Characterisation of cysteine proteases and their catalytic impact on meat myofibril and meat connective tissue proteins

Meat is a major dietary component consumed by many cultures over thousands of years. Research has indicated that meat consumers are willing to pay a premium price for tender meat cuts, however, producing meat with consistent tenderness has been a challenge for the meat industry. Several endogenous meat enzymes such as calpains, cathepsins and caspases; and exogenous cysteine proteases from plant, bacterial and fungal sources have been shown to be capable of hydrolysing meat proteins. However, the composition and the activity of commercial enzyme preparations vary among different suppliers, and to our knowledge the activities of the different available exogenous enzymes have not been comprehensively characterized in the same study before. Therefore, the present study aimed at characterizing several plant and bacterial commercial proteases from Australasian region. This study utilised various synthetic substrates (CBZ-Lys-ONp, BODIPY-FL casein and Azocoll) to characterise comparatively various enzyme extracts from both in-house and commercial preparations [in-house prepared kiwifruit and asparagus enzyme extracts, and commercial enzyme extracts papain (from papaya latex), bromelain (from pineapple fruit), actinidin (from kiwifruit), ginger protease (from ginger rhizome), proteases 31K and 60K (from fungi) and protease G (from bacteria). This study also examined the effect of these exogenous enzyme extracts on meat myofibril and meat connective tissue extracts using 1D-SDS-PAGE in tandem with mass spectrometry, and some preliminary tenderising experiments on meat blocks. Assays with commercially available substrates CBZ-Lys-ONp and BODIPY-FL casein demonstrated that the highest ester bond and amide bond hydrolysing activities were found in the vicinity of pH 5.0-7.0 and at temperatures of 45-75°C for ester bond hydrolysis and 45°C for amide bond hydrolysis. A low level of activity was also found in most enzyme extracts near the meat ageing storage temperature of 4°C. In addition, the hydrolytic activity of a relatively newly discovered asparagus protease from green asparagus stem was also assessed and compared to other well-studied proteases such as papain (from papaya latex), bromelain (from pineapple fruit) and actinidin (from kiwifruit). Kinetic analysis with BODIPY-FL casein as a substrate showed that the maximum velocity (Vmax) and substrate binding affinity (KM) differed significantly between the enzyme extracts. The protease extracts from fungal and bacterial sources appeared to have a relatively higher casein binding affinity (1.3-3 μM) in comparison with the plant protease extracts. Amongst the plant enzyme extracts, the in-house prepared kiwifruit extract had the highest KM of 1.4 μM, while the commercial bromelain enzyme extract had the lowest KM of 0.01 μM. In addition, a significant variation in Azocoll (azo-impregnated collagen) hydrolysis activity was also found amongst the enzyme extracts. The meat myofibril and meat connective tissue protein hydrolysing activity of the enzyme extracts was assessed comparatively with purified meat myofibril and meat connective tissue protein extracts by 1D-SDS-PAGE and mass spectrometry analysis. While some of the enzyme extracts, such as the in-house prepared Kiwifruit, commercial papain and bromelain extracts, hydrolysed myofibril proteins indiscriminately, other proteases, such as the in-house prepared asparagus and commercial fungal and bacterial enzyme extracts, were more selective. Added free cysteine appeared to enhance the activity of most of the enzyme extracts, however, the effect of free cysteine on specific protein hydrolysis varied between the enzyme extracts. Unlike free cysteine, assays with L-ascorbic acid iso-ascorbic acid revealed that these compounds could behave as either activators or inhibitors, depending on the enzyme extract and the concentration of the ascorbic isoform added to the enzyme-substrate mixture. Similar results were also obtained with assays with meat connective tissue proteins. In addition, the effect of meat juice extract components on the hydrolytic activity of the enzyme extracts was preliminarily investigated with Azocoll. Assays with the commercial bromelain extract revealed that inhibiting compounds were found in both unfiltered and filtered (with 10-kDa cut-off limit) fractions of the meat juice extract, indicating that inhibiting compounds of different sizes were present in meat. Preliminary meat tenderisation trials were carried out with meat blocks. The in-house prepared Kiwifruit extract appeared to be capable of hydrolysing meat myofibril proteins as measured by shear force measurement and 1D-SDS-PAGE analysis, however, hydrolysis of intact meat appeared to be more selective than that in the assays with purified myofibril proteins, which was likely related to the increased structural complexity of intact meat and protease inhibitor(s) present in meat. In addition, a lower compression force value was found in meat block samples injected with the kiwifruit extract compared to control samples even after one day of ageing, suggesting that connective tissue proteins were also hydrolysed by the enzyme extract. Moreover, 1D-SDS gels also revealed that various soluble proteins, which contained sarcoplasmic proteins and possibly myofibril solubilised proteins, were degraded during meat ageing with the kiwifruit extract. Further investigations are required to optimise the tenderising effect from other exogenous enzyme extracts