Food Biotechnology Laboratory, Department of Food Sciences and Technology, University of Natural Resources and Life Sciences, Vienna, Austria.
J Agric Food Chem. 2012 Mar 28;60(12):3275-81. doi: 10.1021/jf204915e. Epub 2012 Mar 15.
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.
乳球菌乳亚种 IL1403 的 lnbA 基因编码一种与乳-N-糖苷酶和 N-乙酰-β-D-氨基葡萄糖苷酶具有相似性的多肽。该基因被克隆到表达载体 pET-21d 中，并在大肠杆菌 BL21*（DE3）中过表达。重组纯化酶（LnbA）为单体，分子量约为 37 kDa。用包括 p-硝基苯-N-乙酰-β-D-葡萄糖胺（pNP-GlcNAc）和 p-硝基苯-N-乙酰-β-D-半乳糖胺（pNP-GalNAc）在内的显色底物进行的研究表明，该酶具有 N-乙酰-β-D-氨基葡萄糖苷酶和 N-乙酰-β-D-半乳糖苷酶活性，因此表明该酶是一种 N-乙酰-β-D-己糖苷酶。pNP-GlcNAc 的 K(m)和 k(cat)分别为 2.56 mM 和 26.7 s(-1)，而由于 K(m)非常高（>10 mM），无法确定 pNP-GalNAc 的动力学参数。两种底物的最适温度和 pH 分别为 37°C 和 5.5。在 37°C 和 pH 6.0 下，酶的半衰期为 53 h，但在 50°C 下 30 min 后，酶的活性完全丧失，这意味着该酶的热稳定性相对较低。该酶在 pH 5.5-8 范围内稳定，在 pH 低于 5.5 时不稳定。对天然底物的研究表明，该酶对壳寡糖具有水解活性，但对胶体几丁质或壳聚糖没有活性。未检测到转糖基化产物。总的来说，数据表明 LnbA 的作用可能是降解先前描述的乳球菌几丁质酶系产生的壳寡糖。
