What type of reaction does cellulase catalyze

Here we describe fluorescence detection analysis in microplates of some of the enzymes that catalyze the digestive hydrolysis of polysaccharide polymers into monomeric constituents. While there are a number of ways to produce ethanol, the most cost effective way is through alcoholic fermentation. Figure 1. Schematic representation of the fermentation process. The different technologies used to produce the fermentation sugars necessary for ethanol production are often described as different generations depending on the technology employed.

First generation bio-ethanol production is from fermentable hexose sugars made available by the digestion of starches found in different feed stock sources such as corn or sugar cane. Second generation bioethanol production is the result of the fermentation of lignocellulosic biomass. These feed stocks include: 1 corn stover leaves and stalks of maize ; 2 wheat straw; 3 barley straw; 4 switchgrass; and 5 wood waste products. Depending on the feed stock source, different enzymatic means are used to extract the fermentable saccharides from the polysaccharide polymer.

In order to improve the ethanol yield from these polymers, scientists have turned to the use of specific enzymes from a number of different sources. Using a combination of in vitro digestion, in conjunction with in vivo genetic manipulation of yeast and bacteria strains not only can the digestion efficiency to fermentable sugars be increased, but the ability to ferment monosaccharides other than glucose has been improved. Amylase enzymes catalyze the breakdown of starch into sugars.

Starch is a mixture of amylose and amylopectin polysaccharide polymers. Unlike starch this polymer has no branching. The chain structure forms a stiff extended conformation that has extensive hydrogen bonding with adjacent chains. The result is the formation of microfibrils with high tensile strength and poor water solubility. Cellulase enzymes catalyze the hydrolysis of cellulose into glucose. Cellulase enzymes are produced primarily by fungi, bacteria, and protozoans.

Termites and ruminant herbivores digest cellulose through the action of symbiotic bacteria located in their intestines and ruminating chambers respectively. Xylanase enzymes digest xylan polymers, which are a major constituent of the hemicellulose, into xylose. Xylans are heteropolymers consisting primarily of the pentose sugars xylose and arabinose.

The ability to measure multiple samples simultaneously offers significant advantages in terms of time, expense and effort. The use of microplates is one way that large numbers of samples can be handled with minimal amounts of reagents and time. Especially, as it is recognized as a cost-effective raw material, the useful applications of cellulose in the industrial sector have become much more complex.

This has laid a huge platform for scientists to do cellulose-based research in multidisciplinary approaches. One such area is hydrolysis of cellulose. In nature, this is usually accomplished by cellulases. Cellulase is catalyzing hydrolysis of cellulose. However, cellulase is not a single enzyme.

Fungi, bacteria, and actinomycetes are recorded to be efficient cellulase enzyme producers in the natural environment. These microorganisms must secrete cellulases that are either free or cell surface bound.

Their enzyme production efficiency and the enzyme complex composition are always diverse from each other. Although both aerobic and anaerobic microorganisms produce these enzymes, aerobic cellulolytic fungi, viz. This notion is applied in industries either cellulose is utilized as a raw material or cellulose degradation is a must.

According to recent enzyme market reports, the key areas of the industry where cellulase enzyme is increasingly being applied are healthcare, textile, pulp and paper, detergent, food, and beverages. Its wide application in coffee processing, wine making, and fruit juice production is related to food and beverage segment. In other industrial applications, it is broadly used to produce laundry detergents and cleaning and washing agents. Cellulase is also being highly recognized as an effective alternative to available antibiotics for treatment of biofilms produced by Pseudomonas.

Therefore, the potential of cellulases to fight against antibiotic-resistant bacteria is an amazing trend which will overcome problems in the healthcare sector [ 2 ]. Application of microorganisms or microbial enzymes for pretreatment of lignocellulosic material is currently earning a huge attention of the industry. This is a result of growing interest about depletion of fossil fuel resources in the world which have inspired the production of bioethanol from lignocellulosic biomass through enzymatic hydrolysis [ 3 ].

Lignocellulosic biomass is one of the best options as a low-cost, readily available, eco-friendly raw material. However, it is not found alone. Cellulose is forming lignocellulose in combination with hemicellulose and lignin which finally becomes a compact network structure [ 4 ].

Moreover, it has a crystalline structure which is hard to break down. Therefore, cellulose is insoluble in water and causes limitations in hydrolysis.

That is why it is essential to pretreat lignocellulosic material in industries like bioethanol production. During pretreatment, it will loosen up the crystalline structure and facilitate the degradability to release fermentable sugar forms.

There are several methods available for pretreatment of lignocellulose, viz. Biological pretreatment using cellulolytic microorganisms and their enzymes is found to be the best way of addressing this problem. By all means, cellulase is an enzyme which can cause a huge economic impact. However, there are some considerable bottlenecks of utilizing this enzyme in the industry. For example, the higher cost of cellulase and less catalytic efficiency are especially understood.

Another important point is less understanding of the relationship between hydrolysis mechanisms and molecular structure of the enzyme. This knowledge is important to carry out further improvements in the enzyme to enhance its catalytic activity.

Therefore, this chapter is discussing about the structure and function of cellulase in order to understand its mechanisms of action. The details on current applications of the enzyme have also been summarized here. Furthermore, the efforts have been taken to bring together information on novel biotechnological trends of cellulase. Moreover, it is discussing about possible low-cost, enzymatic pretreatment methods that have been practiced for lignocellulosic materials in order to use it as an efficient raw material to produce bioethanol.

Before moving on to cellulase, it is essential to understand cellulose which is the substrate of cellulase enzyme. This section will provide a short description about cellulose. Cellulose is a linear polysaccharide.

The molecular formula of cellulose is C 6 H 12 O 6 n. It symbolizes the number of glucose subunits connected with each other. This number is varying from hundreds to thousands. Two glucose repeating units together are called cellobiose. It consists of long chains of anhydro-D-glucopyranose units AGU with each cellulose molecule having three hydroxyl groups per AGU with the exception of the terminal ends.

Cellulose has both crystalline and amorphous regions in its structure in various proportions [ 5 ]. Those regions are intertwined to form the structure of cellulose. This crystalline structure is a result of intramolecular and intermolecular hydrogen bonding between glucose monomers in cellulose. These hydrogen bonds construct a huge network that directly contributes to the compact crystal structure of cellulose polymer.

On the other hand, this strong intramolecular and intermolecular hydrogen bond formation leads to poor solubility of cellulose. In plant cell walls, cellulose exists as different levels of structures, i. It is proposed that the macrofibril is composed by the attachment of several newly synthesized elementary fibrils. With the cellular growth, the macrofibrils divide to form individual microfibrils.

Microfibril is consisting of a single elementary fibril. Although elementary fibrils and macrofibrils are composed of mere cellulose, microfibril has noncellulosic polymers like hemicelluloses along with cellulose. It is noted that others consider a microfibril as consisting of a number of elementary fibrils. Microfibril is an elementary fibril associated with noncellulosic polymers. Many such cellulose chains aggregate into bundles called micelles and micelles into microfibrils. Micelles are interconnected with few cellulose fibers.

The plant cell wall structure is stabilized by the macrofibrils. The cross-links between hemicellulose and pectin matrices also support this stabilization process. Lignin is a complex polymer which usually fills the spaces between cellulose and pectin matrices. It forms covalent bonds with hemicellulose. This provides more mechanical strength to the plant cell wall. This structure which is present in plants is collectively called lignocellulose. Other components known as extractives including fats, phenolic, resins, and minerals are also present in lignocellulosic biomass.

Enzymes are known to be very useful in many industrial processes. Their broad applicability has created a significant market demand in the recent years. According to market reports on world enzyme demand , they have recognized several key factors which lead to huge consumer demand for enzymes. Some of them are completely bound with economical advances.

For example, increased per capita income in developing countries causes huge growth in consumer-related industrial applications [ 6 ]. This report also emphasizes the gains in personal incomes in developing countries as the key factor which is supporting growth in demand for enzymes. The development of scientific research on enzymes is mainly based on disciplines such as biotechnology,molecular biology and genetics.

Continued advances in these areas of research, particularly related to DNA manipulation and sequencing, result in extensive increases in enzyme demand worldwide. Cellulase is one such enzyme which earns consecutively increasing demand. Therefore, collection of knowledge about this enzyme is essential for further development of fundamental and applied research on cellulase and for consequent application in human life.

It is produced by fungi, bacteria, actinomycetes, protozoans, plants, and animals. According to Carbohydrate-Active Enzymes database, there is information of the glycoside hydrolase families. Glycoside hydrolases, including cellulase, have been classified into families based on amino acid sequence similarities and crystal structures.

A large number of cellulase genes have now been cloned and characterized. They are found in 13 different families. Furthermore, there are 3D structures of more than 50 cellulases. In the structure of cellulase, there are catalytic modules and non-catalytic modules.

The catalytic modules of cellulases have been classified into numerous families based on their amino acid sequences and crystal structures. Usually, fungal and bacterial cellulase mainly has two or more structural and functional domains. Both aerobic and anaerobic microorganisms are producing this enzyme. Therefore, there are two types of cellulase systems: noncomplex and complex. A noncomplex cellulase system is produced by aerobic cellulolytic microorganisms, and it is a mixture of extracellular cooperative enzymes.

In a noncomplex cellulase system, the common arrangement is joining of a catalytic domain with a cellulose-binding domain CBD.

The enzyme is a multiprotein complex anchored on the surface of the bacterium by non-catalytic proteins that serves to function like the individual noncomplex cellulases but is in one unit. In addition to these two major domains in the cellulase structure, there are some other domains that are present in many cellulases, for instance, S-layer homologous SLH domain, fibronectin-type domains, and NodB-like domain, and there are also other regions of unknown function.

These domains are often connected by Pro and hydroxy amino acid threonine and serine enriched linker sequences. Among all these domains, catalytic and cellulose-binding domains are the most important because they are the domains which are considered participating in hydrolytic mechanisms of the enzyme. Complete cellulose hydrolysis is mediated by the combination of these three main types of enzymes. Endoglucanase usually attacks amorphous areas of cellulose.

The random attack of this enzyme on internal bonds of loosely bound, amorphous areas of cellulose creates new chain ends. These new chain ends are then easily attacked by other types of enzymes. The highest activity of this enzyme usually occurs against soluble cellulose forms or acid-treated amorphous cellulose.

The function of exoglucanase is to produce glucose or cellobiose units by attacking the reducing or nonreducing end of cellulose chains. Endoglucanase is different from exoglucanase because it is usually very active against crystalline cellulose substrates such as Avicel or cellooligosaccharides.

Although an exact mechanism is not yet finalized, fragmentation of cellulose aggregations into short fibers has been observed and reported during the beginning of cellulose hydrolysis prior to releasing any detectable amount of reducing sugars. This is known as amorphogenesis. There are two catalytic mechanisms of cellulases. They are simply introduced as retaining mechanisms and inverting mechanisms.

Cellulases cleave glucosidic bonds by using acid-based catalysis. The catalytic mechanism which occurs depends on the spatial position of the catalytic residues. The retention and inversion of the anomeric configuration of cellulose are the two mechanisms which hydrolyze cellulose. For many decades, cellulases have played a crucial role as biocatalysts. They have shown their potential application in a large number of industries.

Textile, paper and pulp, laundry and detergent, agriculture, medicine, and food and feed industries are some of the major industries which employ microbial cellulases. According to Coherent Market Insights, the textile industry is the dominant market for cellulases in According to most of the enzyme market research reports published in , food and beverages, textile industry, animal feed, and biofuels have been reported to be the major areas of applications.

Furthermore, the reported data showed This same report forecasts that the applications of cellulases will reach million USD by the end of , growing at a compound annual growth rate CAGR of 5. These data suggest that the application of cellulases in industries is drastically rising annually.

Novozymes and DuPont from Denmark are key cellulase enzyme producers supplying these enzymes to the global market for industrial applications. From this point forward, in this chapter, our major effort was to discuss about the current applications of cellulases in major fields that have been listed above. The novel biotechnological trends emerging in those fields while understanding the key areas of research where further studies required also surfaced to an extent.

The textile industry is one of the largest industries in the world. The customer demand for fashion is increasing as they want uniqueness in styles, colors, and the clothes they wear.

There was a significant growth in this industry during the last few decades as a result of this increasing customer demand. This enzyme has now become the third largest group of enzymes used in these applications [ 8 ].

This creates a very competitive market platform for manufacturers that are always looking for environmentally friendly approaches of giving their products a unique look. Cellulase is used for many purposes in the industrial sector. Especially for textile wet processing, biostoning of denim fabric, biopolishing of textile fibers, softening of garments, and removal of excess dye from the fabrics are some of the major applications of this enzyme in the industry.

Fungal cellulases from Trichoderma reesei are the mostly applied enzyme in the textile industry. It is known that plant cell wall degrading enzymes are inducible and under both positive and negative transcriptional control. The authors further propose increased research focus on signal transduction pathways and other potential factors known to influence gene regulation, such as light cycling and intensity.

More recently, Steiger et al. In the modern era of systems biology, the critical mutations conferring superior performance to RUT-C30 and its descendants have been elucidated. As stated above, cre1 regulates catabolite repression in most cellulolytic fungi and is responsible for the strong inhibitory effect glucose has on cellulase production.

However, large-scale enzyme production is usually conducted with more powerful inducers, such as sophorose and lactose. We note that a process has recently been reported which converts glucose rich, biomass derived monosaccharide mixtures to sophorose and other products using enzymatic transglycosylation, providing a more cost-effective source of this inducer.

Indeed, detailed biochemical analyses of Cel7A purified from RUT-C30 broth shows evidence that this enzyme is not glycosylated normally. Proprietary strains of T. Examples of such expression results include the following, in order of highest production: Hormoconis resinae glucoamylase P 0. However, T. Peterson and Nevalainen suggest that the causes of poor heterologous protein expression lie in the incompatibilities of foreign peptides with natural mechanisms of protein recognition and disposal within the cell, known collectively as the unfolded protein response.

In many ways, the concept of direct microbial conversion, also known as consolidated bioprocessing, inspired the early work in T. The molecular cloning era of T. To demonstrate the level of international competition ongoing at this time, we note that four year later workers at VTT reported several landmark accomplishments relevant to this same objective.

Pilot scale L production of T. Many years later, work to express T. In , a notable report describes a systematic study to express, at higher titers than previously reported, active CBH I and II in S.

This concept is, of course, the direct microbial conversion or consolidated bioprocessing process. In the year of this review, Voutilainen et al.

Likely because of the significant resource availability for T. In , Reese and co-workers reported that although many fungi could hydrolyze derivatized celluloses, only a few could grow on crystalline cellulose, such as cotton.

We note that, during the war years, only crude salt and metal ion mediated solvent precipitation of proteins could be applied to protein separations. These methods, developed for fractionating blood plasma proteins for the war effort, were entirely unsuitable for purification of microbial enzymes. This and other postwar developments in protein biochemistry, erupting into play immediately after the war, helped the nascent cellulase biochemistry programs.

Perhaps the most significant of these developments was the work in the s on enhanced protein separation methodology in Uppsala, Sweden. Work by Pederson in Uppsala during this era led to the development of modern size exclusion chromatography with early columns being packed with simple agarose gel spheres. Simple, atmospheric pressure column chromatographic techniques were used in the early and mids to purify cellulases for study.

Enabled by this newly acquired ability to study the action of relatively homogeneous enzyme preparations and measure their respective concentrations accurately, they described in this report the possible role of C 1 enzymes, required for attack on crystalline cellulose, and the more prevalent Cx enzymes, needed for hydrolysis of soluble, derivatized cellulose such as PASC. Characterization of the hypothetical C 1 enzyme lagged behind progress on the Cx enzymes for many years.

One proposal during this time was that C 1 was a protein that decrystallized cellulose by displacing native hydrogen bonds in the microfibril, leading to a more available structure for Cx. In this view, EGs attack amorphous cellulose surface regions of the microfibril, revealing new cleavage sites for exoglucanases. Although a second exoglucanase from T. In , van Tilbeurgh and co-workers were first to describe the systematic multistep chromatographic purification of all key cellulases from T.

A curious observation from this time was that the optimum synergistic ratio of cellulases purified from T. We are not aware that more recent work has confirmed or denied this theory; however, given the challenges now known for removing all traces of EG contamination from CBH I preparations, caution is recommended.

Today, cellulase digestions mechanisms are once again viewed in a sense related to the original concepts from Reese, i.

However, some inconsistencies are apparent given the classical view shown in Figure 9. Reese suggested that C1 was a decrystallizing protein factor, whose function may not be bond-breaking at least, not hydrolytic , but instead possesses an ability to swell or disrupt cellulose, perhaps by intercalating between cellulose chains in the elementary microfibril, the consequence of which is action by other enzymes causing complete digestion to cellobiose. The view shown in Figure 9 actually suggests that Cx is not a single class of enzymes, but the combined action of EGs and exoglucanases that work together to expose the necessary ends of cellulose chains needed to yield simple sugars.

Neither the EGs nor the exoglucanases from fungi cause global decrystallization of cellulose. This hydrolytic enzyme system functions in an ablative manner, peeling one layer at a time. The essential dilemma here is that no single protein is known to dramatically reduce cellulose crystallinity.

Some proteins may cause some local cellulose disruption and morphological changes, for example, the expansins and expansin-like proteins swollenins , but their reported effects on subsequent hydrolytic action are modest.

So, are there protein factors that truly reflect the notions of Reese four decades ago? The relatively recent work to define the action of LPMOs has again posed some new potential answers to this question.

LPMOs were originally classified as fungal GH61 enzymes and nonfungal members of family 33 CBM, but have been reclassified, and are now implicated in the oxidative cleavage of cellulose and other plant polymers.

As reviewed extensively in section 11 , LPMOs are known to act in two reaction schemes on crystalline cellulose, generating oxidized and nonoxidized chain ends Figure Some LPMOs have been shown to oxidize glucose at position C1, releasing lactones that are hydrolyzed to aldonic acids, whereas other enzymes act on the nonreducing end, producing ketoaldoses, or a combination thereof.

Today, the secretome of T. For example, T. Furthermore, as illustrated by Martinez and co-workers, 41 T. Seiboth and co-workers concluded that T. Some caveats to this cocktail paradigm deserve attention. Structurally, some cellulases have binding sites that are intermediate between the closed tunnels of CBHs and the open clefts of EGs.

Functionally, some enzymes have intermediate levels of processivity, depending on the definition of processivity used. However, the term exoglucanase is now essentially obsolete as a class of GH given the early and now more recently confirmed revelation that GH7 CBHs can perform hydrolysis in an endo-initiation fashion.

As suggested above, T. The enzymes shown in Table 2 are those cited currently in CAZy. Some families, for example GHs 6 and 7, contain enzymes with vastly different and mechanistically synergistic activities as described in sections 6 and 7.

Other families, GHs 1 and 3, contain many enzymes with essentially the same activity cleavage of the glycosidic bond in cellobiose ; upon closer inspection, however, some of these enzymes have subtle differences in substrate specificity.

In this case, the EG homologue has substrate tunnel associated peptide loops of shorter length than its CBH counterpart see section 6.

We also note that the EGs found in GH families 5, 7, and 12 act on cellulose to leave the terminal hydroxyl in the retaining configuration. Family GH45 EGs leave the terminal hydroxyl in the inverted configuration. For more comprehensive reviews of the early literature on T. Table 2. Biomass-degrading enzymes work at solid—liquid interfaces, and the concentration of catalytic units at the surface is directly related to the extent of substrate turnover.

Thus, many enzymes that work on polysaccharides are multimodular with catalytic function accomplished by a single or multiple CDs coupled to a binding function via one or more CBMs; these two domains are connected together by linker peptides of varying length and structure. To date, 69 distinct families of CBMs have been discovered and characterized according to the CAZy database family 33 CBMs have been reclassified as oxidative enzymes, as discussed below.

Moreover, CBMs are connected to CDs via linkers, and thus, the roles of these domains are also reviewed. For more general perspectives on CBMs, readers are referred to several reviews from the past decade.

That many GHs contain both binding and catalytic function was first reported in a study by Van Tilbeurgh et al. On the basis of these results, the authors proposed that Tr Cel7A is a multimodular protein with a binding and catalytic function.

This study, along with similar work the same year on two bacterial cellulases from Cellulomonas fimi , solidified the concept that cellulases can exhibit multimodular structures with CBDs. Upon the discovery of additional carbohydrate-binding ligands beyond cellulose, the term CBD was replaced with the broader concept of CBMs.

Kraulis et al. The CBM sequence contains four cysteine residues comprising two disulfide bonds, and all three possible combinations were investigated to determine the most likely pairing. The disulfide bonds are also shown in Figure As discussed below in detail, this flat face is implicated as the binding face to crystalline cellulose.

Type B CBMs exhibit extended grooves or clefts for binding single sugar chains, such as those found in hemicellulose, pectin, or amorphous regions of polymers such as cellulose or chitin. Lastly, type C CBMs are characterized as those that bind mono-, di-, and trisaccharides.

In , three functions were attributed to CBMs: substrate targeting, enhancements to enzyme—substrate proximity, and disruption of the substrate. With the publication of the landmark structural study from Kraulis et al. The CBM was also proposed to enable two-dimensional diffusion of the CBH enzyme on the cellulose surface to enable efficient catalytic performance.

To our knowledge, similar experiments have not yet been conducted for family 1 CBMs. Reinikainen et al. This observation was attributed to a higher extent of glycosylation from yeast expression as observed by a large heterogeneous population of enzymes on a gel. Within the heterologously expressed enzymes, the authors observed that wild-type Cel7A and the YH mutant exhibit roughly equivalent activity, whereas YA and YD result in a significant decrease in activity against crystalline cellulose accompanied by a concomitant decrease in binding affinity, as measured at low temperature.

The PR mutation also resulted in similarly low activity and binding affinity as YA; however, as the authors note, this could be due to structural changes to the CBM given the removal of a proline residue.

This was not clearly resolved until a later study from Reinikainen et al. At a concentration of 1 M MgSO 4 , all mutants performed similarly to the wild-type enzyme on crystalline cellulose, which the authors state suggests that the hydrophobic effect plays a significant role in CBM binding to the substrate. Given the similarity of the PR results to the wild-type enzyme and the reduction in affinity and activity with mutations to Tyr, this led the authors to suggest that the flat, aromatic-containing face of the CBM is likely the binding face to crystalline cellulose.

P16R was the least-affected mutant, similar to results from Reinikainen et al. The 2-D NMR spectroscopy suggests that the Y5A mutation affects the overall compactness of the structure, which was later verified by Mattinen et al.

Table 3. Starting with the Cel7A CBM sequence, the authors made multiple mutations, and found that the Y5W mutation alone can explain much of the differences in binding affinity between the two CBMs Table 4. A subsequent study from Srisodsuk et al. The alignment of aromatic residues on the flat, hydrophilic surface of the CBM is quite similar. The authors also found a nearly linear correlation between activity on Avicel and CBM binding affinity, highlighting the key relationship between CBMs and cellulolytic performance.

Table 4. Linder and Teeri also examined both the binding and reversibility of the Tr Cel7A CBM on crystalline cellulose using a sensitive tritium-labeling approach combined with dilution experiments.

More importantly, they demonstrated that the CBM binding was completely reversible, addressing a question in the literature regarding if CBMs could desorb from cellulose. The authors conclude that the rate of adsorption and desorption of CBMs and the binding affinity to cellulose should be optimally balanced to maximize cellulase activity and minimize nonproductive binding.

The removal of the third disulfide bond in the Cel6A CBM resulted in a decrease in binding affinity and off-rate, although not as drastic as the W7Y mutation, suggesting that the Cel6A CBM rigidity contributes to the CBM-cellulose interaction and that the presence of the tryptophan at the 7-position significantly affects the binding affinity.

This finding is in direct contrast to an influential, earlier study wherein isothermal titration calorimetry experiments on a family 2 CBM also a type A CBM suggested that type A CBM binding is entropically driven. The authors speculate that the potential preference in the Cel6A CBM binding may lead the intact enzyme to bind near reducing ends of cellulose chains.

This finding shed light directly on the type A CBM-cellulose interaction by ascertaining that the protein—ligand binding surface is the flat surface with the glucopyranose rings directly exposed. More recently, Sugimoto et al.

The authors fit their binding isotherms to four binding models, and found that the Hill binding model provided the best fit for CBM-binding to cellulose, with the Langmuir isotherm model also providing a reasonable fit correlation coefficients of 0.

The authors attribute the goodness-of-fit of the Hill model to the explicit treatment of a steric exclusion effect induced by surface crowding of the CBM-fluorescent protein complex and infer that the length and flexibility of the linker domain will dictate the size of the exclusion area.

To date, nearly all studies of family 1 CBMs have either used solid-state peptide synthesis or E. However, detailed mass spectrometry studies from Harrison et al. Given the importance of glycosylation in cellulase activity, as discussed in more detail below in several GH sections, and the sequence conservation of putative O -glycan sites on family 1 CBMs Figure 16 , fungi may employ glycans on CBMs for multiple functions.

In terms of cellulose binding affinity, Taylor et al. The thermodynamic cycle calculations were in quantitative agreement, for example, with the Y5W mutation. From there, the authors predicted that a single mannose at Ser3 could modify the binding affinity by the same order as a tryptophan mutation.

Moreover, the simulations predicted that the addition of mannosylation at different sites would modulate the binding affinity by a significantly different magnitude, suggesting that the location and extent of glycosylation has a major impact on the change in properties. The predictions that glycosylation affects CBM binding affinities were recently tested experimentally.

Two sets of glycoforms for a total of 20 CBMs were synthesized: the first set focused on addition of glycan motifs to single amino acids, namely at Thr1, Ser3, and Ser14 of mono-, di-, and trimannose group, whereas the second set was designed to study the effects of adding glycans to multiple amino acid residues simultaneously, as would be found naturally. For the addition of glycans to single sites, Chen et al. Addition of glycans at Ser14 essentially improved all properties, but not to the extent of Ser3.

For the addition of glycans to multiple sites, single mannosylation at each site was able to increase the binding affinity to BMCC by 7. From a biological perspective, glycan-bearing residues are highly conserved in the 1, 3, and 14 positions on family 1 CBMs, and given the ability to affect multiple, beneficial properties, it is likely that CBM glycosylation is employed commonly by fungi. Given the prevalence of glycosylation in fungal cellulases, this work demonstrates that the study of CBMs should explicitly consider the effects of glycosylation to measure physiologically relevant properties.

Another interesting question related to CBMs is the ability for substrate disruption, as discussed by Boraston et al. The authors suggested that this CBM-cellulose interaction was the result of nonhydrolytic disruption of the substrate.

However, it was not demonstrated that the CBM-treated substrates were subsequently more digestible by reducing-sugar assays nor were cellulose crystallinity measurements conducted. Gao et al. Xiao et al. It is unknown how pretreatment with CBMs affected the conversion at long times or the final yield of reducing sugars from these experiments. Many other CBM effects on fungal cellulase activity and behavior beyond substrate disruption have been investigated.

Hall and co-workers compared the thermal stability of intact Tr Cel7A, the CD alone, and the CBM-linker, the latter two isolated from papain cleavage of the intact enzyme.

Voutilainen et al. A similar study was reported from Kim et al. Although many cellulases have CBMs, there are many examples of fungal and nonfungal cellulases that do not employ them in natural biomass degradation.

There are likely multiple physiological and evolutionary reasons for the lack of CBMs in some cases, such as biomass degradation in organisms that densely pack solids into their digestive organs. The most likely reason for this observation is that, at high solids loadings, the diffusion length for the enzymes to productively bind to substrate is much shorter than in low solids loading digestions, thus precluding the need for CBMs to ensure that the concentration of active enzymes at the substrate interface is high.

For natural systems, high dry-matter content may also preclude the need for CBMs, but a systematic correlation coupled to other environmental and evolutionary considerations has not been conducted to our knowledge. Given the relatively small size of family 1 CBMs coupled to the inherent difficulty in directly studying molecular-level interactions at the heterogeneous cellulose interface, family 1 CBMs have been the subject of computational studies since aimed at further elucidating cellulose—CBM interactions at the molecular level.

It is noteworthy that this simulation was conducted with a previous version of the CHARMM force field, and this behavior has not been observed in subsequent simulations with more updated potentials.

The conservation of an aromatic residue in this position Figure 16 , however, suggests that this aromatic residue is important for a structural or functional reason, and future experimental work will likely shed additional light on this question.

Multiple studies were later reported that suggest how CBMs translate on the cellulose surface. A much more detailed study was later published by Nimlos et al. This study demonstrated that there is a thermodynamic driving force for the Tr Cel7A CBM to translate from hydrophilic surfaces to the preferred hydrophobic face, that the CBM will translate along the hydrophobic surface in both a forward and backward direction with equal probability, and that the flat, hydrophilic face of the CBM is the thermodynamically preferred face for cellulose binding.

Taken together, significant insights have been gained regarding the function of family 1 CBMs, especially accelerated and informed by the initial structural work. Moreover, as is clear from this section as well, most of our collective understanding of family 1 CBMs stems almost solely from the model T. Undoubtedly, there is significant potential for improving cellulase properties via deeper understanding of CBM function in the context of both cellulase performance and stabilization.

Here, we review the characterization of linkers and their putative functions beyond domain connection. We include discussion of nonfungal linkers where appropriate to understanding their general function. Some of the original work related to linker domains and cellulase modularity was conducted with small-angle X-ray scattering SAXS using T.

A second study on Tr Cel7A demonstrated that the enzyme becomes elongated in the presence of xylan from 18 to 22 nm, which was attributed to lengthening in the CBM-linker region. Langsford et al. The properties of the enzymes in terms of kinetics on small molecule substrates such as CMC or p NPC and thermal and pH stabilities were not affected by glycosylation. In the presence of crude protease from C. Cellulase performance in Avicel digestion was not directly compared in the absence of protease.

Overall, this study led Langsford et al. The hinge mutation resulted in similar overall performance of the enzyme relative to the wild-type, but with a reduced binding capacity at high loading.

The mutant removing the entire linker drastically reduced the enzymatic activity toward crystalline cellulose. Boisset et al. From these initial SAXS and light scattering studies, it was not clear if linkers are stiff or flexible.

Receveur and co-workers later reported a comprehensive SAXS study wherein they examined the full-length H. The results for the wild-type enzyme and the shortened-linker variant both demonstrate that the linker volume is quite substantial and extended, suggesting either or both that the glycosylation on the linker provides an excluded volume effect or that the linker is quite flexible, but extended.

The polyproline mutant exhibited a considerable narrowing in the region where the proline insertion was made, suggesting that this region imparted significant local rigidity. The authors interpret their data to suggest that the linker provides the means to separate the CBM and CD and optimize their relative geometry, similar to the interpretation of Srisodsuk et al.

This model is one wherein free energy is gained by compression of the linker during catalysis, which is dissipated by sliding of the CBM. In the aforementioned study from Receveur et al. To overcome this problem and focus only on the linker domain, von Ossowski et al.

To our knowledge, this study marked the first explicit connection of linkers with intrinsically disordered proteins, which has become a large field in the last 10—15 years. Additional studies have been published that include SAXS analysis of intact cellulases with broadly similar conclusions.

Poon et al. A later study from Sammond et al. It has often been mentioned that linkers in cellulase enzymes do not exhibit sequence conservation or high homology to one another. In the aforementioned study from Sammond et al. In doing so, quantitative patterns in linker characteristics begin to emerge, some of which are illustrated in Figure In all cases examined, O -glycosylation was found to be approximately uniformly distributed across the length of linkers, suggesting that it is needed in a distributed manner to protect against proteolysis or to serve additional, unknown functions.

Glycine residues were found to be clustered at the termini of all linkers studied, suggesting the need for flexibility at the junction between ordered domains and the linker, perhaps for orientation during catalytic action.

Taken together, these results begin to suggest that cellulase linkers exhibit properties that are optimized for specific catalytic function, the mechanistic underpinnings for which are mostly unknown.

Detailed characterization of the glycosylation pattern of linkers is essential to understand their behavior. To that end, several in-depth mass spectrometry studies have been conducted to characterize the glycosylation patterns on cellulase linkers. For Tr Cel7A, Harrison et al.

They also found that a mannose residue on the linker exhibited a sulfate group, but the role of this sulfation remains unknown. In , Hui et al. Shortly after, Hui et al. Stals et al. These data, when taken together, further complicate the ability to examine the impact of linker glycosylation in a systematic manner. Many studies have been conducted to understand how linkers behave in isolation or how intact multimodular enzymes behave in solution. Conversely, our collective understanding of linker function during enzymatic action is limited given that cellulases act at a solid—liquid interface, which is inherently difficult to study at the molecular level.

This hypothesis was tested computationally by using a free energy method umbrella sampling by compressing and extending an isolated linker domain over a cellulose slab, which suggested that there was indeed a barrier. Ting et al. Note that the events of processive cellulolytic action are described in detail in sections 6. The rate constant governing hydrolysis and processivity for the CD account for both the work required to decrystallize a single polymer chain and the compression of the linker as the CD moves forward.

The model demonstrates that the maximum enzyme velocity on the surface is reached at intermediate linker stiffness, for a given length. The overall recommendations from this theoretical model are that optimization needs to be conducted not only of the CD hydrolysis rates, as was already known, but also of the linker length and stiffness. Toward further understanding linker behavior during cellulase action, we recently reported a combined computational and experimental study wherein the interaction of the Tr Cel7A and Tr Cel6A enzymes with the surface of cellulose were examined.

Subsequently, the binding affinities to cellulose were experimentally measured of both the glycosylated Tr Cel7A CBM-linker isolated via papain cleavage and the CBM alone produced with solid-state peptide synthesis. These experimental measurements demonstrate that the linker indeed is able to increase the binding affinity to cellulose by a factor of 10 over the CBM alone, as fits with a Langmuir isotherm model, and that it likely does not function as a spring between the two structured domains.

However, there are two types of cellulase systems: noncomplexed and complexed. A noncomplexed cellulase system is the aerobic degradation of cellulose in oxygen.

It is a mixture of extracellular cooperative enzymes. Figure 6.