Studies on the use of purified CBH I for oligosaccharide synthesis

Abstract

The importance of biologically active carbohydrates has been recognized over the last decade. The availability of cheap oligosaccharides for biological activity studies is very reduced. The isolation of these compounds from natural sources is almost impossible, because of their very high specific activity. and consequently very low concentration in nature. As chemical synthesis is a difficult and time consuming, the enzymatic synthesis has been regarded over the last years as a very attractive methodology for oligosaccharide production. The main approach when utilizing glycanases for di- or trisaccharides synthesis has been the transglycosylation reaction. However, the isolation of products is quite complicated. On the other hand, the condensation reaction by reversed hydrolysis activity, which in many cases requires cheaper substrates. has a very low yield. In this work, a purified exoglucanase CBH I from the fungus Trichoderma reesei was analyzed for its reversed hydrolysis activity. The enzyme was purified by conventional methodologies (preparative isoelectric focusing, gel filtration on Sephacryl 100 HR, anionic exchange on a Mono Q column and cationic exchange on a Mono S column), from a commercial cellulase, Cellulast, from Novo. The activity of the purified enzyme on a large set of substrates, such as lichenan, laminarin, filter paper, acid swollen Avicel, xylan and carboxymethylcellulose was characterized, suggesting that it is basically free of contaminant activities. The enzyme was incubated in aqueous media with high sugar concentrations. Several mono- and disaccharides were used, in order to study the enzyme specificity. The obtained products were analyzed in a Dionex chromatographer using a CarboPac PA-100 column. The separated reaction products were analysed by NMR. The yields of the condensation reaction were in several cases considerably high.