A novel method for the degradation of human blood clot by immobilised bromelain using multi-walled carbon nanotube and polyphenol oxidase

Abstract

Pathological blood clot in blood vessels, which often leads to cardiovascular diseases, are one of the most common causes of death in humans. Therefore, enzymatic therapy to degrade blood clots is vital. To achieve this goal, bromelain was immobilized and used for the biodegradation of blood clots. Bromelain was extracted from the pineapple fruit pulp (Ananas comosus) and purified by ion exchange chromatography after precipitation with ammonium sulphate (0-80 %), resulting in a yield of 70%, purification fold of 1.42, and a specific activity of 1175 U/mg. Bromelain was covalently immobilized on functionalized multi-walled carbon nanotubes (MWCNT), with an enzyme loading of 71.35%. The results of the characterization of free and immobilized bromelain demonstrated that the optimum pH for free and immobilized bromelain activity was 7.0, while the pH range of stability was from 5.0 to 8.5 and 4.0 to 9.0, respectively. The optimum temperature for free and immobilized bromelain activity was 45ºC, whereas the stability was 15 to 50°C and 15 to 55°C, respectively. The immobilized bromelain activity was decreased after the fifth reuse, and the storage period of the free and immobilized bromelain was decreased after 6 and 123 days, respectively. Casein was the best substrate-free bromelain, and fibrin was the best substrate for immobilized bromelain. The results of the purification of polyphenol oxidases (PPO) from potatoes by ion exchange chromatography gave a yield of about 54 %, a purification fold of 1.27, and a specific activity of 2804 U/mg. The current study showed that the immobilized bromelain can significantly biodegrade human blood clots in vitro, while the PPO enzyme has no significant effect on blood clots.