Enzymes hydrolysing wood polysaccharides:A progress curve study of oligosaccharide hydrolysis by two cellobiohydrolases and three b-mannanases: Dissertation

Cellulose and hemicelluloses are major structural components of plant cell walls. Cellulases and hemicellulases are the enzymes which hydrolyse these biopolymers to small soluble oligosaccharides in nature. The enzymatic degradation processes are complex because of the need for different enzyme activities in the total degradation of the substrate. These degradation processes have been intensively studied since the 1970s, but there are still many unanswered questions concerning the degradation mechanisms. In the present investigation only pure soluble oligosaccharides, both cello-oligosaccharides and manno-oligosaccharides, were used to study the hydrolysis of substrates catalysed by the cellobiohydrolases, CBHI and CBHII, and the b-mannanases, BMANI and BMANII, of Trichoderma reesei and one b-mannanase of Aspergillus niger. Hydrolysis experiments were performed using oligosaccharides with chain lengths of three to six sugar units. These reactions were monitored as a function of time and analysed by different independent analytical techniques, i.e. high performance liquid chromatography (HPLC), nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS). Experimental kinetic data were evaluated using progress-curve analysis, for which tailor-made computer programs were used. NMR spectroscopy was used to study the stereochemical course of the enzyme reactions in order to determine the stereospecificities of the enzymes. The three b-mannanases used in the study behaved in a similar way and they belong to the same glycosyl hydrolase family. Both HPLC and NMR spectroscopy were used to study the cleavage patterns as well as to produce experimental progress curves for each substrate. According to the HPLC and NMR data, CBHI releases cellobiose from the reducing end of cellotriose, whereas CBHII releases cellobiose from the non-reducing end. Progress curves could be accurately fitted by using tailor-made computer programs which can be applied to different enzymes. The rate and binding constants derived from the progress-curve analysis provide a reliable basis for the evaluation of enzyme kinetics. MS was used to screen the molecular mass composition in the reaction mixture during the hydrolysis. According to the MS analyses, no transglycosylation products were produced during the hydrolysis of cellotriose catalysed by CBHI or in the hydrolysis of mannotriose catalysed by BMANI. Interestingly, it was shown that at the early stage of the hydrolysis of cellotetraose, cellopentaose and cellohexaose, CBHI produced an intermediary product which was one glucose unit longer than the substrate. The b-mannanase BMANI produced a transglycosylation product two mannose units longer than the substrate when mannotetraose, mannopentaose and mannohexaose were used as substrates. Moreover, the rate of transglycosylation was determined for the b-mannanase and was found to be the highest rate of all the reactions studied. Interestingly, the transglycosylation rates of these two retaining enzymes of T. reesei showed that the more open active site of BMANI allows much faster transglycosylation than CBHI