H. J. Huizing

The effect of some environmental factors on the production of L-DOPA by alginate-entrapped cells of Mucuna pruriens.

In-vitro-grown cells of Mucuna pruriens, immobilized in calcium-alginate gels, were able to transform the precursor L-tyrosine into L-dihydroxyphenylalanine (L-DOPA). After the immobilization in alginate the plant cells released 90% of the produced L-DOPA into the medium; supplementation of the medium with calcium inhibited both the transformation of L-tyrosine into L-DOPA and the release of L-DOPA into the medium. Continuous illumination of the beads had a slight beneficial effect on the synthesis of L-DOPA. A simple production medium for the transformation of L-tyrosine into L-DOPA was designed. This medium contained only sucrose and sodium chloride as osmotic stabilizers, a low concentration of calcium chloride for stabilization of the alginate beads, and L-tyrosine as the precursor.

PURIFICATION AND PROPERTIES OF A PHENOL OXIDASE DERIVED FROM SUSPENSION-CULTURES OF MUCUNA-PRURIENS

From cells of Mucuna pruriens, grown in suspension, a monophenol monooxygenase (EC 1.14.18.1) was purified to homogeneity, as deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme appeared to have a native molecular weight of 90000±5000 dalton, and consisted of two subunits, each of 42000±1000 dalton. High-performance liquid chromatography with electrochemical detection for specific measurement of catecholes, was used to determine separately the tyrosinehydroxylating and catecholase activities of the enzyme. For the enzymatic activities, pH optima of, respectively, 7.5 and 5.5–6.5 were found; the effects of some inhibitors on both activities appeared to be different. Michaelis-Menten characteristics for some mono-and o-dihydroxysubstrates were determined.

Production of L-DOPA by cell suspension cultures of Mucuna pruriens

Methods for induction and maintenance of callus as well as cell suspension cultures of Mucuna pruriens are described. The presence of 3- (3, 4-dihy droxyphenyl 1)-L-alanine (L-DOPA) in these cell suspension cultures was demonstrated by means of TLC and HPLC, the latter being equipped with an ultra-violet or a more specific electrochemical detector. Further methods employed were GC, also in combination with mass spectrometry, and thermal desorption chemical ionization mass spectrometry.The endogenous synthesis of 3-(3,4-dihydroxyphenyl)-L-alanine (L-DOPA) by cell suspension cultures of Mucuna pruriens was found to be influenced by several environmental parameters. The nature of the nitrogen source as well as the concentration of nitrogen containing salts, sucrose and phosphate in the culture medium were found to affect the biosynthesis of L-DOPA. Addition of 2, 4-dichlorophenoxyacetic acid to the medium suppressed L-DOPA production; continuous illumination of the cultures had a strong beneficial effect on L-DOPA production. L-DOPA was accumulated intracellularly by the cell suspension cultures. These observations further demonstrate that for certain products of plant cell suspensions product synthesis can be manipulated by a proper selection of specified nutrients.

A chemotaxonomical study of someBoraginaceae: pyrrolizidine alkaloids and phenolic compounds

By means of thin layer chromatography pyrrolizidine alkaloids and phenolic compounds in some members of the familyBoraginaceae, subfamiliesHeliotropioideae andBoraginoideae, were studied. FromOmphalodes verna a main alkaloid was isolated with an isoretronecanol (or stereoisomeric) nucleus. The chemotaxonomical model ofTétényi forBoraginaceae based on fatty acids is generally supported, but relationships betweenHeliotropioideae andCynoglosseae appear to be closer, suggesting parallel developments from common ancesters.

Optimization of the biotransformation of l-tyrosine into l-dihydroxyphenylalanine (DOPA) by alginate-entrapped cells of Mucuna pruriens

The optimization of the biotransformation of l-tyrosine into l-dihydroxyphenylalanine (DOPA), and of formyl-tyrosine into formyl-DOPA by alginate-entrapped cells of Mucuna pruriens is reported. This optimization is discussed in terms of parameters that are relevant for the entrapped cell system (charge of the beads with cells, bead diameter, permeation of the cells) and some parameters that are relevant for the enzymatic transformation (pH, pO2, concentration of ascorbate). The optimization experiments resulted in the description of a biotransformation system which operates at a constant redox potential. With this transformation system a transformation efficiency of 70% could be obtained, at a biotransformation-rate of 435 μmol·h-1·g-1 (DW of cells) at a substrate concentration of 19 mM.

Regeneration of plants from tissue- and cell suspension cultures of Symphytum officinale L. and effect of in vitro culture on pyrrolizidine alkaloid production

Primary calluses were induced from various organs of Symphytum officinale L. (comfrey) plants on solid MS and B5 medium supplemented with plant growth regulators. The callus was further subcultured on B5 medium. Cell suspension cultures were derived from B5 grown calluses by transfer to liquid B5 medium.Calluses as well as cell suspension cultures could be induced to regenerate whole plants on solid MS medium. Plants regenerated from short term cultures were identical with plants from which cultures were initiated in morphology and chromosome number.Production of pyrrolizidine alkaloids ceased on prolonged subculturing of suspensions although polyamines, which might act as precursors, were still detectable. However, regenerated plants produced the original alkaloids.

Induction of phenoloxidase in cell suspension cultures of Mucuna pruriens L: effects on accumulation of L-3,4-dihydroxyphenylalanine and biotransformation capacity

The effects of limitating nitrogen-containing compounds in the medium and of adding the amino-acid analogues p-fluorophenylalanine and ethionine on both phenoloxidase activity and the accumulation of L-3,4-dihydroxyphenylalanine (L-DOPA) are reported for cell suspension cultures of Mucuna pruriens. Nitrogen limitation of the cultures, or the addition of p-fluorophenylalanine or ethionine to the culture medium resulted in an increased phenoloxidase activity. There appeared to be an inverse relationship between phenoloxidase activity and the acccumulation of L-tyrosine into L-DOPA by alginate-entrapped cells occurred at a higher rate when phenoloxidase activity was increased.

Chemotaxonomical investigations of theSymphytum officinale polyploid complex andS. asperum (Boraginaceae): Phytosterols and triterpenoids

From a comparison of phytosterol and triterpenoid patterns of severalSymphytum officinale cytotypes,S. asperum and their interspecific hybrids,S. ×uplandicum, which were obtained from thin layer chromatography and gaschromatography (also in combination with mass spectrometry), the hybrid character of the latter taxon is clearly shown. The specific value of the triterpenoid isobauerenol as a chemotaxonomical marker within this group is discussed in some detail.