A β-N-acetylhexosaminidase from Penicillium oxalicum implicated in its cell-wall degradation

Abstract

High β‐N‐acetylhexosaminidase (EC.3.2.1.52) activity was detected during autolysis of Penicillium oxalicum. Purification of the enzyme to homogeneity yielded an enzyme with a molecular weight of 132 000 Da by gel filtration and 71 900 Da by SDS polyacrylamide gel electrophoresis, suggesting a dimeric structure. The enzyme is an acidic protein with a pl of 5.0. Optimal activity was at pH 4.0 and 40°C, with a Km of 0.80 mmol 1‐1 for p‐nitrophenyl‐β‐N‐acetylglucosaminide and 1.03 mmol 1‐1 for p‐nitrophenyl‐β‐N‐acetylgalactosaminide. The Ki with the competitive inhibitor O‐(2‐acetamido‐2‐deoxy‐D‐glucopyranosylidene) amino‐N‐phenylcarbamate was 1 μmol 1‐1. Hg2+, Ag+ and Fe3+ were effective inhibitors. β‐N‐acetylhexosaminidase hydrolysed chitobiose, chitotriose, chitotetrose and chitopentose to monomer to an extent of 92, 74, 44 and 17% respectively in 40 min. This enzyme, in conjunction with a purified endochitinase from P. oxalicum, hydrolysed a cell‐wall chitin fraction isolated from this fungus, with the production of N‐acetylglucosamine.

Physiological and enzymological characterization of theβ-N-acetylhexosaminidase ofPenicillium chrysogenum

“…Similar to other analogous age-related fungal enzymes (REYES and BYRDE 1973, YAMA-MOTO et al 1985, 1986, REYES et al 1989, RODRÍGUEZ et al 1994, VAN TILBURG and THOMAS 1994), the purified P. chrysogenum HexNAc©ase split PNP-GlcNAc, PNP-GalNAc and N,N'-diacetylchitobiose but in this case no liberation of GlcNAc was detected in the presence of N,N',N''-triacetylchitotriose (Table 2). That is P. chrysogenum HexNAc©ase possessed much narrower substrate specificity for chitin oligomers than other age-related fungal HexNAc'ases and GlcNAc'ases (YAMAMOTO et al 1985, 1986, REYES et al 1989, RODRÍGUEZ et al 1994.…”

“…That is P. chrysogenum HexNAc©ase possessed much narrower substrate specificity for chitin oligomers than other age-related fungal HexNAc'ases and GlcNAc'ases (YAMAMOTO et al 1985, 1986, REYES et al 1989, RODRÍGUEZ et al 1994. Interestingly, a HexNAc©ase with very similar substrate specificity to that of the P. chrysogenum GlcNAc'ase was isolated from growing hyphae of Mucor rouxii (LINDER 1987, RAST et al 1991, MAYER et al 1993).…”

“…In fact, many fungal HexNAc©ases, GlcNAc©ases and chitobiases possess a homodimer structure with M r = 118 000 -150 000 and M r, subunits = 55 000 -74 000 values (MEGA and MATSUSHIMA 1979, YAMAMOTO et al 1985, 1986, OHTAKARA 1988, TAO et al 1990, KOGA et al 1991, ULHOA and PEBERDY 1991, RODRÍGUEZ et al 1994, GEIMBA et al 1998, this paper) but a much higher M r value was reported for the soluble cell-bound Hex-NAc'ase of Sarcoscipha coccinea (M r = 430 000 -450 000, VAFINA and MOLODTSOV 1979), the cytoplasmic/vacuolar GlcNAc'ase of M. rouxii (M r = 320 000, LINDER 1987) and the GlcNAc'ases of Candida albicans which is secreted from the periplasm (GlcNAc©ase A, M r = 350 000, M r, subunit = 85 000; GlcNAc©ase B, M r = 600 000, M r, subunit = 132 000; MOLLOY et al 1994). These observations may be indicative of a higher degree of association for these hydrolases in vivo especially inside the cells.…”

Purification and properties of a chitinase from Penicillium oxalicum autolysates

“…This result can be explained by the second substrate being a polymer, so that its complete degradation could need the action of different enzymes. In fact, this group have reported the synergistic action of this enzyme with a /3-N-acetylhexosarninidase when a chitin fraction of P. oxalicum cell wall was used as substrate (Rodriguez et al 1994). The viscosimetric study showed the highest affinity of the enzyme for carboxymethyl chitin (33.0 U ml-').…”

“…The activity belongs to a lytic complex produced by this fungus, implicated in its cell wall degradation (Copa-Patiiio et al 1990a). A B-glucanase, Bglucosidase and f3-N-acetylhexosaminidase have already been purified and characterized in our laboratory (Copa-Patiiio et al 1989, 1990bRodriguez et al 1994) from this lytic complex.…”