Editing Cellulase

{{Short description|Enzymes that catalyze cellulolysis}}
{{infobox enzyme
| Name       = Cellulase
| EC_number  = 3.2.1.4
| CAS_number = 9012-54-8
| GO_code    = 0008810
| image      = cellulase 1JS4.png
| width      = 260px
| caption    = A cellulase enzyme produced by ''Thermomonospora fusca'', with cellotriose bound in the shallow groove of the catalytic domain
}}

[[Image:1NLRribbon.png|400px|thumb|right|Ribbon representation of the ''Streptomyces lividans'' β-1,4-endoglucanase catalytic domain - an example from the family 12 glycoside hydrolases<ref name="pmid9440876">{{PDB|1NLR}}; {{cite journal | vauthors = Sulzenbacher G, Shareck F, Morosoli R, Dupont C, Davies GJ | title = The ''Streptomyces lividans'' family 12 endoglucanase: construction of the catalytic core, expression, and X-ray structure at 1.75 Å resolution | journal = Biochemistry | volume = 36 | issue = 51 | pages = 16032–9 | date = December 1997 | pmid = 9440876 | doi = 10.1021/bi972407v }}; rendered with [http://pymol.sourceforge.net PyMOL]</ref>]]

'''Cellulase''' ({{EnzExplorer|3.2.1.4}}; systematic name '''4-β-<small>D</small>-glucan 4-glucanohydrolase''') is any of several [[enzymes]] produced chiefly by [[fungi]], [[bacteria]], and [[protozoan]]s that [[catalyze]] [[cellulolysis]], the decomposition of [[cellulose]] and of some related [[polysaccharide]]s:

: Endohydrolysis of (1→4)-β-<small>D</small>-glucosidic linkages in cellulose, lichenin and cereal β-<small>D</small>-glucan

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 [[monosaccharide]]s ("simple sugars") such as β-[[glucose]], or shorter polysaccharides and [[oligosaccharide]]s.  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-β-<small>D</small>-[[glycosidic linkage]]s in cellulose, [[hemicellulose]], [[lichenin]], and cereal [[beta-D-glucan|β-<small>D</small>-glucan]]s. Because cellulose molecules bind strongly to each other, cellulolysis is relatively difficult compared to the breakdown of other polysaccharides such as starch.<ref>{{Citation |title=Cellulose |url=https://www.accessscience.com/content/article/a118200 |access-date=2025-02-24 |language=en |doi=10.1036/1097-8542.118200|url-access=subscription }}</ref>

Most mammals have only very limited ability to digest dietary fibres like cellulose by themselves. In many herbivorous animals such as [[ruminant]]s like cattle and sheep and [[hindgut fermentation|hindgut fermenters]] like horses, cellulases are produced by [[symbiotic]] bacteria. Endogenous cellulases are produced by a few types of [[animals]], such as some [[termite]]s, snails,<ref>{{cite book|vauthors=Bignell DE, Roisin Y, Lo N | title = Biology of termites: a modern synthesis | year = 2011 | publisher = Springer | location = Dordrecht | isbn = 978-9048139767 }}</ref><ref name="pmid9690469">{{cite journal | vauthors = Watanabe H, Noda H, Tokuda G, Lo N | title = A cellulase gene of termite origin | journal = Nature | volume = 394 | issue = 6691 | pages = 330–1 | date = July 1998 | pmid = 9690469 | doi = 10.1038/28527 | bibcode = 1998Natur.394..330W | s2cid = 4384555 }}</ref><ref>{{cite journal | vauthors = Watanabe H, Tokuda G | title = Animal cellulases | journal = Cellular and Molecular Life Sciences | volume = 58 | issue = 9 | pages = 1167–78 | date = August 2001 | pmid = 11577976 | doi = 10.1007/PL00000931 | s2cid = 570164 | pmc = 11337393 }}</ref> and [[earthworm]]s.

Cellulases have also been found in green microalgae (''Chlamydomonas reinhardtii'', ''Gonium pectorale'' and ''Volvox carteri'') and their catalytic domains (CD) belonging to [[Glycoside hydrolase family 9|GH9 Family]] show highest [[sequence homology]] to metazoan endogenous cellulases. Algal cellulases are modular, consisting of putative novel cysteine-rich
carbohydrate-binding modules (CBMs), proline/serine-(PS) rich linkers in addition to putative Ig-like and unknown domains in some members. Cellulase from ''Gonium pectorale'' consisted of two CDs separated by linkers and with a C-terminal CBM.<ref name="guerriero">{{Cite journal |last1=Guerriero |first1=Gea |last2=Sergeant |first2=Kjell |last3=Legay |first3=Sylvain |last4=Hausman |first4=Jean-Francois |last5=Cauchie |first5=Henry-Michel |last6=Ahmad |first6=Irshad |last7=Siddiqui |first7=Khawar |date=2018-06-15 |title=Novel Insights from Comparative In Silico Analysis of Green Microalgal Cellulases |journal=International Journal of Molecular Sciences |language=en |volume=19 |issue=6 |pages=1782 |doi=10.3390/ijms19061782 |doi-access=free|issn=1422-0067 |pmc=6032398 |pmid=29914107}}</ref>

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|endo-1,4-β-<small>D</small>-glucanase]] (β-1,4-glucanase, β-1,4-endoglucan hydrolase, endoglucanase D, 1,4-(1,3;1,4)-β-<small>D</small>-glucan 4-glucanohydrolase), [[carboxymethyl cellulase]] (CMCase), avicelase, [[celludextrinase]], [[cellulase A]], [[cellulosin AP]], [[alkali cellulase]], [[cellulase A 3]], [[9.5 cellulase]], [[celloxylanase]] and [[pancellase SS]]. Enzymes that cleave [[lignin]] have occasionally been called cellulases, but this old usage is deprecated; they are [[lignin-modifying enzyme]]s.

== Types and action ==
Five general types of cellulases based on the type of reaction catalyzed:

*[[Endoglucanase SS|Endocellulases]] (EC 3.2.1.4) randomly cleave internal bonds at amorphous sites that create new chain ends.
* [[Exocellulase]]s or cellobiohydrolases (EC 3.2.1.91) cleave two to four units from the ends of the exposed chains produced by endocellulase, resulting in [[tetrasaccharide]]s<ref name="pmid16006068">{{cite journal | vauthors = Zverlov VV, Schantz N, Schwarz WH | title = A major new component in the cellulosome of ''Clostridium thermocellum'' is a processive endo-β-1,4-glucanase producing cellotetraose | journal = FEMS Microbiology Letters | volume = 249 | issue = 2 | pages = 353–8 | date = August 2005 | pmid = 16006068 | doi = 10.1016/j.femsle.2005.06.037 | doi-access = free }}</ref> or [[disaccharide]]s, such as [[cellobiose]]. Exocellulases are further classified into type I, that work processively from the reducing end of the cellulose chain, and type II, that work processively from the nonreducing end. 
* Cellobiases (EC 3.2.1.21) or [[β-glucosidase]]s hydrolyse the exocellulase product into individual monosaccharides.
* [[Oxidative cellulase]]s depolymerize cellulose by radical reactions, as for instance [[cellobiose dehydrogenase (acceptor)]].
* [[Cellulose phosphorylase]]s depolymerize cellulose using phosphates instead of water.

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 [[food energy|energy]] in fibrous plant material.

== Structure ==

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.<ref>{{cite journal | vauthors = Payne CM, Bomble YJ, Taylor CB, McCabe C, Himmel ME, Crowley MF, Beckham GT | title = Multiple functions of aromatic-carbohydrate interactions in a processive cellulase examined with molecular simulation | journal = The Journal of Biological Chemistry | volume = 286 | issue = 47 | pages = 41028–35 | date = November 2011 | pmid = 21965672 | pmc = 3220501 | doi = 10.1074/jbc.M111.297713 | doi-access = free }}</ref> Additional factors like temperature, pH and metal ions influence the non-covalent interactions between enzyme structure.<ref>{{cite journal | vauthors = Lee YJ, Kim BK, Lee BH, Jo KI, Lee NK, Chung CH, Lee YC, Lee JW | display-authors = 6 | title = Purification and characterization of cellulase produced by ''Bacillus amyoliquefaciens'' DL-3 utilizing rice hull | journal = Bioresource Technology | volume = 99 | issue = 2 | pages = 378–86 | date = January 2008 | pmid = 17320379 | doi = 10.1016/j.biortech.2006.12.013 | bibcode = 2008BiTec..99..378L }}</ref> The ''Thermotoga maritima'' species make cellulases consisting of 2 β-sheets (protein structures) surrounding a central catalytic region which is the active-site.<ref name="onlinelibrary.wiley.com">{{cite journal | vauthors = Cheng YS, Ko TP, Wu TH, Ma Y, Huang CH, Lai HL, Wang AH, Liu JR, Guo RT | display-authors = 6 | title = Crystal structure and substrate-binding mode of cellulase 12A from ''Thermotoga maritima''' | journal = Proteins | volume = 79 | issue = 4 | pages = 1193–204 | date = April 2011 | pmid = 21268113 | doi = 10.1002/prot.22953 | s2cid = 23572933 }}</ref> 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.<ref name="onlinelibrary.wiley.com"/> Cellulases produced by the species ''Coprinopsis cinerea'' consists of seven protein strands in the shape of an enclosed tunnel called a β/α barrel.<ref>{{cite journal | vauthors = Liu Y, Yoshida M, Kurakata Y, Miyazaki T, Igarashi K, Samejima M, Fukuda K, Nishikawa A, Tonozuka T | display-authors = 6 | title = Crystal structure of a glycoside hydrolase family 6 enzyme, CcCel6C, a cellulase constitutively produced by ''Coprinopsis cinerea'' | journal = The FEBS Journal | volume = 277 | issue = 6 | pages = 1532–42 | date = March 2010 | pmid = 20148970 | doi = 10.1111/j.1742-4658.2010.07582.x | s2cid = 6338050 | doi-access = free }}</ref> 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 ===

In many bacteria, cellulases in vivo are complex enzyme structures organized in [[Supramolecular assembly|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 exocellulases 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.<ref>{{cite journal | vauthors = Tsai SL, DaSilva NA, Chen W | title = Functional display of complex cellulosomes on the yeast surface via adaptive assembly | journal = ACS Synthetic Biology | volume = 2 | issue = 1 | pages = 14–21 | date = January 2013 | pmid = 23656322 | doi = 10.1021/sb300047u | citeseerx = 10.1.1.701.5515 }}</ref>

Multidomain cellulases are widespread among many taxonomic groups, however, cellulases from anaerobic bacteria, found in cellulosomes, have the most complex architecture consisting of different types of modules. For example, ''Clostridium cellulolyticum'' produces 13 GH9 modular cellulases containing a different number and arrangement of catalytic-domain (CD), carbohydrate-binding module (CBM), dockerin, linker and Ig-like domain.<ref name="pmid24451379">{{cite journal | vauthors = Ravachol J, Borne R, Tardif C, de Philip P, Fierobe HP | title = Characterization of all family-9 glycoside hydrolases synthesized by the cellulosome-producing bacterium ''Clostridium cellulolyticum'' | journal = The Journal of Biological Chemistry | volume = 289 | issue = 11 | pages = 7335–48 | date = March 2014 | pmid = 24451379 | pmc = 3953250 | doi = 10.1074/jbc.M113.545046 | doi-access = free }}</ref>

The cellulase complex from ''[[Trichoderma reesei]]'', for example, comprises a component labeled C1 (57,000 [[dalton (unit)|dalton]]s) that separates the chains of crystalline cellulose, an endoglucanase (about 52,000 daltons), an exoglucanase (about 61,000 dalton), and a β-glucosidase (76,000 daltons).<ref name=worth>Worthington Biochemical Corporation (2014), [http://www.worthington-biochem.com/cel/default.html Cellulase]. Accessed on 2014-07-03</ref>

Numerous "signature" sequences known as [[dockerin]]s and [[cohesin]]s have been identified in the [[genome]]s of bacteria that produce cellulosomes. Depending on their [[amino acid sequence]] and [[tertiary structures]], cellulases are divided into clans and families.<ref name="pmid9818257">{{cite journal | vauthors = Bayer EA, Chanzy H, Lamed R, Shoham Y | title = Cellulose, cellulases and cellulosomes | journal = Current Opinion in Structural Biology | volume = 8 | issue = 5 | pages = 548–57 | date = October 1998 | pmid = 9818257 | doi = 10.1016/S0959-440X(98)80143-7 }}</ref>

Multimodular cellulases are more efficient than free enzyme (with only CD) due to synergism because of the close proximity between the enzyme and the cellulosic substrate. CBM are involved in binding of cellulose whereas glycosylated linkers provide flexibility to the CD for higher activity and protease protection, as well as increased binding to the cellulose surface.<ref name="guerriero" />

== Mechanism of cellulolysis ==
[[Image:Types of Cellulase2.png|thumb|center|500px|class=skin-invert-image|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).]]

[[Image:Cellulase Mech.jpg|thumb|center|500px|class=skin-invert-image|Mechanistic<ref>{{Cite book|chapter-url=http://pubs.rsc.org/en/content/chapterhtml/2015/bk9781782621133-00001?isbn=978-1-78262-113-3|chapter=Chapter 1. Conversion of Biomass into Sugars|last1=Bhaumik|first1=Prasenjit|last2=Dhepe|first2=Paresh Laxmikant|date=2015-01-01|publisher=Royal Society of Chemistry|pages=1–53|language=en|doi=10.1039/9781782622079-00001|title=Biomass Sugars for Non-Fuel Applications|series=Green Chemistry Series|isbn=978-1-78262-113-3}}</ref> details of beta-glucosidase activity of cellulase]]

== Uses ==
Cellulase is used for commercial food processing in [[coffee]]. It performs [[hydrolysis]] of cellulose during drying of [[coffee bean|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. 

===Paper and pulp===
Cellulases have a wide varierty of applications in the paper and pulp industry. In the production and recycling processes cellulases can be applied to improve [[Debarking (lumber)|debarking]], [[pulping]], [[bleaching]], [[drainage]] or [[deinking]].<ref>{{cite book | title = New and Future Developments in Microbial Biotechnology and Bioengineering: Microbial Cellulase System Properties and Applications |chapter = Chapter 13: Cellulase in pulp and paper industry | last1=Singh | first1=S|last2=Singh|first2=V|last3=Aamir|first3=M|last4=Dubey|first4=M|last5=Patel|first5=J|last6=Upadhyay|first6=R|last7=Gupta|first7=V|date= 2 Aug 2016 |doi=10.1016/B978-0-444-63507-5.00013-7}}</ref> 

The use of cellulase can also improve the quality of the paper. Cellulases affect the fiber morphology, which may lead to improved fibre-fibre bonding, resulting in increased fibre cohesion.<ref name ="Kudah">{{cite journal|journal=Enzyme Research|title=Microbial Cellulases and Their Industrial Applications |last1=Kuhad|first1=R.C.|last2=Gupta|first2=R.|last3=Singh|first3=A.|date=2011|volume=10|pages=6065–6072|doi=10.4061/2011/280696|doi-access=free }}</ref> Additional effects on the paper may include increased tensile strength, higher bulk, porosity and tissue softness.

===Pharmaceutical===
Cellulase is used in medicine as a treatment for phytobezoars, a form of cellulose [[bezoar]] found in the human [[stomach]], and it has exhibited efficacy in degrading polymicrobial bacterial [[biofilm]]s by hydrolyzing the β(1-4) glycosidic linkages within the structural, matrix exopolysaccharides of the [[extracellular polymeric substance]] (EPS).<ref>{{cite journal | vauthors = Fleming D, Rumbaugh KP | title = Approaches to Dispersing Medical Biofilms | journal = Microorganisms | volume = 5 | issue = 2 | pages = 15 | date = April 2017 | pmid = 28368320 | pmc = 5488086 | doi = 10.3390/microorganisms5020015 | doi-access = free }}</ref><ref>{{cite journal | vauthors = Fleming D, Chahin L, Rumbaugh K | title = Glycoside Hydrolases Degrade Polymicrobial Bacterial Biofilms in Wounds | journal = Antimicrobial Agents and Chemotherapy | volume = 61 | issue = 2 | pages = AAC.01998–16 | date = February 2017 | pmid = 27872074 | pmc = 5278739 | doi = 10.1128/AAC.01998-16 | url = https://zenodo.org/record/896345 }}</ref>

===Textiles===
Various uses of cellulases in the textile industry include [[biostoning]] of jeans, [[polishing]] of textile fibres, softening of garments, removal of excess dye or the restoration of colour brightness.

===Agriculture===
Cellulases can be used in the agricultural sector as a plant pathogen and for disease control. It is also applied to enhance seed germination and improvement of the root system, and may lead to improved soil quality and recude the dependence on mineral fertilisers.<ref name = "Kudah" />

== Measurement ==
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. 
* '''DNSA Method''' Cellulase activity was determined by incubating 0.5 ml of supernatant with 0.5 ml of 1% carboxymethylcellulose (CMC) in 0.05M citrate buffer (pH 4.8) at 50&nbsp;°C for 30 minutes. The reaction was terminated by the addition of 3 ml dinitrosalicylic acid reagent. Absorbance was read at 540&nbsp;nm.<ref>{{cite journal | vauthors = Jasani H, Umretiya N, Dharajiya D, Kapuria M, Shah S, Patel J | title = Isolation, optimization and production of cellulase by ''Aspergillus niger'' from agricultural waste. | journal = Journal of Pure and Applied Microbiology | date = June 2016 | volume = 10 | issue = 2 | pages = 1159–66 | url = https://www.researchgate.net/publication/304714901 }}</ref>

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.<ref>{{cite journal | vauthors = Umezurike GM | title = The cellulolytic enzymes of ''Botryodiplodia theobromae'' Pat. Separation and characterization of cellulases and β-glucosidases | journal = The Biochemical Journal | volume = 177 | issue = 1 | pages = 9–19 | date = January 1979 | pmid = 106849 | pmc = 1186335 | doi = 10.1042/bj1770009 }}</ref> 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 ===

The lower DP cello-oligosaccharides (DP2-6) are sufficiently soluble in water to act as viable substrates for cellulase enzymes.<ref>{{cite journal | vauthors = Telke AA, Zhuang N, Ghatge SS, Lee SH, Ali Shah A, Khan H, Um Y, Shin HD, Chung YR, Lee KH, Kim SW | display-authors = 6 | title = Engineering of family-5 glycoside hydrolase (Cel5A) from an uncultured bacterium for efficient hydrolysis of cellulosic substrates | journal = PLOS ONE | volume = 8 | issue = 6 | pages = e65727 | year = 2013 | pmid = 23785445 | pmc = 3681849 | doi = 10.1371/journal.pone.0065727 | bibcode = 2013PLoSO...865727T | doi-access = free }}</ref> 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 [[Ion chromatography|IC]] methods to gain valuable information on the substrate requirements of a particular cellulase enzyme.

=== Reduced cello-oligosaccharide substrates ===

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.<ref>{{cite journal | vauthors = Nelson N | year = 1944 | title = A photometric adaptation of the Somogyi method for the determination of glucose | journal = J. Biol. Chem. | volume = 153 | issue = 2 | pages = 375–80 | doi = 10.1016/S0021-9258(18)71980-7 | doi-access = free }}</ref><ref>{{cite journal | vauthors = Smogyi M | title = Notes on sugar determination | journal = The Journal of Biological Chemistry | volume = 195 | issue = 1 | pages = 19–23 | date = March 1952 | doi = 10.1016/S0021-9258(19)50870-5 | pmid = 14938350 | doi-access = free }}</ref>

=== Dyed polysaccharide substrates ===
<ref>{{cite journal | vauthors = McCleary BV | title = New chromogenic substrates for the assay of alpha-amylase and (1 leads to 4)-β-D-glucanase | journal = Carbohydrate Research | volume = 86 | issue = 1 | pages = 97–104 | date = November 1980 | pmid = 6159974 | doi = 10.1016/s0008-6215(00)84584-x }}</ref>

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 ===
[[File:Use of enzyme coupled reagents for the measurement of endo-cellulase.svg|thumb|center|550px|class=skin-invert-image|Colourimetric and fluorimetric cellulase substrates can be used in the presence of ancillary β-glucosidase for the specific measurement of ''endo''-cellulase activity]]
New reagents have been developed that allow for the specific measurement of ''endo''-cellulase.<ref>{{cite journal | vauthors = McCleary BV, Mangan D, Daly R, Fort S, Ivory R, McCormack N | title = Novel substrates for the measurement of endo-1,4-β-glucanase (endo-cellulase) | journal = Carbohydrate Research | volume = 385 | pages = 9–17 | date = February 2014 | pmid = 24398300 | doi = 10.1016/j.carres.2013.12.001 }}</ref><ref>{{cite journal | vauthors = Mangan D, McCleary BV, Liadova A, Ivory R, McCormack N | title = Quantitative fluorometric assay for the measurement of endo-1,4-β-glucanase | journal = Carbohydrate Research | volume = 395 | pages = 47–51 | date = August 2014 | pmid = 25038461 | doi = 10.1016/j.carres.2014.05.002 }}</ref> 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 ==
* [[Cellulose 1,4-beta-cellobiosidase]], an efficient cellulase
* [[Cellulase unit]], a unit for quantifying cellulase activity

== References ==
{{reflist}}

== Further reading ==
{{refbegin}}
* {{cite book | vauthors = Chapin FS, Matson PA, Mooney HA | title = Principles of terrestrial ecosystem ecology | year = 2002 | publisher = Springer | location = New York | isbn = 978-0-387-95439-4 | url = http://www.crc.uqam.ca/Publication/Principles%20of%20terrestrial%20ecosystem%20ecology.pdf | access-date = 2014-07-04 | archive-url = https://web.archive.org/web/20160305123505/http://www.crc.uqam.ca/Publication/Principles%20of%20terrestrial%20ecosystem%20ecology.pdf | archive-date = 2016-03-05 | url-status = dead }}
* The Merck Manual of Diagnosis and Therapy, Chapter 24
* {{cite journal | vauthors = Deka D, Bhargavi P, Sharma A, Goyal D, Jawed M, Goyal A | title = Enhancement of Cellulase Activity from a New Strain of Bacillus subtilis by Medium Optimization and Analysis with Various Cellulosic Substrates | journal = Enzyme Research | volume = 2011 | pages = 151656 | year = 2011 | pmid = 21637325 | pmc = 3102325 | doi = 10.4061/2011/151656 | doi-access = free }}
* {{cite journal | vauthors = Zafar M, Ahmed S, Khan MI, Jamil A | title = Recombinant expression and characterization of a novel endoglucanase from ''Bacillus subtilis'' in ''Escherichia coli'' | journal = Molecular Biology Reports | volume = 41 | issue = 5 | pages = 3295–302 | date = May 2014 | pmid = 24493451 | doi = 10.1007/s11033-014-3192-8 | s2cid = 203374 }}

{{refend}}

{{Authority control}}
{{Glycoside hydrolases}}

[[Category:Carbohydrate metabolism]]
[[Category:Cellulose]]
[[Category:Enzymes]]