Monday, August 13, 2012
Columbia Hall, Terrace Level (Washington Hilton)
Many cellulolytic organisms secrets large population of free carbohydrate binding modules (CBMs), which are not components of catalytic cellulases. It has been hypothesized that these modules may destabilize, swell or lose crystalline structure of polysaccharides thereby assisting enzymatic cleavage of them. In the present study CBM33-2 and family 5 endoglucanase (Cel5), from Hahella chejuensis KCTC 2396 were cloned, expressed, purified and characterized in E.coli. The CBM33-2 is 60kDa consisting of two domains, CBM33 and CBM2. The endoglucanase (Cel5) is a metalloenzyme, having molecular weight 67kDa with N terminal family -5 Glycoside Hydrolase (GH5) and two CBM6 modules at C terminal. TLC analysis of hydrolysis products indicates processive cleavage of cellulosic substrates. CBM33-2 showed binding with broad range of cellulosic substrates and chitin. The optimum pH and temp for binding was 6.5 and 45ºC. It enhanced cellulolytic activity of endoglucanase Cel5 on insoluble substrates by 3-5 folds. Scanning Electron Microscope observation of cellulosic substrate treated with CBM33-2 showed loosening, swollening, and destabilization of crystalline structure of cellulose. X-ray diffraction analysis indicated 8-10% reduction in crystallinity index thus improved cellulase adsorption by 5-10% .CBM33-2 also presented 50% enhancement in chitinase activity of Streptomyces griseus chitinase. This report provides distinct approach for study of the free CBMs and the synergistic action of it in degradation of crystalline polysaccharides.
This work was supported by a grant from the International Collaborative R&D Program of Knowledge Economy Technology Innovation Program, MKE, and a grant (NRF-2010-C1AAA001-0029084) from the National Research Foundation, MEST, and BK21 Program of Korea.