title: Heterologous expression and characterization of an N-acetyl-β-D-hexosaminidase from Lactococcus lactis ssp. lactis IL1403.
abstract: The lnbA gene of Lactococcus lactis ssp. lactis IL1403 encodes a polypeptide with similarity to lacto-N-biosidases and N-acetyl-β-D-hexosaminidases. The gene was cloned into the expression vector pET-21d and overexpressed in Escherichia coli BL21* (DE3). The recombinant purified enzyme (LnbA) was a monomer with a molecular weight of approximately 37 kDa. Studies with chromogenic substrates including p-nitrophenyl N-acetyl-β-D-glucosamine (pNP-GlcNAc) and p-nitrophenyl N-acetyl-β-D-galactosamine (pNP-GalNAc) showed that the enzyme had both N-acetyl-β-D-glucosaminidase and N-acetyl-β-D-galactosaminidase activity, thus indicating that the enzyme is an N-acetyl-β-D-hexosaminidase. K(m) and k(cat) for pNP-GlcNAc were 2.56 mM and 26.7 s(-1), respectively, whereas kinetic parameters for pNP-GalNAc could not be determined due to the K(m) being very high (>10 mM). The optimal temperature and pH of the enzyme were 37 °C and 5.5, respectively, for both substrates. The half-life of activity at 37 °C and pH 6.0 was 53 h, but activity was completely abolished after 30 min at 50 °C, meaning that the enzyme has relatively low temperature stability. The enzyme was stable in the pH 5.5-8 range and was unstable at pH below 5.5. Studies with natural substrates showed hydrolytic activity on chito-oligosaccharides but not on colloidal chitin or chitosan. Transglycosylation products were not detected. In all, the data suggest that LnbA's role may be to degrade chito-oligosaccharides that are produced by the previously described chitinolytic system of L. lactis.
authors.affiliation: Food Biotechnology Laboratory, Department of Food Sciences and Technology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Hoang Anh
authors.initials: HA
authors.lastname: Nguyen
authors.forename: Thu-Ha
authors.initials: TH
authors.lastname: Nguyen
authors.forename: Vladimír
authors.initials: V
authors.lastname: Křen
authors.forename: Vincent G H
authors.initials: VG
authors.lastname: Eijsink
authors.forename: Dietmar
authors.initials: D
authors.lastname: Haltrich
authors.forename: Clemens K
authors.initials: CK
authors.lastname: Peterbauer
mesh_terms: D002686:Chitin / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013294:Lactococcus lactis / Q000201:enzymology* / Q000235:genetics; D009844:Oligosaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D009844:Oligosaccharides; D011994:Recombinant Proteins; D002686:Chitin; D001619:beta-N-Acetylhexosaminidases