The reserves of the so-called ‘fossil fuels’ are getting depleted continuously. Without energy,our future is doomed. Second-generation or advanced bioethanol is a potential remedy to this energy crisis. It avoids the “food vs. fuel dilemma” (suffered by first-generation or conventional biofuels) by extracting energy from agricultural waste (i.e. stump and stover). The main constituents of the non-food substrates are cellulose, hemicellulose and lignin, though it contains some amounts of pectin and peptides.
The procedure to get bioethanol, starting from waste materials, has several steps. First, wehave to choose a suitable feedstock substrate, preferably with low crystallinity or ‘recalcitrance’. Biotechnology is being used to produce recombinant plants which intrinsically possess loosely packed wood. Second, the feedstock undergoes pretreatment. Optimum protocols of physical and chemical pretreatment are developed by precise biophysical methods. Third, enzyme cocktail is used to break down the biopolymers present in the substrate into simpler building blocks (i.e. oligomers and monomers). As the building blocks are mostly carbohydrates, this process is called ‘saccharification’ (Latinsaccharum means ‘sugar’). Fourth, the simpler sugars are fermented by cellular(e.g. using baker’s yeast) or cell-free (i.e. using extracted or synthetic enzymes) system. Fifth and last, the fermented broth is purified.
The cocktail mentioned in the third step involves many hydrolases (enzymes that catalyse the breakdown of the glycosidic bonds between sugar subunits due to reaction with water) namely cellulases, hemicellulases and ligninases. However, the processis not as smooth as it seems. The substrates are often highly crystalline,whereas the hydrolases mentioned previously work best if the substrate is loosely packed. What now? Luckily, scientists have discovered an interesting family of enzymes which cleave the glycoside bonds within the chains of crystalline polysaccharides! Yeah, crystallinity of the substrate is not an obstacle to this family called Lytic polysaccharide monooxygenase (LPMO), because its members have got a flat substrate-binding surface as opposed to a pocket or crevice as seen in canonical enzymes. LPMOs are oxido-reductases that make the job of the fellow hydrolases easy, by chopping the crystalline substrate into loosely packed forms.
The structure of these types of enzymes is generally elucidated by NMR spectroscopy, X-ray crystallography and computational approaches. A typical LPMO, in its nativestate, has a single copper atom in +2 oxidation state at the catalytic site. It has a conserved ‘T’-shaped structure (namely the histidine brace) made of 2-histidine residues coordinated to Cu. The copper centre and its ligands play acrucial role in the catalysis, as one would expect for a monooxygenase-metallo enzyme. It does not carry its own personal cofactor, so it has to dependon external electron donors in order to cleave and oxidize the substrate. Thedonor can be some small molecule (e.g. ascorbate, hydroquinone) or some enzyme(e.g. cellobiose dehydrogenase). Activity of various LPMOs on theircorresponding substrates is measured with the help of various types of mass spectrometry and chromatography. The flat substrate binding surface is easierto monitor, making the LPMOs of special interest to the bioinorganic chemists.We know very little about the exact catalytic mechanism of LPMOs. We are yet to understand the factors governing their regio-selectivity (i.e. which cleavage product will be oxidized) and substrate-specificity (e.g. discrimination between binding cellulose and binding starch).
We are even clueless regarding the natural co-substrate of LPMOs: is it dioxygen (O2) or is it hydrogen peroxide (H2O2). But this is exactly how science works, isn’t it?These and several other questions will eventually be answered through intense interdisciplinary research. Welcome on board!
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