Hansol Kim

© 2015 Walker et al.Background: Carbohydrate binding modules bind polysaccharides and help target glycoside hydrolases catalytic domains to their appropriate carbohydrate substrates. To better understand how CBMs can improve cellulolytic enzyme reactivity, representatives from each of the 18 families of CBM found in Ruminoclostridium thermocellum were fused to the multifunctional GH5 catalytic domain of CelE, which can hydrolyze numerous types of polysaccharides including cellulose, mannan, and xylan. Since CelE is a cellulosomal enzyme, none of these fusions to a CBM previously existed. Results: CelEcc-CBM fusions were assayed for their ability to hydrolyze cellulose, lichenan, xylan, and mannan. Several CelEcc-CBM fusions showed enhanced hydrolytic activity with different substrates relative to the fusion to CBM3a from the cellulosome scaffoldin, which has high affinity for binding to crystalline cellulose. Additional binding studies and quantitative catalysis studies using nanostructure-initiator mass spectrometry were carried out with the CBM3a, CBM6, CBM30, and CBM44 fusion enzymes. In general, and consistent with observations of others, enhanced enzyme reactivity was correlated with moderate binding affinity of the CBM. Numerical analysis of reaction time courses showed that CelEcc-CBM44, a combination of a multifunctional enzyme domain with a CBM having broad binding specificity, gave the fastest rates for hydrolysis of both the hexose and pentose fractions of ionic-liquid pretreated switchgrass. Conclusion: We have shown that fusions of different CBMs to a single multifunctional GH5 catalytic domain can increase its rate of reaction with different pure polysaccharides and with pretreated biomass. This fusion approach, incorporating domains with broad specificity for binding and catalysis, provides a new avenue to improve reactivity of simple combinations of enzymes within the complexity of plant biomass.

© 2015. American Geophysical Union. All Rights Reserved. The impacts of droughts on the Amazon ecosystem have been broadly discussed in recent years, but a comprehensive understanding of the consequences is still missing. In this study, we show evidence of a fragile hydrological equilibrium in the western Amazon. While drainage systems located near the equator and the western Amazon do not show water deficit in years with average climate conditions, this equilibrium can be broken during drought events. More importantly, we show that this effect is persistent, taking years until the normal hydrological patterns are reestablished. We show clear links between persistent changes in forest canopy structure and changes in hydrological patterns, revealing physical evidence of hydrological mechanisms that may lead to permanent changes in parts of the Amazon ecosystem. If prospects of increasing drought frequency are confirmed, a change in the current hydroecological patterns in the western Amazon could take place in less than a decade.