Aktivitas kitooligomer hasil reaksi enzimatik terhadap proliferasi sel limfosit dan sel kanker

Abstract

Local chitin waste from crab industries can be used as a source for production of chitooligomers which has important biological activity. The aims of this research were to evaluate activities chitooligomers produced by enzymatic hydrolysis upon proliferation of lymphocyte and cancer cells. The chitosanase enzyme was obtained from thermophilic bacterium Bacillus licheniformis MB2 isolated from Tompaso Manado. The medium for producing the enzyme contained 1% colloidal chitosan and the enzyme was harvested after seven days of incubation at 55oC, The free cell supernatant were heated at 60 oC for 20 minutes. The heat stable protein enzyme was coagulated with 80% saturated ammonium sulphate and purified using hydrophobic interaction chromatography with butyl sepharose gel. The enzyme of 0.005, 0.0085, 0.10 dan 0,17 IU/mg chitosan were use on 1% soluble chitosan substrate with 85 and 90 % degree deacetylation to produce chitooligomers through incubation at one and three hours. The reaction products were analyzed and fractionated using HPLC. The effect of chitooligomers hidrolysate on normal cells and cancer cells was evaluated using lymphocyte cells previously isolated from human blood and cancer cells line : KR4 (lymphablastoid B), K-562 (chronic myelogenous leukimia), HeLa (epythel carcinoma cervix), and A549 (lung carcinoma ). The inhibition of cancer cells proliferation was visualized by spectrophotometer after addition of MTT reagent. At concentration of 17 ìg/ml the chitooligomers inhibited the cancer cells KR4 by 12.27%, K562 by 20.58%, HeLa by 31.72%, and A549 by 22.70%. In general, hidrolysate and fractionated chitooligomers (trimer to hexamer) showed better anticancer activity than 2-Bromo deoxy uridine used as positive control at similiar concentration. The inhibition 60% of lymphocyte cells was found when treated with chitooligomers hidrolysate from chitosan with 70% degree deacetylation. Fractionated chitooligomers (trimer to hexamer) 20% inhibited the lymphocytes. However, chitooligomers hidrolysate from chitosan with 85 and 90% degree deacetylation enhanced lymphocyte proliferation about 184.54%. K562, HeLa and A549 cancer cells treated with chitooligomers appeared to undergo apoptosis by 35%, 27%, and 8% respectively as shown by fluorescent microscopy. Disrupted membrane was found on cells treated with chitooligomers as observed by scanning electron mycroscope.