Cello‐Oligosaccharide Hydrolysis by Cellobiohydrolase II from Trichoderma Reesei

Abstract

The hydrolysis of soluble cello-oligosaccharides, with a degree of polymerisation of 4-6, catalysed by cellobiohydrolase I1 from Trichoderma reesei was studied using 'H-NMR spectroscopy and HPLC. The experimental progress curves were analysed by fitting numerically integrated kinetic equations, which provided cleavage patterns and kinetic constants for each oligosaccharide. This analysis procedure accounts for product inhibition and avoids the initial slope approximation. No glucose was detected at the beginning of the reaction indicating that only the internal glycosidic linkages are attacked. For cellotetraose only the second glycosidic linkage was cleaved. For cellopentaose and cellohexaose the second and the third glycosidic linkage from the non-reducing end were cleaved with approximately equal probability. The degradation rates of these cello-oligosaccharides, 1 -12 s-' at 27"C, are about 10-100 times faster than for the 4-methylumbelliferyl substituted analogs or for cellotriose. No intermediate products larger than cellotriose were released. The degradation rate for cellotetraose were higher than its off-rate, which accounts for the processive degradation of cellohexaose. A high cellohexaose/enzyme ratio caused slow reversible inactivation of the enzyme. sites of cleavage, hydrolysis stereochemistry, kinetic and binding constants as well as in in vitro mutagenesis studies [6-81. These studies have been performed mostly using 4-methylumbelliferyl p-D-glycosides derived from cellotriose, cellotetraose and cellopentaose. The binding data have been obtained by fluorescence titration techniques, which have provided association constants also for cello-oligosaccharides by means of competition experiments [9, 101. NMR spectroscopy is well suited to follow the stereochemistry of saccharides 11 I] and we have recently used it together with progress curve analysis to study the hydrolysis of cellotriose by 7: reesei CBHII [12]. Replacing the initial-rate approximation by the complete progress curve analysis was highly advantageous, since it allows the elucidation of more complex mechanisms and reduces the amount of enzyme needed .In the present work the hydrolysis of cellotetraose, cellopentame and cellohexaose by CBHII has been studied by means of proton NMR spectroscopy and HPLC while using progress curves to analyse the kinetic data.Correspondence to V. Harjunpaa, VTT Chemical Technology, P. 0.

Toward an aggregated understanding of enzymatic hydrolysis of cellulose: Noncomplexed cellulase systems

“…Thus, the specific activity of CBH2 on cellohexaose at 27jC (k cat = 14 s À1 ; Harjunpaa et al, 1996;Koivula et al, 1998Koivula et al, , 2002) is quite comparable to that for Aspergillus awamori glucoamylase on maltohexaose (G 6 ) at 45jC (49 s À1 ; Fierobe et al, 1998), particularly when the different measurement temperatures are considered. The 3.5-fold higher value of k cat observed for glucoamylase at 45jC relative to CBH at 27jC is very close to what would be expected based on the widely observed trend of doubled activity for every 10jC increase in temperature (Godfrey and West, 1996).…”

Processivity, Synergism, and Substrate Specificity of Thermobifida fusca Cel6B

“…A different ratio, G2/(G1 ϩ G3) was used to measure processivity for T. reesei exocellulase Cel7A (32) as G1 was assumed to be released only from the initial attack (G3 hydrolysis by Cel7A was not addressed in this study). High processivity means that on May 9, 2018 by guest http://aem.asm.org/ the enzyme has been optimized for the movement of a cellulose chain in the active site, but this change can reduce hydrolysis activity on easily diffusible soluble substrates (8), which is in agreement with the slow hydrolysis of G6 by the N282A mutant enzyme. Although the individual mutant enzymes had higher activity with FP than the wild-type enzyme, mixtures of these enzymes with T. fusca Cel5A did not show increased synergism with FP.…”

Aromatic Residues in the Catalytic Center of Chitinase A from Serratia marcescens Affect Processivity, Enzyme Activity, and Biomass Converting Efficiency

“…Kinetic experiments on two processive enzymes (the cellobiohydrolase CBHII and ChiB) using oligomeric substrates have shown that the "sticky" character of the binding gives an off rate, k off , that is much lower than k cat (Ref. 57 and Norberg et al, 3 respectively). In terms of processivity, low off rates may be translated into a higher probability for sliding rather than dissociation of the substrate.…”