## 3 Results and Discussion
### Substrate Specificity toward 4 NP-\(\beta\)-Glc/GalNAc and 2 NP-\(\beta\)-Glc/GalNAc  
\(\beta\)-\(N\)-acetylhexosaminidasees are characterized by their inherent dual catalytic activity, which allows them to act over two substrates differing in the configuration of the  
Figure 3: Effect of pH (**A**) and temperature (**B**) on the specific activity of Hex. Reaction conditions: reaction volume 2 mL, 4-NP-\(\beta\)-GalNAc concentration 4 mM, enzyme activity 0.2-0.3 U. The pH profile was investigated at 37 \({}^{\circ}\)C, and the temperature profile was investigated at pH 4.5. Figure 2: Time course of the extracellular Hex activity of PcHex3437, PcHex3210, PcHex1, and PcHex62558 in the culture medium containing 0.5% \(N\)-acetylglucosamine as the inducer. hydroxyl group on C-4 of the glycan, which may be \(N\)-acetyllglucosamine (GlcNAc) or \(N\)-acetylgalactosamine (GalNAc). This specificity characteristic is commonly tested on substrates such as 4 NP-\(\beta\)-GalNAc and 4 NP-\(\beta\)-GlcNAc, which are largely accepted by a wide range of \(\beta\)-\(N\)-acetylhexosaminidases. Moreover, in this study, we used nitrophenyl glycosides with a nitro group binding to the phenyl group in position 2, namely, 2 NP-\(\beta\)-GalNAc and 2 NP-\(\beta\)-GlcNAc, because the GlcNAcase/GalNAc case ratio may also be affected by the aglycon structure [17]. In general, \(\beta\)-\(N\)-acetylhexosaminidases prefer D-glucose structures, with the GlcNAcase-to-GalNAc case ratio ranging from 1.5 to 4.0 [3]. As shown in Table 2, Hex produced by _P. crustosum_ strains showed higher specific activity toward 2-nitrophenyl substrates, which indicated their preference for 2-nitrophenyl aglycon over 4-nitrophenyl, apart from the strain PcHex3437, for which the difference was not very significant. Concerning the preferences for glucose or galactose, Hex isolated from all four strains of _P. crustosum_ exhibited a GalNAcase/GlcNAcNAc case ratio similar to that of various species of _Penicillium_, namely, 0.9 for _Penicillium chrysogenum_[18], 1.2 for _Penicillium funcilosum_, or 1.15 for _Penicillium purpurrogenum_[19], but still lower than that of _P. oxalicum_ strains, for which the GalNAcase/GlcNAcNAc case ratio usually ranged from 1.4 to 2.3 [19,20]. The determination of the preference for GalNAcase/GlcNAcNAc case is a delicate matter, as the preference not only depends on the producing strain but also varies during microorganism cultivation and can be influenced by the presence of some inorganic salts or organic solvents [19]. Kinetic constants describing the hydrolysis of the substrates 4 NP-\(\beta\)-GalNAc, 4 NP-\(\beta\)-GalNAc, 2 NP-\(\beta\)-GalNAc, and 2 NP-\(\beta\)-GlcNAc were determined for all four PcHex. The kinetics of the hydrolysis of 4 NP-\(\beta\)-GalNAc, 4 NP-\(\beta\)-GalNAc, 2 NP-\(\beta\)-GalNAc, and 2 NP-\(\beta\)-GlcNAc was described using the Michaelis-Menten equation. The kinetic parameters \(\mathrm{k_{cat}}\) (catalytic constant), and \(\mathrm{K_{m}}\) (Michaelis constant) were determined using nonlinear regression from the initial reaction rate versus substrate concentration data using MATLAB R2022a (lsqnonlin procedure). When comparing the \(\mathrm{k_{cat}/K_{m}}\) values shown in Figure 4, the most efficient catalyst overall was PcHex1, with the highest \(\mathrm{k_{cat}/K_{m}}\) values for almost all tested substrates, except for 2 NP-\(\beta\)-GlcNAc. The catalytic efficiency of PcHex1 was more than threefold higher than that of PcHex3437. Three of the four PcHex catalyzed the hydrolysis of 4 NPGlcNAcNAc the most efficiently. Only the strain PcHex3210 operated most efficiently with the substrate 2 NP-\(\beta\)-GlcNAc.