## 2 The Principles and Enzymes Used in EAE
### Types and Activity of Hydrolytic Enzymes
#### 2.2.3 Hemicellulases  
Hemicellulases is a common name for several enzymes with hemicellulose activity, e.g., xylanases, glucuronidases, arabinofuranosidases, galactosidases, mannanases, and acetyl or feruloyl esterases. These classes of hemicellulases can hydrolyse different structures of hemicellulose [30].  
The properties of hemicellulases depend on their origin. Interestingly, different organisms are able to produce different sets of enzymes, which may act synergistically [30; 42]. Xylan is a structure that occurs in the largest amount in hemicellulose; hence, endoxylanases and xylosidases are the most important enzymes. Endoxylanases (endo-g-1,4-xylanase/1,4-g-p-xylan xylanohydrolase) decompose glycosidic bonds releasing small oligosaccharides. Xylosidases (1,4-g-p-xylan xylohydrolase) hydrolyse \(\beta\)-1,4-bonds, which leads to the release of xylose from xylooligosaccharides. Another enzymatic compound from the hemicellulose family is \(\beta\)-mannanase (endo-1,4-\(\beta\)-mannanase/1,4-\(\beta\)-p-mannananohydrolase), which decomposes hemicelluloses with a dominant number of mannans. Due to this action, it releases short mannooligomers [30]. Subsequently, mannooligomers can be hydrolysed another enzyme--\(\beta\)-mannosidase (\(\beta\)-D-mannosidase/1,4-\(\beta\)-D-mannoside mannohydrolase), which leads to the release of mannose. \(\alpha\)-L-arabinofuranosidase, in turn, belongs to debranching enzymes, which \"cut off\" side groups or substituents [30]. \(\alpha\)-D-glucuronidases cleave \(\alpha\)-1,2- bonds between the main chain in glucuronoxylan and glucuronic acid residues, which may lead to debranching of glucuronoxylans [43]. Another enzyme, i.e., acetyl xylan esterase, is an important agent used for enzymatic hydrolysis of lignocellulosic materials, since it removes O-acetyl groups from acetylxylan, supporting or enhancing xylanase activity [44]. Among hemicellulases, there is \(\alpha\)-1-p-galactoside galactohydrolase, which hydrolyses \(\alpha\)-1,6 bonds from the main chain and releases alpha-1,6-linked-p-galactopyranosyl substituents. The list of hemicellulose enzymes also comprises phenolic acid esterases: feruloyl esterases and \(p\)-couamaryl esterases, which hydrolyse ester bonds between arabinose and monomers of ferulic acid as well as bonds between arabinose and \(p\)-couamaric acid [30].  
Depending on the origin of the enzyme, there may be different optimal pH and temperature values. As reported by [PERSON] et al., fungal xylanase is more effective at pH values ranging from 3.5 to 6.5 and temperatures of 40-60 \({}^{\circ}\)C [45]. In turn, bacterial xylanases are more effective at pH 5.0-8.0 and in the temperature range of 50-80 \({}^{\circ}\)C [46].  
Hemicellulases are produced abundantly by microorganisms, but fungi and thermophilic bacteria, e.g., _Aspergillus_ sp., _Trichoderma_ sp., _Bacillus_ sp., and _Cellibrio_ sp., are the most essential for commercial use [42]. Interestingly, _Clostridium_ bacteria are able to produce a complex of cellulases and hemicellulases. The enzymes are also produced by _Penicillium_ and _Rhizopus_ sp. These enzymes are used in the bakery and beverage industries. They can replace oxidative substances and can also be used to clarify juices. They are also used in the textile industry and for production of prebiotic oligosaccharides [30; 42]. _Aspergillus niger_ hemicellulose is a commercially available product. It is in a powder form and its specific activity is estimated at 0.3-3.0 unit/mg solid. According to the manufacturer, one unit will produce a relative fluidity change of 1 per 5 min using locust bean gum as a substrate at pH 4.5 and 40 \({}^{\circ}\)C. Importantly, the enzyme should be stored at a temperature of \(-20\)\({}^{\circ}\)C (www.sigmaaldrich.com; accessed on 18 January 2022).