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  • Enzyme activity loss during the hydrolysis


    Enzyme activity loss during the hydrolysis process has traditionally been associated with thermal, mechanical, and/or chemical mechanisms (Okino et al., 2013, Ye et al., 2012, Zhang et al., 2010). Due to the recent discovery of enzyme activity loss due to interaction with substrate, it is important to understand the relative contributions of enzyme–substrate interactions and all combined environmental sources (the net result of all contributions of the processing environment other than the substrate) to activity loss. The following experiments were designed to address this objective. First, activity loss was determined and normalized as a function of substrate loading, and compared with cellulase incubated without substrate. Then, three independent metrics were compared to quantify the relative extents of inactivation: (1) Relative activity loss from enzyme–substrate interactions and from environmental sources. (2) The apparent guanylyl cyclase and residual activity of enzyme following interaction with substrate and from environmental inactivation mechanisms. (3) The inactivation rate constant due to enzyme–substrate interactions and due to environmental sources.
    Results and discussion
    Conclusions Relative extents of activity loss from enzyme–substrate interactions and from all combined environmental mechanisms were compared. Three independent metrics were quantified to demonstrate this: (1) Relative extents of inactivation, where the majority effect in activity loss was shown to be due to enzyme–substrate interactions. (2) The apparent half-life of enzyme following interaction with substrate was 1.37–11.01h, compared to 21.5h for all combined environmental inactivation. (3) The inactivation rate constant for enzyme–substrate interactions () was about 46 times higher compared to the inactivation rate constant of all combined environmental mechanisms (). In conclusion, enzyme–substrate interactions contributed more towards the overall inactivation than all combined environmental mechanisms.
    Acknowledgements The authors thank Dr. Andrew N. Lane of the James Graham Brown Cancer Center, University of Louisville, for his helpful discussions and technical advice and Genencor International, Inc. for providing the Spezyme CP cellulase. This work was funded by the United States Department of Energy, award number DE-FC36-046014221.
    Introduction Eleven million tonnes of waste are produced yearly by the European pulp and paper industry (Monte et al., 2009). Approximately 70% of these originate from manufacturing tissue paper from recovered fibre, leading to the generation of considerable amounts of deinking sludge (150 kg dry solids/t paper manufactured), which must be properly managed to avoid negative effects on the environment (Deviatkin et al., 2016). Deinking sludge (DIS) exists as a mixture of short cellulosic fibers and inorganic fillers, such as calcium carbonate and china clay, and residual chemicals dissolved in water (Likon and Saarela, 2012). DIS originating from printed recycle mills is high in ash content compared to sludge originating from corrugated recycle mills and virgin pulp mills (Boshoff et al., 2016). Traditional methods of DIS management include landfilling, landspreading, composting, incineration and pyrolysis, utilisation as construction material and landfill capping material (Likon and Trebše, 2012). However, due to high moisture content some of these recovery methods, such as incineration and pyrolysis, are expensive for large amounts of sludge while the environmental impact of others is questionable due to the possibility of hazardous substances leaking into the environment. As a result, numerous possibilities for biological valorisation of paper sludge waste, including its fermentation and anaerobic digestion are currently being explored (Gottumukkala et al., 2016). These aim for efficient microbial transformation of cellulose waste into bioethanol (Boshoff et al., 2016), biomethane (Mohan et al., 2016), biohydrogen or other value added chemicals (Liguori and Faraco, 2016).