enzyme activity the cultivation medium (without cells) was used after centrifugation (15,000 g, 10 min at 4°C). Enzyme assayEnzyme activity was determined by a modified method of RUBIO et al. (2002) using sucrose as a substrate. The reaction mixture contained a suitable amount of enzyme (0.2-0.4 ml) and 0.4 mM sucrose in Mc Ilvaine buffer pH 4.5 in a final volume of 2 ml. Enzyme activity was determined at 30°C for 30 to 60 min. The control contained temperature-inactivated enzyme (100°C, 10 min). The enzyme activity was expressed in katals. Proteins were determined by the method of BRADFORD (1976) using bovine serum albumin as a standard protein. The glucose content released by the enzyme was determined by the method of TRINDER (1969). RESULTS AND DISCUSSIONSynthetic chromogenic substrates are very suitable for biochemical and histochemical studies of hydrolytic enzymes. In the last decades, several methods for the determination of hydrolytic enzymes have been developed. Various naturally occurring chromogenic substrates may be used for these purposes (KIM et al. 2002;MIČIETA et al. 2002). p-nitrophenyl-β-Dfructofuranoside and naphthyl-β-D-fructofuranoside were not synthesised and only a natural substrate -sucrose was used for the study of sucrase.Therefore only sucrose was used to study the activity of intracellular and extracellular (acid soluble) sucrase. The highest specific activity of the studied enzyme was recorded on day 8 of cultivation (Table 2) in undifferentiated cells. Homogenised cell suspension cultures and culture medium alone after 8-day cultivation were used to assay the activity of intrac...

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Study of immobilized and extracellular invertase of lemon balm

et al. 2008

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Lactase in Immobilized Cells of Watermelon

Cited by 6 publications

References 12 publications

Identification and determination of aminopeptidase activities secreted by lemon balm

Identification and determination of aminopeptidase activities secreted by lemon balm

Identification of sucrase activity in cell suspension and culture medium of melon

Study of immobilized and extracellular invertase of lemon balm

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