**4.2 Cellulase catalysis mechanism**

The endogilanase randomly acts to cut the 1,4-glycosidic bonds so that the cellulose chain has a new end. Endoglucanases produced by bacteria, fungi, animals and plants have different catalyst modules. In fungi, the endoglucanase produced generally has a catalytic module without carbohydrate-binding module (CBM), while the endogluanase in bacteria is generally supplemented with CBM. CBM is generally located at the N or C terminus in the cellulose structure and functions as a binding site between enzymes and an insoluble substrate, allowing cellulase to break down the crystalline domain regions of cellulose. Most cellulases have an enzyme active site in the form of clefts that allow cellulases to bind and break the cellulose chains to produce glucose, soluble cellodextrins and insoluble cellulose fragments. Some endoglucanases can also act gradually to hydrolyze the crystalline domain of cellulose, which results in the main product being cellobiose or cellodextrin [20].

Exogucanases are known to work specifically at the ends of the cellulose chains and produce the main products in the form of cellobiose and glucose. Exoglucanase can effectively act on the crystalline domain structure of cellulose. Cellobiohydrolase (CBH) is one of the most widely produced exoglucanases. CBH is generally produced by bacteria and fungi, with a variety of different catalyst modules. A recognized significant CBH structure is a tunnel structure formed from two surface loops on the active site of the enzyme. The tunnel-shaped active site of

**135**

*The Nanocellulose Fibers from Symbiotic Culture of Bacteria and Yeast (SCOBY) Kombucha...*

exoglucanase makes the cellulose hydrolysis process unique. In the mechanism of hydrolysis of exoglucanase, the cellulose chain enters the tunnel, where the active side of the enzyme recognizes the end of the cellulose chain and hydrolyzes the 1,4-glycosidic bonds at the end of the cellulose chain. In general, exoglucanase and endoglucanase have an enzyme folding side, the difference in the folding structure

B-glucosidase (BG) is an exogluanase that does not contain CBM and functions to hydrolyze cellubiose and cellodextrin into glucose. BG acts as an enzyme that lowers the level of cellubiose in the substrate which can act as a CBH inhibitor and endogilanase. BG is known to be produced by bacteria, fungi, plants and animals. In aerobic fungi, it is known that BG is produced extracellularly, while in bacteria BG is produced intracellally and is maintained in the cytoplasm. BG has a pocketshaped enzyme active site, which allows the enzyme to bind to non-reducing glucose units and hydrolyze cellobiose and cellodextrin to glucose [22].

The success of the cellulase enzyme to carry out cellulose hydrolysis is influenced by several factors, including the degree of water swelling, the level of crystal-

Water content in cellulose is an important factor that can affect the performance of the cellulase enzyme. Cellulose which has a low DWS level tends to be dry and has a narrow surface area due to shrinkage. The ability of cellulose to swell and shrink is influenced by the nature of the solvent used. Solvents with non-polar characteristics generally find it difficult to swell cellulose structures and increase the surface area, whereas solvents with polar characteristics are known to swell cellulose structures very well. The swollen structure of cellulose has a wider surface area, which allows the cellulase to more easily penetrate the multiple sides of the cellulose [23].

The degree of crystallinity of cellulose is known to play a role in determining the rate of hydrolysis of cellulase enzymes. This factor is motivated by the data regarding amorphous cellulose which is degraded more quickly to cellobiose, compared to crystalline cellulose. This data is used by researchers as a form of confirmation of cellulase performance, where the increased crystallization data of a cellulose treated with cellulase, indicates good cellulase activity. This theory is believed by looking at the data that the cellulase first hydrolyzes amorphous cellulose and converts it into a crystalline form [23, 24]. SCOBY kombucha is known to consist of 37% crystalline

Crystallization of cellulose also affects the adsorption rate of cellulase enzymes on cellulose. It is known that cellulose with a higher degree of crystallization has a lower enzyme adsorption rate. The crystalline structure of cellulose generally inhibits penetration of the hydrolase system, CBM and other enzyme components [23].

The synergistic effect of cellulase is one of the important factors in the hydrolysis of cellulose. This synergistic effect can occur on the performance of endogluanase-endogluanase, endogluanase-exogluxase, exogilanase-exogilanase,

linity, and the enzymatic synergistic effect that can occur on cellulase.

*DOI: http://dx.doi.org/10.5772/intechopen.96310*

of the two is only in the active side of the enzyme [21].

**4.3 Factors affecting cellulase performance**

*4.3.1 Degree of water swelling (DWS)*

*4.3.2 Degree of crystallinity*

*4.3.3 Enzyme synergistic effects*

structure and 63% nanofibril structure [22].

*The Nanocellulose Fibers from Symbiotic Culture of Bacteria and Yeast (SCOBY) Kombucha... DOI: http://dx.doi.org/10.5772/intechopen.96310*

exoglucanase makes the cellulose hydrolysis process unique. In the mechanism of hydrolysis of exoglucanase, the cellulose chain enters the tunnel, where the active side of the enzyme recognizes the end of the cellulose chain and hydrolyzes the 1,4-glycosidic bonds at the end of the cellulose chain. In general, exoglucanase and endoglucanase have an enzyme folding side, the difference in the folding structure of the two is only in the active side of the enzyme [21].

B-glucosidase (BG) is an exogluanase that does not contain CBM and functions to hydrolyze cellubiose and cellodextrin into glucose. BG acts as an enzyme that lowers the level of cellubiose in the substrate which can act as a CBH inhibitor and endogilanase. BG is known to be produced by bacteria, fungi, plants and animals. In aerobic fungi, it is known that BG is produced extracellularly, while in bacteria BG is produced intracellally and is maintained in the cytoplasm. BG has a pocketshaped enzyme active site, which allows the enzyme to bind to non-reducing glucose units and hydrolyze cellobiose and cellodextrin to glucose [22].

#### **4.3 Factors affecting cellulase performance**

The success of the cellulase enzyme to carry out cellulose hydrolysis is influenced by several factors, including the degree of water swelling, the level of crystallinity, and the enzymatic synergistic effect that can occur on cellulase.

#### *4.3.1 Degree of water swelling (DWS)*

*Nanofibers - Synthesis, Properties and Applications*

of about 3-4 nm in length and 70–80 nm in width [16].

it can change the size of cellulose to nanocellulose.

**4.2 Cellulase catalysis mechanism**

**4.1 Screening method for cellulase producing bacteria**

Cellulose I is synthesized to form cellulose complexes that are linked to one another outside the cytoplasmic membrane, while cellulose II is formed to resemble free cellulose fibers outside the cytoplasmic membrane. The structure content of cellulose I and II will affect the tensile strength, polymerization rate and crystallinity index of cellulose fibers. The microfibrils produced by *Acetobacter xylinum* have dimensions

Cellulase is an enzyme that can degrade cellulose by breaking the 1,4-glycosidic

bonds in cellulose polymers. Naturally, cellulases can be obtained in nature as metabolites of microbial metabolism, such as bacteria and fungi. Microbes that can produce cellulase enzymes usually have habitats in the soil, where these microbes play a role in the degradation of cellulose in plants. Cellulase is known to be one of the most widely used enzymes in the industrial sector, such as bio-stoning in the textile industry, extraction of fruit and vegetable juices in the food industry, and bleaching in the paper industry [18]. In the manufacture of nanocellulose, cellulase can also be used to degrade the structure of cellulose fibrils into crystalline, so that

Screening of bacteria that can produce cellulase can be done using CMC (Carboxymehylcellulose) medium. CMC is a cellulose derivative which is commonly used as a thickener or stabilizer in the industrial field [19]. The composition of the CMC medium includes CMC, yeast extract, MgSO4, NH4H2PO4, and KCl. Bacteria that are thought to be able to produce cellulase are cultivated first on CMC agar medium, under certain conditions. The growth of bacterial colonies on CMC medium can be an early marker of cellulase activity in bacteria. Qualitative confirmation of cellulase activity in bacteria can be done by testing 1% Congo Red and 1 M NaCl. The formation of a clear zone that occurred around the bacterial colony after testing the Congo Red can be used as a qualitative positive result of cellulase activity in bacteria. The formation of this clear zone indicates that bacteria can hydrolyze cellulase contained in the medium to simple sugar (glucose) [18].

The endogilanase randomly acts to cut the 1,4-glycosidic bonds so that the cellulose chain has a new end. Endoglucanases produced by bacteria, fungi, animals and plants have different catalyst modules. In fungi, the endoglucanase produced generally has a catalytic module without carbohydrate-binding module (CBM), while the endogluanase in bacteria is generally supplemented with CBM. CBM is generally located at the N or C terminus in the cellulose structure and functions as a binding site between enzymes and an insoluble substrate, allowing cellulase to break down the crystalline domain regions of cellulose. Most cellulases have an enzyme active site in the form of clefts that allow cellulases to bind and break the cellulose chains to produce glucose, soluble cellodextrins and insoluble cellulose fragments. Some endoglucanases can also act gradually to hydrolyze the crystalline domain of cellulose, which results in the main product being cellobiose or cellodextrin [20]. Exogucanases are known to work specifically at the ends of the cellulose chains and produce the main products in the form of cellobiose and glucose. Exoglucanase can effectively act on the crystalline domain structure of cellulose. Cellobiohydrolase (CBH) is one of the most widely produced exoglucanases. CBH is generally produced by bacteria and fungi, with a variety of different catalyst modules. A recognized significant CBH structure is a tunnel structure formed from two surface loops on the active site of the enzyme. The tunnel-shaped active site of

**134**

Water content in cellulose is an important factor that can affect the performance of the cellulase enzyme. Cellulose which has a low DWS level tends to be dry and has a narrow surface area due to shrinkage. The ability of cellulose to swell and shrink is influenced by the nature of the solvent used. Solvents with non-polar characteristics generally find it difficult to swell cellulose structures and increase the surface area, whereas solvents with polar characteristics are known to swell cellulose structures very well. The swollen structure of cellulose has a wider surface area, which allows the cellulase to more easily penetrate the multiple sides of the cellulose [23].

#### *4.3.2 Degree of crystallinity*

The degree of crystallinity of cellulose is known to play a role in determining the rate of hydrolysis of cellulase enzymes. This factor is motivated by the data regarding amorphous cellulose which is degraded more quickly to cellobiose, compared to crystalline cellulose. This data is used by researchers as a form of confirmation of cellulase performance, where the increased crystallization data of a cellulose treated with cellulase, indicates good cellulase activity. This theory is believed by looking at the data that the cellulase first hydrolyzes amorphous cellulose and converts it into a crystalline form [23, 24]. SCOBY kombucha is known to consist of 37% crystalline structure and 63% nanofibril structure [22].

Crystallization of cellulose also affects the adsorption rate of cellulase enzymes on cellulose. It is known that cellulose with a higher degree of crystallization has a lower enzyme adsorption rate. The crystalline structure of cellulose generally inhibits penetration of the hydrolase system, CBM and other enzyme components [23].

#### *4.3.3 Enzyme synergistic effects*

The synergistic effect of cellulase is one of the important factors in the hydrolysis of cellulose. This synergistic effect can occur on the performance of endogluanase-endogluanase, endogluanase-exogluxase, exogilanase-exogilanase,

**Figure 6.**

*Cellulase action mechanism; CBH: Cellobiosehydrolase/Exoglukanase; EG: Endoglukanase, NR: Non Reductor; R: Reductor [26].*

or endogilanase/exogilanase with CBM simultaneously (**Figure 6**). Several studies have shown that the performance of the endogucanase-exogilanase enzyme can occur synergistically and produce good cellulose hydrolysis products. Other studies have shown that the use of cellulases that have the same cutting-edge (endo-endogluanase or exogluanase) can make the two enzymes inhibit each other [25].
