PROLYL 4-HYDROXYLASE FROM INVITRO-CELL CULTURES

Fifteen cell suspension strains cultivated in vitro were screened for the highest cell wall hydroxyproline content. Differences in hydroxyproline content were observed in different plant families as well as in different strains of a same family. The suspension of soybean Glycine max cv. Mandarin showing the highest hydroxyproline content was chosen as a source of prolyl 4-hydroxylase (EC 1.14.11.2.) for purification. Kinetics of hydroxyproline content and prolyl 4-hydroxylase specific activity were similar, with a maximum at 14 days after subculture. Elicitation of cell suspensions by arachidonic acid leads to an increase in 30% of activity. Triton X-100 was the most effective detergent among four non-ionic detergents tested for solubilizing the soybean membrane-bound prolyl 4-hydroxylase from microsomal fractions. This enzyme, unstable at room temperature and at 4 °C even with the addition of enzyme co-factors (ascorbic acid and ferrous ions), can be stored for several months at -80°C. Prolyl 4-hydroxylase was purified from microsomal fractions through chromatography on DEAE Sephadex A-25 and poly L-proline-Sepharose affinity columns. Comparison of prolyl 4-hydroxylase activities from different forms of soybean (cell suspensions, cell suspensions elicited with arachidonic acid, calluses, whole plant) shows that activity is highest in the cell suspensions. © 1990, Gustav Fischer Verlag, Stuttgart. All rights reserved.