## 3 Results and Discussion
### Substrate Specificity toward Nonconventional Substrates
The observation of the ability to catalyze the hydrolysis of chlorinated derivatives of other than \(N\)-acetylated saccharides is so interesting that it would be useful to focus future research on verifying this ability using \(\beta\)-\(N\)-acetylhexosaminidaseases in a highly purified form and, this way, exclude the presence of contaminating activities of other enzymes those presence in pre-purified preparations cannot be excluded with certainty. Separation of an Anomeric Mixture of 4 MUl-\(\alpha\)/\(\beta\)-GalNAc by Selective Hydrolysis  
Fungal \(\beta\)-\(N\)-acetylhexosaminidaseases can selectively hydrolyze \(\beta\)-anomer in a mixture of \(\alpha\)- and \(\beta\)-substituted GalNAc derivatives. It is a convenient method for preparing \(\alpha\)-anomers of synthetic substrates suitable for the rapid identification and quantification of specific enzymes. This method is favorable because the enzymes are inexpensive and well-characterized, and the reaction is carried out under mild reaction conditions. After the hydrolysis reaction, \(\alpha\)-anomer can be easily separated from the reaction mixture by extraction [11]. This is the first study to report the ability of hexosaminidaseases to efficiently hydrolyze \(\beta\)-anomer of 4 MU-GalNAc in a chemically prepared anomeric mixture of 4 MU-\(\alpha\)/\(\beta\)-GalNAc (Scheme 1). The main product of this hydrolysis reaction, namely, 4 MU-\(\alpha\)-GalNAc, is a fluorogenic synthetic substrate, which allows the sensitive determination of \(\alpha\)-N-acetylgalactosaminidase (\(\alpha\)-NAGA), an important marker monitored in clinical practice. It \begin{table}
\begin{tabular}{c c c c} \hline \hline  & \multicolumn{3}{c}{**Specific Activity (U/mg Protein)**} \\ \hline  & **4 MU-\(\beta\)-GalNAc** & **X-\(\beta\)-GalNAc** & **2 Cl-4 NP-\(\beta\)-Gal** \\ \hline PcHex1 & 15.4 \(\pm\) 1.2 & 0.181 \(\pm\) 0.004 & 3.32 \(\pm\) 0.29 \\ PcHex3437 & 5.67 \(\pm\) 0.51 & 0.171 \(\pm\) 0.025 & 3.97 \(\pm\) 0.30 \\ PcHex3210 & 5.05 \(\pm\) 0.47 & 0.221 \(\pm\) 0.019 & 2.082 \(\pm\) 0.009 \\ PcHex62558 & 5.89 \(\pm\) 0.42 & 0.287 \(\pm\) 0.025 & 2.50 \(\pm\) 0.21 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Enzyme hydrolysis of 4 MU-\(\beta\)-GalNAc, X-\(\beta\)-GalNAc, and 2 Cl-4 NP-\(\beta\)-Gal. is naturally produced by the human body and is required by cells to degrade glycolipids and glycoproteins. Genetic mutations in the NAGA gene can cause a loss of enzyme activity, leading to a serious disease called Schindler's disease [28]. For the separation of the anomeric mixture, we chose the hexosaminidase from the _P. crustosum_ strain PcHex1, which showed the highest specific activity toward the substrate 4 MU-\(\beta\)-GalNAc (Table 2). The hydrolysis reaction was carried out under standard conditions in citrate-phosphate buffer (pH 4.5). After 4 h, 99.1% of the \(\beta\)-anomer was hydrolyzed (Figure 6). The reaction mixture consisted of \(\alpha\)-anomer of 4-MU-GalNAc, 4-MU aglycone, and \(N\)-acetylgalactosamine. After double-solvent extraction with ethyl acetate, 98% of 4-MU was removed from the reaction mixture, leaving 91% of the \(\alpha\)-anomer in the aqueous phase along with \(N\)-acetylgalactosamine. The aqueous phase was subsequently extracted thrice with n-butanol. The total yield of 4 MU-\(\alpha\)-GalNAc extracted into n-butanol was 90%. As for further simplification of the process, the buffer in the reaction medium was replaced with distilled water. The 4 MU-\(\alpha\)/\(\beta\)-GalNAc nanometer mixture was thus dissolved in distilled water, and the pH of the mixture was adjusted to pH 4.5 at the beginning of the reaction and was then controlled during hydrolysis. The enzymatic activity of Hex and the product yield remained the same. The improved procedure for the preparation of \(\alpha\)-anomeric derivatives of glycosides enables the effective production of 4 MU-\(\alpha\)-GalNAc in a buffer-free environment, thus, minimizing the impurity content in the final product.