The discovery of lytic polysaccharide monooxygenases currently classified as CBM33 and GH61 may represent a revolution in enzymatic biomass processing, although further work is needed to establish their full potential. From a scientific point of view these enzymes are interesting because they represent a novel type of enzymatic activity. From an applied point of view these enzymes are of interest because they may speed up enzymatic conversion of biomass, thus reducing enzyme loads and processing times. Reported effects on chitinase activity are huge [8
], whereas reported effects on cellulose activity are less [9
] but still significant and of considerable commercial value. One of the best known commercial cellulose preparations today, Cellic CTec2 produced by Novozymes, contains extra GH61s that contribute to this product’s improved performance compared to its predecessors [55
]. Although industrial knowledge on these enzymes remains mostly invisible to the outside world, it would seem that accumulated research on CBM33s and GH61s still is quite limited. It is thus likely that further improvements in enzymatic biomass conversion through use of these enzymes will emerge in the coming years.
The classifications of CBM33s and GH61s as carbohydrate-binding modules and glycoside hydrolases, respectively, are clearly wrong and will thus change in the near future. Although the two enzyme families show strong similarities, it remains to be seen how similar they really are. Mechanistically, there may be differences, as indicated by the production of C4 and perhaps even C6-oxidized sugars by some of the GH61s described so far. Also, all GH61 structures published so far show methylation of the N-terminal histidine in the active site [13
]. Such methylation has not been observed in CBM33s and, to the best of our knowledge, not in bacteria in general [13
Another issue concerns possible variation in substrate specificity. The multiplicity of genes, especially in the case of GH61s, the large sequence variation (e.g. see [18
]) and, in the case of CBM33s, the huge variation in modular structure, all suggest that different substrates are targeted. In addition to the various forms and crystal faces of chitin and cellulose, other complex and ordered structures may be targeted such as junction zones or other carbohydrate aggregates in tightly inter-linked hemicellulose-cellulose chains [83
]. The physiological data available for some CBM33s certainly support the idea of a wider substrate range within this family.
It is important to note that so far, there are no indications that these novel lytic polysaccharide monooxygenases act on single chains, which in an experimental context would mean soluble oligosaccharides. In this sense these novel enzymes differ dramatically from classical glucanases, which need to position single chains in their active sites grooves, clefts or tunnels [48
], and which normally are active on soluble oligosaccharides. Such glucanases may have additional CBMs providing affinity for crystalline surfaces [86
]. Since CBM33s and GH61s have extended substrate-binding surfaces, one may wonder how extended and ordered the substrate surfaces need to be. It is conceivable that other plant polysaccharides as well as perhaps even the more complex of the glycans found in glycoproteins, contain enough “surface” (i.e. expanding beyond a single chain), to interact with certain CBM33s and/or GH61s. All in all, we consider it likely that CBM33s and GH61s acting on biomass structures such as xylan, mannan and starch will be discovered in the near future.
While the recent finding of lytic polysaccharide monooxygenases represents a major advance in the development of better enzyme technology for biomass conversion, more is likely to come. Proteins such as “swollenins” [87
] and expansins [89
] have for long been known to affect cellulose and other plant polysaccharides but their use in biomass processing has not yet been fully explored. Certain CBMs may have roles beyond mere substrate-binding and they might have some sort of substrate-disrupting effect, thus increasing accessibility to glucanases [74
]. Careful engineering of CBMs and appending them to certain glucanases may yield possibilities that so far have remained under-explored. Finally, current massive studies on the microbiomes of herbivores reveal a plethora of potentially relevant biomass-converting enzymes that need further attention and that seem to include cellulolytic enzyme systems unlike the systems known so far [90
Production of biofuels via enzymatic depolymerization of non-food plant polysaccharides currently receives massive attention and the first commercial production facilities are being built [93
]. One major reason for current progress is the drastic reduction in enzyme costs that commercial producers have achieved over the past decade. More improvement is needed though, since enzyme costs remain high and, for some tougher substrates, prohibitive [4
]. The developments described above open new avenues for further development of enzyme technology in the field. Interestingly, the novel CBM33 and GH61 enzymes do something very different to their substrates than well known hydrolytic enzymes and their implementation may thus require novel thinking. For example, while glucanases need thermochemically pretreated substrates with disrupted crystallinity and sufficiently accessible single polymer chains, these novel enzymes may handle more compact and inaccessible materials. Thus, further implementation of the possibilities offered by CBM33s and GH61s may not only affect the costs of the enzymatic saccharification step but may also direct further optimization of the preceding pretreatment step and process design in general.