Cellulase

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Cellulase
Cellulase 1JS4.png
A cellulase enzyme produced by Thermomonospora fusca, with cellotriose bound in the shallow groove of the catalytic domain
Identifiers
EC number3.2.1.4
CAS number9012-54-8
Databases
IntEnzIntEnz view
BRENDABRENDA entry
ExPASyNiceZyme view
KEGGKEGG entry
MetaCycmetabolic pathway
PRIAMprofile
PDB structuresRCSB PDB PDBe PDBsum
Gene OntologyAmiGO / QuickGO
Ribbon representation of the Streptomyces lividans beta-1,4-endoglucanase catalytic domain - an example from the family 12 glycoside hydrolases[1]

Cellulase is any of several enzymes produced chiefly by fungi, bacteria, and protozoans that catalyze cellulolysis, the decomposition of cellulose and of some related polysaccharides. The name is also used for any naturally occurring mixture or complex of various such enzymes, that act serially or synergistically to decompose cellulosic material.

Cellulases break down the cellulose molecule into monosaccharides ("simple sugars") such as beta-glucose, or shorter polysaccharides and oligosaccharides. Cellulose breakdown is of considerable economic importance, because it makes a major constituent of plants available for consumption and use in chemical reactions. The specific reaction involved is the hydrolysis of the 1,4-beta-D-glycosidic linkages in cellulose, hemicellulose, lichenin, and cereal beta-D-glucans. Because cellulose molecules bind strongly to each other, cellulolysis is relatively difficult compared to the breakdown of other polysaccharides such as starch.[2]

Most mammals have only very limited ability to digest dietary fibres such as cellulose by themselves. In many herbivorous animals such as ruminants like cattle and sheep and hindgut fermenters like horses, cellulases are produced by symbiotic bacteria. Cellulases are produced by a few types of animals, such as some termites and snails.[3][4][5]

Several different kinds of cellulases are known, which differ structurally and mechanistically. Synonyms, derivatives, and specific enzymes associated with the name "cellulase" include endo-1,4-beta-D-glucanase (beta-1,4-glucanase, beta-1,4-endoglucan hydrolase, endoglucanase D, 1,4-(1,3,1,4)-beta-D-glucan 4-glucanohydrolase), carboxymethyl cellulase (CMCase), avicelase, celludextrinase, cellulase A, cellulosin AP, alkali cellulase, cellulase A 3, 9.5 cellulase, and pancellase SS. Enzymes that cleave lignin have occasionally been called cellulases, but this old usage is deprecated; they are lignin-modifying enzymes.

Types and action[edit]

Five general types of cellulases based on the type of reaction catalyzed:

Avicelase has almost exclusively exo-cellulase activity, since avicel is a highly micro-crystalline substrate.

Within the above types there are also progressive (also known as processive) and nonprogressive types. Progressive cellulase will continue to interact with a single polysaccharide strand, nonprogressive cellulase will interact once then disengage and engage another polysaccharide strand.

Cellulase action is considered to be synergistic as all three classes of cellulase can yield much more sugar than the addition of all three separately. Aside from ruminants, most animals (including humans) do not produce cellulase in their bodies and can only partially break down cellulose through fermentation, limiting their ability to use energy in fibrous plant material.

Structure[edit]

Most fungal cellulases have a two-domain structure, with one catalytic domain and one cellulose binding domain, that are connected by a flexible linker. This structure is adapted for working on an insoluble substrate, and it allows the enzyme to diffuse two-dimensionally on a surface in a caterpillar-like fashion. However, there are also cellulases (mostly endoglucanases) that lack cellulose binding domains.

Both binding of substrates and catalysis depend on the three-dimensional structure of the enzyme which arises as a consequence of the level of protein folding. The amino acid sequence and arrangement of their residues that occur within the active site, the position where the substrate binds, may influence factors like binding affinity of ligands, stabilization of substrates within the active site and catalysis. The substrate structure is complementary to the precise active site structure of enzyme. Changes in the position of residues may result in distortion of one or more of these interactions.[7] Additional factors like temperature, pH and metal ions influence the non-covalent interactions between enzyme structure.[8] The Thermotoga maritima species make cellulases consisting of 2 beta-sheets (protein structures) surrounding a central catalytic region which is the active-site.[9] The enzyme is categorised as an endoglucanase, which internally cleaves β-1,4 -glycosydic bonds in cellulose chains facilitating further degradation of the polymer. Different species in the same family as T. Maritima make cellulases with different structures.[9] Cellulases produced by the species Coprinopsis Cinerea consists of seven protein strands in the shape of an enclosed tunnel called a beta/alpha barrel.[10] These enzymes hydrolyse the substrate carboxymethyl cellulose. Binding of the substrate in the active site induces a change in conformation which allows degradation of the molecule.

Cellulase complexes[edit]

In many bacteria, cellulases in-vivo are complex enzyme structures organized in supramolecular complexes, the cellulosomes. They can contain, but are not limited to, five different enzymatic subunits representing namely endocellulases, exocellulases, cellobiases, oxidative cellulases and cellulose phosphorylases wherein only endocellulases and cellobiases participate in the actual hydrolysis of the β(1→ 4) linkage. The number of sub-units making up cellulosomes can also determine the rate of enzyme activity.[11]

The cellulase complex from Trichoderma reesei, for example, comprises a component labeled C1 (57,000 daltons) that separates the chains of crystalline cellulose, an endoglucanase (about 52,000 daltons), an exoglucanase (about 61,000 dalton), and a beta-glucosidase (76,000 daltons).[12]

Numerous "signature" sequences known as dockerins and cohesins have been identified in the genomes of bacteria that produce cellulosomes. Depending on their amino acid sequence and tertiary structures, cellulases are divided into clans and families.[13]

Mechanism of cellulolysis[edit]

The three types of reaction catalyzed by cellulases:1. Breakage of the noncovalent interactions present in the amorphous structure of cellulose (endocellulase) 2. Hydrolysis of chain ends to break the polymer into smaller sugars (exocellulase) 3. Hydrolysis of disaccharides and tetrasaccharides into glucose (beta-glucosidase).
Mechanistic[14] details of beta-glucosidase activity of cellulase

Uses[edit]

Cellulase is used for commercial food processing in coffee. It performs hydrolysis of cellulose during drying of beans. Furthermore, cellulases are widely used in textile industry and in laundry detergents. They have also been used in the pulp and paper industry for various purposes, and they are even used for pharmaceutical applications. Cellulase is used in the fermentation of biomass into biofuels, although this process is relatively experimental at present. Medically, Cellulase is used as a treatment for phytobezoars, a form of cellulose bezoar found in the human stomach, and it has exhibited efficacy in degrading polymicrobial bacterial biofilms by hydrolyzing the β(1-4) glycosidic linkages within the structural, matrix exopolysaccharides of the extracellular polymeric substance (EPS).[15][16]

Measurement[edit]

As the native substrate, cellulose, is a water-insoluble polymer, traditional reducing sugar assays using this substrate can not be employed for the measurement of cellulase activity. Analytical scientists have developed a number of alternative methods.

Viscometric substrates[edit]

A viscometer can be used to measure the decrease in viscosity of a solution containing a water-soluble cellulose derivative such as carboxymethyl cellulose upon incubation with a cellulase sample.[17] The decrease in viscosity is directly proportional to the cellulase activity. While such assays are very sensitive and specific for endo-cellulase (exo-acting cellulase enzymes produce little or no change in viscosity), they are limited by the fact that it is hard to define activity in conventional enzyme units (micromoles of substrate hydrolyzed or product produced per minute).

Cellooligosaccharide substrates[edit]

The lower DP cello-oligosaccharides (DP2-6) are sufficiently soluble in water to act as viable substrates for cellulase enzymes.[18] However, as these substrates are themselves 'reducing sugars', they are not suitable for use in traditional reducing sugar assays because they generate a high 'blank' value. However their cellulase mediated hydrolysis can be monitored by HPLC or IC methods to gain valuable information on the substrate requirements of a particular cellulase enzyme.

Reduced cellooligosaccharide substrates[edit]

Cello-oligosaccharides can be chemically reduced through the action of sodium borohydride to produce their corresponding sugar alcohols. These compounds do not react in reducing sugar assays but their hydrolysis products do. This makes borohydride reduced cello-oligosaccharides valuable substrates for the assay of cellulase using traditional reducing sugar assays such as the Nelson-Symogyi method.[19][20]

Dyed polysaccharide substrates[21][edit]

These substrates can be subdivided into two classes-

  • Insoluble chromogenic substrates: An insoluble cellulase substrate such as AZCL-HE-cellulose absorbs water to create gelatinous particles when placed in solution. This substrate is gradually depolymerised and solubilised by the action of cellulase. The reaction is terminated by adding an alkaline solution to stop enzyme activity and the reaction slurry is filtered or centrifuged. The colour in the filtrate or supernatant is measured and can be related to enzyme activity.
  • Soluble chromogenic substrates: A cellulase sample is incubated with a water-soluble substrate such as azo-CM-cellulose, the reaction is terminated and high molecular weight, partially hydrolysed fragments are precipitated from solution with an organic solvent such as ethanol or methoxyethanol. The suspension is mixed thoroughly, centrifuged, and the colour in the supernatant solution (due to small, soluble, dyed fragments) is measured. With the aid of a standard curve, the enzyme activity can be determined.

Enzyme coupled reagents[edit]

Colourimetric and fluorimetric cellulase substrates can be used in the presence of ancillary β-glucosidase for the specific measurement of endo-cellulase activity

Recently, new reagents have been developed that allow for the specific measurement of endo-cellulase.[22][23] These methods involve the use of functionalised oligosaccharide substrates in the presence of an ancillary enzyme. In the example shown, a cellulase enzyme is able to recognise the trisaccharide fragment of cellulose and cleave this unit. The ancillary enzyme present in the reagent mixture (β-glucosidase) then acts to hydrolyse the fragment containing the chromophore or fluorophore. The assay is terminated by the addition of a basic solution that stops the enzymatic reaction and deprotonates the liberated phenolic compound to produce the phenolate species. The cellulase activity of a given sample is directly proportional to the quantity of phenolate liberated which can be measured using a spectrophotometer. The acetal functionalisation on the non-reducing end of the trisaccharide substrate prevents the action of the ancillary β-glucosidase on the parent substrate.

See also[edit]

References[edit]

  1. ^ PDB: 1NLR​; Sulzenbacher G, Shareck F, Morosoli R, Dupont C, Davies GJ (1997). "The Streptomyces lividans family 12 endoglucanase: construction of the catalytic core, expression, and X-ray structure at 1.75 Å resolution". Biochemistry. 36 (51): 16032–16039. doi:10.1021/bi972407v. PMID 9440876.; rendered with PyMOL
  2. ^ Barkalow, David G.; Whistler, Roy L. "Cellulose". AccessScience, McGraw-Hill.
  3. ^ Bignell DE, Roisin Y, Lo N (2011). Biology of termites: a modern synthesis. Dordrecht: Springer. ISBN 978-9048139767.
  4. ^ Watanabe H, Noda H, Tokuda G, Lo N (1998). "A cellulase gene of termite origin". Nature. 394 (6691): 330–1. doi:10.1038/28527. PMID 9690469.
  5. ^ Watanabe, H.; Tokuda, G. (August 2001). "Animal cellulases". Cellular and Molecular Life Sciences. 58 (9): 1167–1178. doi:10.1007/PL00000931.
  6. ^ Zverlov VV, Schantz N, Schwarz WH (2005). "A major new component in the cellulosome of Clostridium thermocellum is a processive endo-beta-1,4-glucanase producing cellotetraose". FEMS Microbiol. Lett. 249 (2): 353–8. doi:10.1016/j.femsle.2005.06.037. PMID 16006068.
  7. ^ Payne, Christina M.; Bomble, Yannick J.; Taylor, Courtney B.; McCabe, Clare; Himmel, Michael E.; Crowley, Michael F.; Beckham, Gregg T. (25 November 2011). "Multiple Functions of Aromatic-Carbohydrate Interactions in a Processive Cellulase Examined with Molecular Simulation". Journal of Biological Chemistry. 286 (47): 41028–41035. doi:10.1074/jbc.M111.297713. ISSN 0021-9258. PMC 3220501.
  8. ^ Lee, You-Jung; Kim, Bo-Kyung; Lee, Bo-Hwa; Jo, Kang-Ik; Lee, Nam-Kyu; Chung, Chung-Han; Lee, Young-Choon; Lee, Jin-Woo (1 January 2008). "Purification and characterization of cellulase produced by Bacillus amyoliquefaciens DL-3 utilizing rice hull". Bioresource Technology. 99: 378–386. doi:10.1016/j.biortech.2006.12.013.
  9. ^ a b Cheng, Ya-Shan; Ko, Tzu-Ping; Wu, Tzu-Hui; Ma, Yanhe; Huang, Chun-Hsiang; Lai, Hui-Lin; Wang, Andrew H.-J.; Liu, Je-Ruei; Guo, Rey-Ting (1 April 2011). "Crystal structure and substrate-binding mode of cellulase 12A from Thermotoga maritima". Proteins: Structure, Function, and Bioinformatics. 79 (4): 1193–1204. doi:10.1002/prot.22953. ISSN 1097-0134.
  10. ^ Liu, Yuan; Yoshida, Makoto; Kurakata, Yuma; Miyazaki, Takatsugu; Igarashi, Kiyohiko; Samejima, Masahiro; Fukuda, Kiyoharu; Nishikawa, Atsushi; Tonozuka, Takashi (1 March 2010). "Crystal structure of a glycoside hydrolase family 6 enzyme, CcCel6C, a cellulase constitutively produced by Coprinopsis cinerea". FEBS Journal. 277 (6): 1532–1542. doi:10.1111/j.1742-4658.2010.07582.x. ISSN 1742-4658.
  11. ^ Tsai, Shen-Long; DaSilva, Nancy A.; Chen, Wilfred (18 January 2013). "Functional Display of Complex Cellulosomes on the Yeast Surface via Adaptive Assembly". ACS Synthetic Biology. 2 (1): 14–21. doi:10.1021/sb300047u.
  12. ^ Worthington Biochemical Corporation (2014), Cellulase. Accessed on 2014-07-03
  13. ^ Bayer EA, Chanzy H, Lamed R, Shoham Y (1998). "Cellulose, cellulases and cellulosomes". Curr. Opin. Struct. Biol. 8 (5): 548–57. doi:10.1016/S0959-440X(98)80143-7. PMID 9818257.
  14. ^ Bhaumik, Prasenjit; Dhepe, Paresh Laxmikant (2015-01-01). Chapter 1. Conversion of Biomass into Sugars. Royal Society of Chemistry. pp. 1–53. doi:10.1039/9781782622079-00001.
  15. ^ Fleming, Derek; Rumbaugh, Kendra P. (2017-04-01). "Approaches to Dispersing Medical Biofilms". Microorganisms. 5 (2): 15. doi:10.3390/microorganisms5020015. PMC 5488086. PMID 28368320.
  16. ^ Fleming, Derek; Chahin, Laura; Rumbaugh, Kendra (February 2017). "Glycoside Hydrolases Degrade Polymicrobial Bacterial Biofilms in Wounds" (PDF). Antimicrobial Agents and Chemotherapy. 61 (2): AAC.01998–16. doi:10.1128/AAC.01998-16. ISSN 1098-6596. PMC 5278739. PMID 27872074.
  17. ^ Umezurike, GM (Jan 1979). "The cellulolytic enzymes of Botryodiplodia theobromae Pat. Separation and characterization of cellulases and beta-glucosidases". Biochem J. 177 (1): 9–19. doi:10.1042/bj1770009. PMC 1186335. PMID 106849.
  18. ^ Telke, AA; Zhuang, N; Ghatge, SS; Lee S-H, Ali Shah A; et al. (2013). "Engineering of Family-5 Glycoside Hydrolase (Cel5A) from an Uncultured Bacterium for Efficient Hydrolysis of Cellulosic Substrates". PLoS ONE. 8 (6): e65727. doi:10.1371/journal.pone.0065727. PMC 3681849. PMID 23785445.
  19. ^ Nelson, N. (1944). "A photometric adaptation of the Somogyi method for the determination of glucose". J. Biol. Chem. 153: 375–80.
  20. ^ Somogyi, M. (1952). "Notes on Sugar Determination". J. Biol. Chem. 195: 19–23.
  21. ^ McCleary, BV (Nov 1980). "New chromogenic substrates for the assay of alpha-amylase and (1 leads to 4)-beta-D-glucanase". Carbohydr Res. 86 (1): 97–104. doi:10.1016/s0008-6215(00)84584-x. PMID 6159974.
  22. ^ McCleary, BV; Mangan, D; Daly, R; Fort, S; Ivory, R; McCormack, N. (Feb 2014). "Novel Substrates for the Measurement of endo-1,4-β-Glucanase (endo-cellulase)". Carbohydr Res. 385: 9–17. doi:10.1016/j.carres.2013.12.001.
  23. ^ Mangan, D; McCleary, BV; Liadova, A; Ivory, R; McCormack, N. (Aug 2014). "Quantitative fluorometric assay for the measurement of endo-1,4-β-glucanase". Carbohydr Res. 395: 47–51. doi:10.1016/j.carres.2014.05.002.

Further reading[edit]

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