Cathepsin D, a muscle proteinase, participates in lysosomally mediated protein degradation in vivo. This enzyme has been proposed to play a significant role in the postmortem proteolysis process apparently associated with tenderisation. The lack of data on the postmortem characteristics of ostrich meat, especially on the ageing process and its influence on meat tenderness, called for an investigation into this process. There is no data available for purified ostrich cathepsin D, and the aim of this study was, therefore, to isolate, purify and characterise cathepsin D from ostrich skeletal muscle and subsequently investigate the possible role that it may have in the tenderisation process of meat. Cathepsin D was successfully isolated and purified from ostrich skeletal muscle using pepstatin A-agarose chromatography. The purified enzyme was composed of two subunits (14 and 29kDa). The amino acid composition as well as the N-terminal amino acid sequence of both subunits were determined. Kinetic parameters (Km and Vm), thermodynamic parameters (Ea, ∆H, ∆S and ∆G) and functional characteristics (effect of pH, temperature and various inhibitors on cathepsin D activity) were determined and are reported in this study. Ostrich muscle cathepsin D showed a pH optimum of 4 and a temperature optimum of 45°C. The activity of cathepsin D was strongly inhibited by pepstatin A and DTT. Purified ostrich cathepsin D displayed kinetic and functional properties similar to previously reported values from various species. The effect of storage on the activity of cathepsin D was investigated over a 30 day period. It was established that substantial postmortem cathepsin D activity remained throughout the storage period, to implicate cathepsin D, fulfilling a possible role in meat maturation.

The ostrich is becoming an important source of meat for humans in developed and developing countries. This study was conducted to purify and characterize myofibrilbound serine protease (MBSP) and its endogenous inhibitor (MBSPI) from skeletal muscle of the ostrich. It is well documented that MBSP is tightly bound to myofibrils and its endogenous inhibitor has been purified from the same tissue of other studied mammalian species. Literature supports an association of MBSP and its endogenous inhibitor with the degradation of myofribrillar proteins, resulting in the softening of muscle that lead to the conversion of muscle into meat with the control of the inhibitor. MBSP was successfully dissociated from washed myofibrils by 40 percent ethylene glycol at pH 8.5. Following centrifugation, MBSP was partially purified in two chromatographic steps, namely Toyopearl Super Q 650S and p-aminobenzamidine-Agarose. On the other hand, MBSPI was fractionated from the sarcoplasmic fraction using 75 percent ammonium sulfate saturation, followed by centrifugation and partially purified by three chromatographic steps, namely Toyopearl Super Q 650S, Superdex 200 and HiTrap SP HR. Ostrich MBSP was physicochemically and kinetically characterized, while MBSPI was only physicochemically characterized. Ostrich MBSP revealed an Mr of 21 kDa, cleaving synthetic fluorogenic substrates specifically at the carboxyl side of arginine residues. Optimum pH and temperature of ostrich MBSP were 8.0 and 40˚C, respectively. Kinetic parameters (Km and Vmax values) were calculated from Lineweaver-Burk plots. The characteristics of ostrich MBSP were compared to the values obtained for commercial bovine trypsin in this study, as well as that obtained for MBSP from various fish species and mouse. The results suggest that ostrich MBSP is a trypsin-like serine protease, thereby confirming the existence of MBSP in ostrich skeletal muscle. Partially purified ostrich MBSPI (Mr 17 kDa) (one form) shares 100 percent identity to myoglobin from the same species, while 2 other forms of MBSPI (Mr values of 35 and 36 kDa) exhibited high sequence identity to glyceraldehyde 3- phosphate dehydrogenase (GAPDH) (76 percent) from human and rat.

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. / Land and Food Systems, Faculty of / Graduate

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LD2668 .T4 1948 D5 / Master of Science

A heat stable protease was identified as the cause of textural degradation in cooked arrowtooth flounder (Atheresthes stomias) muscle. Maximum proteolytic activity in the fish muscle was observed between 55°C and 60°C and myosin heavy chain appeared to be the primary substrate for the enzyme. Degradation of this myofibrillar protein at 55°C was extremely rapid and myosin heavy chain was completely hydrolyzed to peptide fragments smaller than actin, while actin itself was unaffected. A single strand 32kD proteolytic enzyme was extracted from the muscle and purified 125-fold. The enzyme was stable to freezing for up to 6 months. Activity of the semi-purified enzyme at 55°C was optimal against casein between pH 6.0 and 7.0. Sulfhydryl reagents p-chloromercuriphenylsulfonic acid, iodoacetate, iodoacetamide and cystatin were effective in inhibiting enzyme activity in casein assays. The serine protease inhibitors phenylmethylsulfonylfluoride and trypsin-chymotrypsin inhibitor appeared to activate enzyme activity against casein. Adenosine triphosphate was also an activator. Arrowtooth flounder was then considered as a raw material for surimi, since the surimi process provides for repeated washing of the minced muscle and a final mixing step during which inhibitory substances can be conveniently added. Arrowtooth muscle was monitored at all stages of surimi production. There was no evidence of myosin degradation on sodium dodecyl sulphate polyacrylamide electrophoretic gels at any time during surimi production or during the preparation of samples for testing. However, when the washed mince was incubated at 55°C, 12% residual proteolytic activity was observed. This level was sufficient to degrade the myosin component of surimi gels prepared from the control surimi to which no inhibitors had been added. The food grade substances tested for proteolytic inhibition were bovine blood plasma powder, egg white powder, whey protein concentrate, carrageenan and crude α₂-macroglobulin. Addition of plasma and/or egg white powders to control surimi resulted in a product that was comparable to pollock in functional properties as measured by gel strength, expressible moisture and fold tests. Electrophoretic comparison of surimi made with 1.0% or 2.0% plasma powder or egg white with surimi produced with 0.1% or 0.2% α₂-macroglobulin suggested that the plasma and egg white contributed gel enhancing effects in addition to protease inhibition. Carrageenan was not effective as either a protease inhibitor or gel enhancer. / Graduation date: 1992

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The main site for protein degradation along the endosomal pathway is believed to be the late endosome. Lysosomes are thought to be storage organelles that, when necessary, inject proteases into the late endosome. It was hypothesised that differences in the lumenal redox environments between the two organelles could be responsible for their functional differences. In an attempt to quantify this potential difference, the lysosomal cysteine protease cathepsin B was isolated by an improved purification procedure. Several intracellular reducing agents were used to activate cathepsin B, the most effective being cysteine. Cysteine was used to activate cathepsin B under various pH conditions in order to model endosomal conditions. An inverse relationship was found between the pH and the concentration of cysteine required to activate cathepsin B. This suggested that cathepsin B may have an optimal redox potential. In order to determine this potential, cysteinexystine redox buffers were made up and used in determination of the activity of the enzyme against a synthetic and a whole protein substrate (haemoglobin). No distinct redox potential could be determined using either substrate, but it was found that cystine stimulated proteolysis of haemoglobin. A similar stimulatory effect was observed for cathepsin D and papain hydrolysis of haemoglobin. This effect is possibly due to the ability of cystine to promote substrate structure, effectively increasing the substrate concentration. These findings and other results obtained from the literature have been used to create a model of how proteolysis may be regulated along the endosomal system. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1999.