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Process Biochemistry

The saccharide spectrum, as a distribution of fractions of different molecular mass, of sixteen beers was determined by ultracentrifugation using filters with cut-offs of 1, 5, 10 and 50 kDa. The saccharide concentrations in the filtrates were determined by density measurements. The saccharide composition was examined through HPAEC-PAD. The results were compared with the values of classic features of beers. The newly developed method provides additional information of the beers and is a simple and fast tool for exploring the effect of the saccharide spectrum on the industrial characteristics. The results revealed that similar top fermentation beers and similar lager beers have different saccharide spectra.

Background: Cel6A is one of the two cellobiohydrolases produced by Trichoderma reesei. The catalytic core has a structure that is a variation of the classic TIM barrel. The active site is located inside a tunnel, the roof of which is formed mainly by a pair of loops.Results: We describe three new ligand complexes. One is the structure of the wild-type enzyme in complex with a nonhydrolysable cello-oligosaccharide, methyl 4-S-β-cellobiosyl-4-thio-β-cellobioside (Glc)2-S-(Glc)2, which differs from a cellotetraose in the nature of the central glycosidic linkage where a sulphur atom replaces an oxygen atom. The second structure is a mutant, Y169F, in complex with the same ligand, and the third is the wild-type enzyme in complex with m-iodobenzyl β-D-glucopyranosyl-β(1,4)-D-xylopyranoside (IBXG).Conclusions: The (Glc)2-S-(Glc)2 ligand binds in the -2 to +2 sites in both the wild-type and mutant enzymes. The glucosyl unit in the -1 site is distorted from the usual chair conformation in both structures. The IBXG ligand binds in the -2 to +1 sites, with the xylosyl unit in the -1 site where it adopts the energetically favourable chair conformation. The -1 site glucosyl of the (Glc)2-S-(Glc)2 ligand is unable to take on this conformation because of steric clashes with the protein. The crystallographic results show that one of the tunnel-forming loops in Cel6A is sensitive to modifications at the active site, and is able to take on a number of different conformations. One of the conformational changes disrupts a set of interactions at the active site that we propose is an integral part of the reaction mechanism.

Trichoderma reesei degrades native cellulose utilizing a set of cellulolytic enzymes dominated by two cellobiohydrolases, CBHI and CBHII. These enzymes exhibit the typical two domain architecture of fungal cellulases, and both act primarily as exoglucanases liberating cellobiose from the ends of the polymeric cellulose chains. The three dimensional structures of the catalytic domains of CBHI and CBHII revealed that their active sites are situated in tunnels formed by long loops on the enzymes surface. The active sites of homologous endoglucanases lack these loops and have more open active sites permitting catalytic activity in the internal positions of cellulose chains. Site-directed mutagenesis and structural studies have identified the key catalytic residues of both CBHI and CBHII. Similarly, the primary interaction surface of the cellulose-binding domain has been defined and residues responsible for its tight binding to cellulose identified.