Murata Takeomi, Amarume Satoshi, Hattori Takeshi, Tokuyama Shinji, Tokuyasu Ken, Kawagishi Hirokazu, Usui Taich
Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan.
Biochem Biophys Res Commun. 2005 Oct 21;336(2):514-20. doi: 10.1016/j.bbrc.2005.08.123.
We report a novel enzyme from the culture filtrate of Amycolatopsis orientalis, that endoglycosidically releases an N-acetyllactosamine-repeating unit (Galbeta1,4GlcNAcbeta1,3Galbeta1,4GlcNAc, LN2) from a synthetic chromogenic substrate Galbeta1,4GlcNAcbeta1,3Galbeta1,4GlcNAcbeta-pNP (1). The enzyme activity was purified by 80% saturated ammonium sulfate precipitation followed by gel filtration and affinity chromatography. The enzyme splits 1, Galbeta1,4GlcNAcbeta-pNP (2), GlcNAcbeta1,3Galbeta1,4GlcNAcbeta-pNP (3), and GlcNAcbeta1,4GlcNAcbeta-pNP (4) into the corresponding oligosaccharides and p-nitrophenol. The catalytic efficiencies (k(cat)/K(m)) for compounds 1, 2, and 4 were 0.6, 0.05, and 13, respectively. Compound 4 acts as a fairly good substrate for the enzyme, and LN2-releasing activity was inhibited by 4 and GlcNAcbeta1,4GlcNAcbeta1,4GlcNAcbeta-pNP (7), indicating that this enzyme activity is derived from a kind of chitinase. The enzyme hydrolyzed 1 by a mechanism leading to retention of the anomeric configuration. This is the first report of a N-acetyllactosamine-repeating unit releasing enzyme.
我们报道了一种来自东方拟无枝酸菌培养滤液的新型酶，该酶能从合成生色底物Galβ1,4GlcNAcβ1,3Galβ1,4GlcNAc（LN2）的Galβ1,4GlcNAcβ1,3Galβ1,4GlcNAcβ-pNP（1）中内切糖苷释放出N-乙酰乳糖胺重复单元。通过80%饱和度的硫酸铵沉淀，随后进行凝胶过滤和亲和层析对该酶活性进行了纯化。该酶能将1、Galβ1,4GlcNAcβ-pNP（2）、GlcNAcβ1,3Galβ1,4GlcNAcβ-pNP（3）和GlcNAcβ1,4GlcNAcβ-pNP（4）分别裂解为相应的寡糖和对硝基苯酚。化合物1、2和4的催化效率（k(cat)/K(m)）分别为0.6、0.05和13。化合物4是该酶相当好的底物，且LN2释放活性受到4和GlcNAcβ1,4GlcNAcβ1,4GlcNAcβ-pNP（7）的抑制，表明该酶活性源自一种几丁质酶。该酶通过导致异头构型保留的机制水解1。这是关于N-乙酰乳糖胺重复单元释放酶的首次报道。
