Study On Enzymological Characterization And Anti-cancer Effect Of Tenebrio Fibrinolyric Proteins

Tenebrio molitor containing rich protein and other nutritional ingredients, was a kind of precious edible insect resources,so Tenebrio molitor had been used widely in food, health care products. medicine and feed and so on.Adds on the advantages of strong survivability and adaptiveness, quick growth, low cultivation cost, Tenebrio molitor had already become the third fastest growing worm-raising industry in the world only after silkworms-raising and beekeeping. We had already proved that one kind of active protein extracted from Tenebrio molitor in vivo, worked very well in dissolving the thrombosis. However, cancer and cardiovascular disease were currently the world's two great life killer,so we hoped that through further studies on this active protein,we could provide more complete basic experimental data, for later discover of an efficient, safe and low-cost drugs.A novel of fibrinolytic protein named Fibrinolyric Tenebrio Proteins (TFP), was separated and purified from Tenebrio molitor by the methods of homogenate leaching mention, salting, anion exchange and glucan gel filtering method. Fiber tablet method showed TFP had good fibrinolytic activity;The natural electrophoresis prooved its purity of a single component; The relative molecular weight was about 56.1 kD measuered by the degeneration electrophoresis;Protein concentration was 6.061 mg·mL-In order to further understand the enzymological characterization, this paper measured different Michaelis constant(Km),by the effect of TFP on three kinds of substrates (BAEE, BAPNA and casein). Using BAEE as the substrate.the Km was 0.182 mmol/L,which was significantly less than the Km(1.138 mmol/L),when BAPNA was used as the substrate.However, when casein was used as the substrate, the Km wasn't measured out. The action of TFP on BAEE showed sterase activity; The action of TFP on BAPNA showed activity of non-peptide bond amide; The action of TFP on casein showed peptidase activity,so we reached a conclusion:esterase activity>Activity of non-peptide bond amide>Peptidase activity.Simulating intestinal environment-partial alkaline, TFP wasn't degradated by trypsin,and could be bowel soluble preparations. To determine its enzymological classification.we measured the inhibition of fibrinolytic activity of TFP by PMSF.the inhibitor of serine proteinase.and the results showed that 5mmol/L PMSF could fully inhibit TFP fibrinolytic activity,and the inhibition was positively correlated with concentration of PMSF. Meanwhile,the test results of IR showed that the structure of TFP and trypsin were very similar, and trypsin belonged to serine protease, thus we preliminarily inferred TFP also belonged to serine protease based on their strict conservative with protease active site in their species evolutionary.This paper also maked preliminary study of the anti-cancer effects of TFP by culturing animal cells in vitro, and the results of MTT method detection showed that TFP had obvious inhibition on the tumor cells A549,but had no apparent effect on H22 cells. In addition, comparing the inhibition of TFP and cisplatin(anti-cancer drugs) on tumor cells A549, it was concluded that the fomer worked rapidly, and could achieve good inhibition on tumor growth in short time.which was the incomparable advantages of other anti-cancer drugs. Maybe both mechanism was different and sill need further research.