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Lytic polysaccharide monooxygenases (LPMOs) are mononuclear copper enzymes that act in synergy with glycoside hydrolases to saccharify the most abundant polysaccharides in nature. Both O 2 and H 2 O 2 can be cosubstrates for LPMOs. The Lentinus similis LPMO (LsAA9A) has previously been shown to oxidatively cleave oligosaccharides when supplied with ascorbate as a reductant. This study demonstrates that LsAA9A is unable to complete the catalytic cycle and cleave cellulose without H 2 O 2 . Instead, cellooligomers efficiently prevent the slow continuous oxidation of ascorbate taking place under ambient conditions in the absence of a substrate. LsAA9A specifically cleaves cellooligomers in a fast and stoichiometric reaction with H 2 O 2 as a cosubstrate. However, the product profile contains more non-oxidized saccharides than anticipated by the generally accepted LPMO reaction scheme. The scission of glucosidic bonds and oxidation of the saccharide therefore appear not to be directly coupled. This was confirmed by the complete absence of oxidized products under anoxic conditions. A mechanism is proposed involving the hydrolysis of a cellulosic radical formed by a H 2 O 2 -derived caged hydroxyl radical. In addition, LsAA9A catalyzes another stoichiometric reaction with excess H 2 O 2 to oxidize ascorbate in the absence of cellulose. Ascorbate is not a cosubstrate for the reaction leading to the scission of glucosidic bonds by LsAA9A.

A novel electrochemical enzyme biosensor was developed for real-time detection of cellulase activity when acting on their natural insoluble substrate, cellulose. The enzyme biosensor was constructed with pyranose dehydrongease (PDH) from Agaricus meleagris that was immobilized on the surface of a carbon paste electrode, which contained the mediator 2,6-dichlorophenolindophenol (DCIP). An oxidation current of the reduced form of DCIP, DCIPH2, produced by the PDH-catalyzed reaction with either glucose or cellobiose, was recorded under constant-potential amperometry at +0.25V (vs. Ag/AgCl). The PDH-biosensor was shown to be anomer unspecific and it can therefore be used in kinetic studies over broad time-scales of both retaining- and inverting cellulases (in addition to enzyme cocktails). The biosensor was used for real-time measurements of the activity of the inverting cellobiohydrolase Cel6A from Hypocrea jecorina (HjCel6A) on cellulosic substrates with different morphology (bacterial microcrystalline cellulose (BMCC) and Avicel). The steady-state rate of hydrolysis increased towards a saturation plateau with increasing loads of substrate. The experimental results were rationalized using a steady-state rate equation for processive cellulases, and it was found that the turnover for HjCel6A at saturating substrate concentration (i.e. maximal apparent specific activity) was similar (0.39-0.40s(-1)) for the two substrates. Conversely, the substrate load at half-saturation was much lower for BMCC compared to Avicel. Biosensors covered with a polycarbonate membrane showed high operational stability of several weeks with daily use.

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes of industrial and biological importance. In particular, LPMOs play important roles in fungal lifestyle. No inhibitors of LPMOs have yet been reported.In this study, a diverse library of 100 plant extracts was screened for LPMO activitymodulating effects. By employing protein crystallography and LC-MS, we successfully identified a natural LPMO inhibitor.Extract screening revealed a significant LPMO inhibition by methanolic extract of Cinnamomum cassia (cinnamon), which inhibited LsAA9A LPMO from Lentinus similis in a concentration-dependent manner. With a notable exception, other microbial LPMOs from families AA9 and AA10 were also inhibited by this cinnamon extract. The polyphenol cinnamtannin B1 was identified as the inhibitory component by crystallography. Cinnamtannin B1 was bound to the surface of LsAA9A at two distinct binding sites: one close to the active site and another at a pocket on the opposite side of the protein. Independent characterization of cinnamon extract by LC-MS and subsequent activity measurements confirmed that the compound inhibiting LsAA9A was cinnamtannin B1.The results of this study show that specific natural LPMO inhibitors of plant origin exist in nature, providing the opportunity for future exploitation of such compounds within various biotechnological contexts.

We have measured activity and substrate affinity of the thermostable cellobiohydrolase, Cel7A, from Rasamsonia emersonii over a broad range of temperatures. For the wild type enzyme, which does not have a Carbohydrate Binding Module (CBM), higher temperature only led to moderately increased activity against cellulose, and we ascribed this to a pronounced, temperature induced desorption of enzyme from the substrate surface. We also tested a "high affinity" variant of R. emersonii Cel7A with a linker and CBM from a related enzyme. At room temperature, the activity of the variant was similar to the wild type, but the variant was more accelerated by temperature and about two-fold faster around 70°C. This better thermoactivation of the high-affinity variant could not be linked to differences in stability or the catalytic process, but coincided with less desorption as temperature increased. Based on these observations and earlier reports on moderate thermoactivation of cellulases, we suggest that better cellulolytic activity at industrially relevant temperatures may be attained by engineering improved substrate affinity into enzymes that already possess good thermostability. K E Y W O R D SArrhenius equation, Cel7A, cellulase, enzyme inactivation, interfacial enzyme activity, optimal temperature 1 | INTRODUCTION One of the most important concepts in technical applications of enzymes is the bell-shaped relationship between temperature and activity, and in the simplest interpretation, this reflects a balance between two independent processes (Laidler & Peterman, 1979). At moderate temperatures, where the enzyme is stable, the rate increases with temperature in same way as other chemical reactions. We will call this thermoactivation of the enzyme process, and in many cases, it follows an exponential course in concurrence with the canonical Arrhenius equation. In practical terms, thermoactivation may be quantified by the so-called Q 10 -value, which is the fractional growth in reaction rate upon a 10°C increment in temperature. As long as the enzyme is stable, thermoactivation corresponding to Q 10 about two has been commonly reported for enzyme reactions (Elias, Wieczorek, Rosenne, & Tawfik, 2014) although widely differing values are also known (Wolfenden, Snider, Ridgway, & Miller, 1999). At higher temperatures, enzyme inactivation (reversible or irreversible) becomes significant. This diminishes thermoactivation, and ultimately leads to a rapid decline in activity as the enzyme denatures. The resulting maximum in activity defines the optimal temperature, T opt . Although poorly defined because it depends on experimental conditions (e.g., duration of the assay), this parameter has proven practical in comparative discussions of enzymes for technical applications.The bell-shaped course of temperature-activity curves has influenced engineering strategies for industrial enzymes. Thus, a feasible way to speed up a certain enzyme application is to engineer variants with increased stability. This will limit activity lo...