Proteolytic Activity, Plant

Lipase (Microbial) Activity for Medium- and Long-Chain Fatty Acids, (S3)105 Lysozyme Activity, (S3)106 Maltogenic Amylase Activity, 804 Milk-Clotting Activity, 805 Pancreatin Activity, 805 Pepsin Activity, 807 Phospholipase A2 Activity, 808 Phytase Activity, 808 Plant Proteolytic Activity, 810 Proteolytic Activity, Bacterial (PC), 811 Proteolytic Activity, Fungal (HUT), 812 Proteolytic Activity, Fungal (SAP), 813 Pullulanase Activity, 814 Trypsin Activity, 814 Enzyme Assays, 786 Enzyme-Hydrolyzed (Source) Protein,... 

Papain is a cysteine protease isolated from the latex of the immature fruit and leaves of the plant Carica papaya. It consists of a single 23.4 kDa, 212 amino acid polypeptide, and the purified enzyme exhibits broad proteolytic activity. Although it can be used as a debriding agent, it is also used for a variety of other industrial processes, including meat tenderizing and for the clarification of beverages. 

Plant proteolytic enzymes are cysteine proteases, that is, they have a cysteine that is critical for the catalytic activity. Plant proteases are thought to function by a mechanism reminiscent of that shown for chy-motrypsin (fig. 8.11). Propose a structure for the acyl-enzyme intermediate that would exist for plant proteases. 

Proteases are regulated in vivo by autoinhibition (as zymogens), proteolytic activation, turnover and by endogenous protease inhibitor proteins. The following brief sketch of protease complexity is accompanied by succinct reference to the physiological context and hence the potential pharmacological relevance of plant-derived protease inhibitors to be described later in this review. 

Proteolytic activity in the juice of the pineapple plant Ananas comosus) was first reported in 1879 (7). More recently, the juice from the stem of the pineapple plant was shown to be a rich source of stem bromelain. This name is used to distinguish the enzyme from another which is derived from the fruit (8, 9). Mature pineapple stems are collected by special harvesting machines. The juice is pressed by special mills and then filtered. Most commercial preparations of bromelain have been precipitated from the stem juice by acetone. 

S. Ota, K. Horie, F. Hagino, C. Hashimoto, and H. Date. Fractionation and some properties of the proteolytically active components of bromelains in the stem and the fruit of the pineapple plant. J. Biochem. 71 817 (1972). 

In the raw state, mature soybeans and many other plant foodstuffs contain protease Inhibitors that diminish the proteolytic activities of trypsin and chymotrypsln in the Intestinal tract, cause pancreatic hypertrophy and suppress growth. Trypsin Inhibitors (TI) account for about 40% of the pancreatic hypertrophic effect and growth-inhibitory capacity of raw soy proteins. The resistance of the raw undenatured protein to tryptic digestion accounts for the remaining 60%. The practical significance of residual TI activity In heat-processed soy protein products and the biochemical effects of other protease Inhibitors have been reviewed (40). 

For an enzyme to be placed into one of these categories it must first be fully characterized. Since only a few plant proteinases have been characterized (see Ref. 2 for details) the general term proteinase will be used here for an enzyme with undefined proteolytic activity. 

Borchers R (1965) Proteolytic activity of rumen fluid in vitro. J Anim Sci 24 1033-1038 Briskin DP (2000) Medicinal plants and phytomedicines. Linking plant biochemistry and physiology to human health. Plant Physiol 124 507-514 Broderick GA, Balthrop JE (1979) Chemical inhibition of amino acid deamination by ruminal microbes in vitro. 1 Anim Sci 49 1101-1111... 

Methods for eliminating bitter peptides in partial protein hydrolysates are known, but they cause a significant loss of essential amino acids. These procedures usually include additional enzymatic hydrolysis under controlled conditions (a shorter time for the hydrolysis leads to higher peptides that are not bitter) and a selection of suitable proteases, such as aminopeptidases, carboxypeptidases and some other proteases. Enzymes of plant and microbial origin have been successfully used for this purpose. For example, the intracellular peptidases from Lactococcus lactis ssp. cremoris and Brevibacterium linens, which have high proteolytic activity, successfully hydrolyse bitter peptides in cheeses. 

Further studies (2) revealed that a soluble phospholipase activity in potato leaves could be stimulated to the same degree (30-50%) by either calmodulin or protein kinase (-t-ATP). Two other plant enzymes, quinate NAD+ oxidoreductase (3) and isofloridoside-phosphate synthase (4), have also been shown to be similarly stimulated by both calmodulin and protein phosphorylation. However, the latter enzyme was also shown to be stimulated to an even greater degree by proteolysis with trypsin or chymotrypsin (4). This study was undertaken to investigate whether the phospholipase activity in potato leaves may also respond to proteolytic activation. 

The only other plant enzyme which has been reported to be stimulated by the same three treatments is isofloridoside-phosphate synthase (4) as previously described. Those authors presented evidence for the presence of an endogenous acid protease which could stimulate the enzyme. They also proposed (4) that calmodulin and protein kinase somehow stimulated this protease which in turn proteolytically-activated the isofloridoside-phosphate synthase. Such a mechanism is also conceivable for the potato leaf phospholipase. The only other plant enzyme which has been reported to be stimulated by proteolytic activation is a glucan synthase in soybean cells (6). It is also interesting to note that an animal phospholipase, pancreatic phospholipase A, is activated by the proteolytic removal of a heptapeptide from the ammo terminus (7). 

PPT application results in a 50% decrease in GS levels in leaf tissues glutamate dehydrogenase levels are enhanced but, as intracellular ammonia levels rise, the amination reaction catalyzed by glutamate dehydrogenase insufficiently compensates for the inhibition of GS. Nitrate reductase is reduced, but proteolytic activity is unaffected. A wide-ranging survey of GS levels in plants reported a 70-fold variation in extractable GS levels there was also considerable variation in the ratios of the cytosolic and chloroplastic isozymes. The kinetics of inhibition by PPT, however, were very similar, and there was no apparent correlation between natural GS levels and herbicide susceptibility. PPT uptake and transport therefore seem to be the determining factors in natural herbicide sensitivity. 

Mammals, fungi, and higher plants produce a family of proteolytic enzymes known as aspartic proteases. These enzymes are active at acidic (or sometimes neutral) pH, and each possesses two aspartic acid residues at the active site. Aspartic proteases carry out a variety of functions (Table 16.3), including digestion pepsin and ehymosin), lysosomal protein degradation eathepsin D and E), and regulation of blood pressure renin is an aspartic protease involved in the production of an otensin, a hormone that stimulates smooth muscle contraction and reduces excretion of salts and fluid). The aspartic proteases display a variety of substrate specificities, but normally they are most active in the cleavage of peptide bonds between two hydrophobic amino acid residues. The preferred substrates of pepsin, for example, contain aromatic residues on both sides of the peptide bond to be cleaved. 

H. Murata, J. L. McEvoy, A. Chatterjee, A. Collmer, A. K. Chatterjee. Molecular cloning of an aepA gene that activates production of extracellular pectolytic, cellulolytic, and proteolytic enzymes in Erwinia carotovora subsp. carotovora. Mol. Plant Micr. Interact. 4 239 (1991). 

Peroxidase activity has long been associated with extracts of plant tissue and the crystalline enzyme from horse radish root has been studied in extenso, particularly in regard to its mechanism of action (11). Plants also contain ferredoxin and various specialized cytochromes, both of which substances play an essential role in photosynthesis (95, 96). Agavain, a crystalline proteolytic enzyme from the leaves of Agave,... 

In the quest to find other plants that are suitable as bioreactors, various monocoty-ledonous and dicotyledonous species have been tested. These include corn [16], rice and wheat [17], alfalfa [18], potato [19, 20], oilseed rape [21], pea [22], tomato [23] and soybean [24]. The major advantage of cereal crops is that recombinant proteins can be directed to accumulate in seeds, which are evolutionar specialized for storage and thus protect proteins from proteolytic degradation. Recombinant proteins are reported to remain stable in seeds for up to five months at room temperature [17] and for at least three years at refrigerator temperature without significant loss of activity [25]. In addition, the seed proteome is less complex than the leaf proteome, which makes purification quicker and more economical [26]. 

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