## 3 Results and Discussion
### Substrate Specificity toward Nonconventional Substrates  
The specific activities of the purified PcHex were measured with three different nonconventional substrates: 4-methylumbeliferryl-2-acetamido-2-deoxy-\(\beta\)-D-galactopyranoside (4 MU-\(\beta\)-GalNAc), 5-bromo-4-chloro-3-indolyl-N-acetyl-\(\beta\)-D-galactopyranoside (X-\(\beta\)-GalNAc), and 2-chloro-4-nitrophenyl-\(\beta\)-D-galactopyranoside (2 Cl-4 NP-\(\beta\)-Gal; Figure 5). They belong to the group of synthetic fluorogenic/chromogenic enzyme substrates, and the properties of Hex acting on these substrates have not yet been studied. Most of the fluorogenic substrates contain a fluorescent coumarin heterocyclic, such as 4-methylumbelliferone (4-MU), which is often used, for example, for the detection of glycosidases and phosphatases [21]. The fluorescence of 4-MU is strongly dependent on pH and reaches the maximum at pH 9-10 [22]. Derivatives of 4-methylumbelliferryl provide a more sensitive detection method than nitrophenyl glycosides, which are frequently used for carbohydrate-active enzymes [23]. 4-Methylumbelliferryl-\(N\)-acetyl-\(\beta\)-D-glucosamine has been successfully used for the early detection of fungal growth on various cultural heritage materials before it was visible to the human eye [24]. PcHex1 (15.38 U/mg protein) showed the highest specific activity against 4-MUGalNAc with 98% hydrolysis after 120 min. The other three enzymes showed threefold lower activity, approximately 5 U/mg protein (Table 3). Figure 4: Kinetic parameter analysis describing hydrolysis of 4 NP-\(\beta\)-Gal/GlcNAc and 2 NP-\(\beta\)-Gal/GlcNAc by PcHex. X-\(\beta\)-GalNAc is a synthetic substrate consisting of \(N\)-acetylgalactopyranoside and aglycone 5-bromo-4-chloro-indolxyl, which is commonly referred to as X. Indoxyl substrates are very popular among chromogenic substrates because of their intensive color, which appears when two indoxyl molecules dimerize under oxidative conditions. This compound is insoluble, which provides highly localized labeling of the target. Probably the best-known indoxyl substrate is 5-bromo-4-chloro-3-indolyl \(\beta\)-D-galactopyranoside (X-Gal), which is used in molecular biology applications to distinguish nonrecombinant transform colonies (blue) from recombinant colonies carrying the Lac Z gene (white) [25]. The hydrolysis of X-\(\beta\)-GalNAc was catalyzed by all four purified PcHex, although their specific activity toward this substrate was significantly lower than that toward the other two substrates. The highest specific activity was observed for the enzyme PcHex 62558 (0.28 U/mg protein), with an almost 94% degree of hydrolysis after 22 h.  
Testing hydrolysis reactions using hexosaminidase with 2 Cl-4 NP-\(\beta\)-Gal as the substrate has not yet been described in the literature, although this substrate was used to measure the level of salivary \(\alpha\)-amylase in athletes [26] and to analyze the correlation between pain and salivary \(\alpha\)-amylase levels during dental treatment in children with Down syndrome [27]. During the hydrolysis of 2 Cl-4 NP-\(\beta\)-Gal, 2-chloro-4-nitrophenol is released, which causes a more intense yellow color than that generated by 4-nitrophenol. Hydrolysis of this substrate was surprising for us because, unlike other substrates assessed in this study, 2 Cl-4 NP-\(\beta\)-Gal lacks the \(N\)-acetyl group on galactopyranoside. This group is usually crucial for \(\beta\)-\(N\)-acetylhexosaminidaseases, as they are sensitive to its modification. However, fungal \(\beta\)-\(N\)-acetylhexosaminidaseases tolerate certain \(N\)-acyl-modified substrates that have sterical changes at C-2 (shorter or longer acyls, a hydroxyl group instead of a hydrogen atom) [17]. The hexosaminidaseePcHex3437 could hydrolyze this substrate easily (enzyme activity: 3.97 U/mg), with the degree of hydrolysis reaching 96% in 30 min of reaction, similar to PcHex1.