Partial Purification and Characterization of Mannanase Enzyme from Bacillus pumilus

Abstract

Bacillus pumilus isolate is one of bacterial collection from Biotechnology Culture Collection (BTCC) LIPI, isolated from Pari island waters was examined to determine the ability of bacteria to degrade mannan from Locust Bean Gum (LBG). Based on qualitative analysis, this isolate showed the abilities to degrade mannan while the quantitative analysis showed the highest mannanase activity was obtained on the third day of incubation time at 3,2 U/mL with 0,9 U/mg specific activity. Initial purification using ultrafiltration 5000 WMCO showed mannanase activity at 5,1 U/mL with 1,44 U/mg specific activity. Enzyme purification by gel filtration produced a peak in fraction no.31 with mannanase activity at 4,2 U/mL with 4,00 U/mg specific activity. Analysis of hydrolitic products by thin layer chromatography showed that the main products from Locust Bean Gum are manobiose, manotetraose and manohexaose. Maximum activity of the mannanase enzyme produced by B. pumilus isolate occurred at pH 5,0 and temperature of 40°C. This enzyme has 30,17 kDa single protein band when running with SDS PAGE with Michaelis Menten (Km) and maximum velocity (Vmax) at 0,076 mg/mL and 0,02 U/mg respectively.