###### Abstract  
\(\beta\)-\(N\)-acetylhexosaminidases have great potential in applied biocatalysis owing to their ability to act on a wide range of natural and modified substrates. In this work, \(\beta\)-\(N\)-acetylhexosaminidases from four _Penicillin crustosum_ strains (PcHex) were studied. The production strains showed the highest enzymatic activity in the culture medium after 11-14 days of cultivation. The specific activity of the isolated and purified PcHex of hydrolysis of 4-nitrophenyl-N-acetyl-\(\beta\)-D-galactopyranoside was 15-20 U/mg protein. All isolated \(\beta\)-\(N\)-acetylhexosaminidases showed similar pH-activity profiles, with the optimum pH being 4.0-5.0 and the optimum temperature being 40 \({}^{\circ}\)C-50 \({}^{\circ}\)C. Apart from standard substrates, two synthetic substrates (5-bromo-4-chloro-3-indolyl-N-acetyl-\(\beta\)-D-galactoside and 2-chloro-4-nitrophenyl-\(\beta\)-D-galactopyranoside) were tested and successfully hydrolyzed using \(\beta\)-\(N\)-acetylhexosaminidases programics. Maximum activity toward the fluorogenic substrate 4-methylumbelliferyl-\(N\)-acetyl-\(\beta\)-D-galactopyranoside was observed for enzyme preparations from PcHex (15.38 U/mg protein) isolated from swab samples of books in the Slovak National Museum in Martin (Slovak Republic) and stored in our laboratory. The same enzyme preparation was used for the selective hydrolysis of \(\beta\)-anomer of 4 MU-GalNAc from anomeric mixture of 4 MU-\(\alpha\)/\(\beta\)-GalNAc. Thus, pure \(\alpha\)-anomer (with the total yield being 90%) was separated from the mixture, which suggests the application potential of these enzymes.  
Penicillin crustosum; \(\beta\)-\(N\)-acetylhexosaminidases; biochemical characterization; synthetic glycosides; chemo-enzymatic synthesis +
Footnote †: journal: _Sci._  
Footnote †: journal: _Sci.  
To expand the current group of natural \(\beta\)-\(N\)-acetylhexosaminidaseases, we investigated the production and properties of fungal \(\beta\)-\(N\)-acetylhexosaminidaseases from _Penicillium crustosum_ that have not been studied so far. The fungus _P. crustosum_ was first isolated by [PERSON] in 1930 [4]. Since then, it has been extensively used as an active producer of pectorolytic and cellulolytic enzymes [5], a novel lipase [6], and schizophrenia degrading \(\beta\)-glucanase [7]. Moreover, _P. crustosum_ has been used for the production of secondary metabolites such as roqueforting, penitrem, viridicatols, terrestricic acid, and anddrastin [8]. However, to the best of our knowledge, none of the previous studies have reported the production of \(\beta\)-\(N\)-acetylhexosaminidaseases by _P. crustosum_. [PERSON] et al. [9] assessed active glucanases and glucosidases from 92 strains of fungi from various marine habitats, one of which was _P. crustosum_, which, however, showed no \(\beta\)-\(N\)-acetylhexosaminidase activity [9]. Thus far, the potential application of Hex in the production of \(\alpha\)-anomers from synthetic substrates by enzymatic hydrolysis of the anomeric mixture has been described in the literature only twice, that is, once using bacterial Hex in the preparation of \(\alpha\)-anomer from GlcNAc [10], and the other, using fungal Hex in the preparation of 4-nitrophenyl-\(N\)-acetyl-\(\alpha\)-D-galactopyranoside (4-NP-\(\alpha\)-GalNAc), which is our previous study [11].  
In this research, we aimed to explore the cultivation conditions, biochemical properties, and substrate specificity of \(\beta\)-\(N\)-acetylhexosaminidaseases isolated from four _P. crustosum_ (henceforth referred to as PcHex) strains. The ability of PcHex to hydrolyze modified glycosides used as synthetic substrates was also investigated, which has not yet been reported. Furthermore, the purified enzyme was used in the specific hydrolysis of b-anomer from chemically synthetized anomeric mixture of the fluorogenic substrate 4-methylumbelliferyl-\(N\)-acetyl-\(\alpha\)/\(\beta\)-D-galactopyranoside (4 MU-\(\alpha\)/\(\beta\)-GalNAc).