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Proteolytic activity in plant tissue and cell suspension culture

University of British Columbia

Proteolytic enzymes are common in plants but are usually specifi to endogenous protein. Plant proteases with specificities applicable to the food industry include papain, ficin and bromelain. Other plants have been used in traditional methods of food preparation for their proteolytic action on food components. The following species were investigated for propagation in tissue culture: Carica papaya, Ficus carica, Cynara cardunculus, Galium verum, Circium arvense, Dieffenbachia amoena, D. picta and Ananas comosus. Tissues of the first five of these demonstrated proteolytic activity by clearing of milk turbidity in agar medium. Commercial papain and ficin preparations are currently obtained from latex of immature papaya and fig fruit, respectively. This investigation was conducted, in part, to determine the feasibility of producing these two enzymes by the in vitro cell culture technique. Standard method of aseptic seed germination and leaf tissue excision were employed for callus initiation. Cell suspension cultures derived from callus were maintained in B5 medium at 28 °C in darkness. Proteolytic activity was determined by a modification of the Food Chemicals Codex method for papain and protein content was determined by Bradford's dye-binding, method. Production of protein and protease varied among cell cultures, but could be influenced by changes to some nutritional factors. Fig cells were grown in medium supplemented with single amino acids in the presence of either nitrate or ammonia as a source of inorganic nitrogen. All nitrate-based media produced higher yields of cell dry weight than ammonia-based media. Glutamic and aspartic acids were most stimulatory growth, protein accumulation and protease activity of fig cells. Skimmed milk, added at 3% (v/v), was a highly effective growth stimulant, and also resulted in higher protein and protease levels than the amino acids. Fresh casein and whey, added individually, produced similar results to skimmed milk. Citric acid, added at the level found in the 3% milk supplement, also caused stimulation of fig cell growth, protein synthesis and protease activity not significantly different from skimmed milk. It appears that nitrogen accumulation and reduction in fig cells may have been limited by an energy requirement which could be satisfied with the addition of citric acid or milk whey to the basal medium.

Text

2010-03-30

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http://hdl.handle.net/2429/23130

Food Science

University of British Columbia

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