Abstract
β-glucosidases (BGs) from Aspergillus fumigatus, Aspergillus niger, Aspergillus oryzae, Magnaporthe grisea, Neurospora crassa, and Penicillium brasilianum were purified to homogeneity, and investigated for their (simultaneous) hydrolytic and transglycosylation activity in samples with high concentrations of either cellobiose or glucose. The rate of the hydrolytic process (which converts one cellobiose to two glucose molecules) shows a maximum around 10–15 mM cellobiose and decreases with further increase in the concentration of substrate. At the highest investigated concentration (100 mM cellobiose), the hydrolytic activity for the different enzymes ranged from 10% to 55% of the maximum value. This decline in hydrolysis was essentially compensated by increased transglycosylation (which converts two cellobiose to one glucose and one trisaccharide). Hence, it was concluded that the hydrolytic slowdown at high substrate concentrations solely relies on an increased flow through the transglycosylation pathway and not an inhibition that delays the catalytic cycle. Transglycosylation was also detected at high product (glucose) concentrations, but in this case, it was not a major cause for the slowdown in hydrolysis. The experimental data was modeled to obtain kinetic parameters for both hydrolysis and transglycosylation. These parameters were subsequently used in calculations that quantified the negative effects on BG activity of respectively transglycosylation and product inhibition. The kinetic parameters and the mathematical method presented here allow estimation of these effects, and we suggest that this may be useful for the evaluation of BGs for industrial use.
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Acknowledgement
We thank Torben Lund and Jacob Krake for the help with the HILIC-ESI-MS setup. This work was supported by funds from the Danish Agency for Science, Technology, and Innovation (Grant no. 2104-07-0028) and the Department of Energy (Award Number DE-FC36-08GO18080).
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The online resource shows in detail how Eqs. 3 and 4 is derived and how SAS is used to obtain the maximum likelihood values of the kinetic parameters. It also shows how calculation of glucose, cellobiose, and triose as a function of time could be determined using a reaction scheme which includes hydrolysis and transglycosylation of cellobiose as well as hydrolysis of triose and product inhibition by glucose.
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Bohlin, C., Praestgaard, E., Baumann, M.J. et al. A comparative study of hydrolysis and transglycosylation activities of fungal β-glucosidases. Appl Microbiol Biotechnol 97, 159–169 (2013). https://doi.org/10.1007/s00253-012-3875-9
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DOI: https://doi.org/10.1007/s00253-012-3875-9