## 2 The Principles and Enzymes Used in EAE
### Types and Activity of Hydrolytic Enzymes
#### 2.2.1 Cellulaes  
Cellulae is not a single enzyme; in fact, it is a complex of enzymes, which act synergistically. The complex comprises at least three major subclasses of enzymes such as endoglucases (endo-1,4-\(\beta\)-\(\mathrm{p}_{\mathrm{T}}\)-glucanane), exoglucanases (cellobiohydrolases; 1,4-\(\beta\)-\(\mathrm{p}_{\mathrm{T}}\)-glucan cellobiohydrolase), and \(\beta\)-glucosidasees (cellobiase, \(\beta\)-\(\mathrm{p}_{\mathrm{T}}\)-glucose-glucohydrolase) [28; 29]. Endoglucanases have an ability to randomly hydrolyse \(\beta\)-1,4-glycosidic bonds inside the molecule and, consequently, release a long chain of cello-oligosaccharides. These enzymes act on the cellulose amorphous region. Exoglucanases break cello-oligosaccharides at the end of the chain and release glucose and cellobiose. Cellobiase catalyses the conversion of cellobiose into glucose [29; 30].  
Cellulaes are used in a number of industrial processes, e.g., wastewater treatment, the bakery, textile, brewing, paper, and food industries, wine production, and the production of bioethanol. These enzymes are naturally produced abundantly by microorganisms and cultures of higher fungi, e.g., _Trichoderma reesei_, _Aspergillus niger_, _Trichoderma viride_, _Penicillium purpurrogenum_, _Penicillium echinulatum_, _Pleutrotus vorida_, _Pleutrotus cornucopiae_, and _Bacillus pumilus_[31; 32; 33]. Interestingly, the activity of an enzyme at specific temperatures and pH values depends on the organism that produced this enzyme. For example, cellulase obtained from _Penicillium purpurrogenum_, _Pleutrotus vorida_, and _Pleutrotus cornucopiae_ exhibited maximum activity at a temperature of 50 \({}^{\circ}\)C. The optimum pH value for fungal cellulase is 4.5. However, cellulase from _Aspergillus phoenicus_ has its maximum activity at 4.8-5.5 pH [28].  
Trichoderma resei, also known as Hypocrea jecorina, is considered the most efficient producer of cellulase for industrial applications [28]. The enzyme is produced through submerged fermentation of a selected strain of the fungus and can be bought as an aqueous solution for laboratory research. Cellulase obtained from _T. reesei_ is able to decompose cellulose into glucose, cellobiose, and other glucose polymers as well. According to the producer, its specific activity is estimated at more than 700 units/g, and its density is 1.10-1.30 g/mL. The optimum values of pH and temperature are estimated to be 6 and 52 \({}^{\circ}\)C, respectively (www.sigmaaldrich.com; access on 18 January 2022).  
The other cellulase that is quite popular is cellulase from Aspergillus niger. The enzyme is available in the form of powder, and its specific activity is estimated at more than 0.3 units/mg. Cellulase from Aspergillus niger hydrolyses endo-1,4-g\({}_{\text{-D}}\)-glycosidic linkages in several types of polysaccharides. The stable pH range of the enzyme is 5.0-10.0 and the optimal pH for its activity is 6.0-7.0. It has been established that the enzyme obtained from _A. niger_ cultivated in a liquid culture medium with rice bran and yeast extract is thermally stable, and the optimal temperature at pH 6.0 is 70 \({}^{\circ}\)C [34]. However, the investigations conducted in 2020 by [PERSON] et al. revealed that cellulase obtained from _A. niger_ cultured on _Arachis hypogaea_ shells had an optimum pH and temperature of 4 and 40 \({}^{\circ}\)C, respectively [35]. This may indicate that the activity of specific enzymes depends on the producing species and culture conditions. Aspergillus sp. cellulase obtained through submerged fermentation can also be bought as an aqueous solution, and its specific activity reaches over 1000 units/g.  
The growing interest in the potential and efficient applications of cellulases influences the direction of scientific research. Many new studies are focused on improving the production of cellulase by different organisms, e.g., bacteria [29; 36].