Proteolytic and Clot Lysis Activity Screening of Crude Proteases Extracted from Tissues and Bacterial Isolates of Holothuria Scabra

Abstract

Cardiovascular diseases (CVDs) are still the leading mortality causes in the last decades. The ailments are multifactorial characterized by excessive clot (thrombus) formation in the blood vessels. Thrombus could be degraded through thrombolysis mechanism by plasmin activated by various fibrinolysis agents including, urokinase, nattokinase, or streptokinase. However, the use of these agents is restricted by relatively high cost, short half-life, allergic reaction, and bleeding effects. The search for more economical and safer thrombolytic (clot lysis) agents are essential to address the underlying problem in CVD therapy. Among Holothurians, H. scabra has been known to have the highest protein content making it ideal substrate for protease enzymes including fibrinolytic types with clot lysis properties. However, isolation of a proteases with antithrombotic activities either from tissue or from bacteria of H. scabra has not been reported. This study aimed to screen proteolytic and clot lysis activities of crude protease extracts from tissue and bacteria isolated from fermented intestine of H. scabra. Crude protease of tissue of H. scabra was extracted by cold centrifugation, which activity was measured using UV-spectrophotometer. Crude protease of proteolytic bacteria selected by clear zones on skim milk agar (SMA) medium was isolated from nutrient broth (NB). Extracts showing proteolytic activity were subjected to gravimetry-based clot lysis test. As results, crude proteases isolated directly from the H. scabra’s tissue showed low proteolytic activities, thus were no proceed to clot lysis activity test. Crude protease extracted bacteria could show both proteolytic and clot lysis activities. In conclusion, based on this screening study, intestine of H. scabra is a rich source of proteolytic bacteria, some of them could produce crude enzymes showing competitive thrombolysis activities with Nattokinase. Thus, they appeared to have more potentials to be developed as thrombolysis agent than those directly extracted from the organism’s tissue.

Optimization of Crude Protease Production from Bacillus thuringiensis HSFI-12 and Thrombolytic Activity Its Enzyme Dialysate

“…The aim was to determine whether bacteria have the potential to produce proteases or not. The presence of proteolytic activity is indicated by the appearance of a clear zone around the bacteria [13,16] as shown in Figure 6. The clear zone formed around the colony was due to the ability of bacterial protease to degrade the casein substrate contained in the skim milk agar media.…”

“…After obtaining a single isolate resulting from bacterial purification on NA media, the next step was to test the production of protease enzymes in Skim Milk Agar (SMA) producer media. Bacteria growing on NA media were scratched on SMA media and incubated for 24 h at 37 ℃ and then observed the formation of a clear zone around the colony growth [13,16,23].…”

“…Tube 1 was added to 100 L of PBS (negative control), tube 2 was added to 100 L of Nattokinase (positive control), tubes 3 -6 were added to 100 L of pure enzyme. The samples were then incubated at 37 ℃ overnight [13,23,29].…”

“…Sources of protease enzymes include plants (43.85 %), bacteria (18.09 %), fungi (15.08 %), animals (11.15 %), algae (7.42 %) and viruses (4.41 %) [11,12]. Isolation of bacteria from the digestive organs of sea cucumbers to obtain bacterial protease to be used as a thrombolytic agent had been conducted by Hidayati et al [13]. One the most promising bacterial isolates producing thrombolytic protease obtained was strain HSFI-12.…”

“…Crude enzyme Holothuria scabra Fermented Intestine-12 (HSFI-12) isolate had shown higher thrombolytic ability than that of Nattokinase as positive control [13]. Meanwhile, incubation time greatly affect the growth of enzyme producing bacteria [14].…”