Li H, Morimoto K, Katagiri N, Kimura T, Sakka K, Lun S, Ohmiya K
School of Biotechnology, Southern Yangtze University, 170 Huihe Road, Wuxi 214036, China.
Appl Microbiol Biotechnol. 2002 Dec;60(4):420-7. doi: 10.1007/s00253-002-1129-y. Epub 2002 Oct 18.
A beta- N-acetylglucosaminidase gene ( nag3A) from Clostridium paraputrificum M-21 was cloned in Escherichia coli. The nag3A gene consists of an open reading frame of 1,239-bp, encoding 413 amino acids with a deduced molecular weight of 45,531 Da. Nag3A is a single domain enzyme containing a family 3 glycoside hydrolase catalytic domain. Nag3A was purified from recombinant E. coli and characterized. The enzyme hydrolyzed chitooligomers such as di- N-acetylchitobiose, tri- N-acetylchitotriose, tetra- N-acetylchitotetraose, penta- N-acetylchitopentaose, hexa- N-acetylchitohexaose, ball-milled chitin, and synthetic substrates such as 4-methylumbelliferyl N-acetyl beta- D-glucosaminide [4-MU-(GlcNAc)], but had no activity at all against p-nitrophenyl-beta- D-glucoside, p-nitrophenyl-beta- D-xyloside, or p-nitrophenyl-beta- D-galactosamine. The enzyme was optimally active at 50 degrees C and pH 7.0, and the apparent K(m) and V(max) values for 4-MU-(GlcNAc) were 7.9 micro M and 21.8 micro mol min(-1) mg protein(-1), respectively. SDS-PAGE, zymogram, and immunological analyses suggested that this enzyme is induced by ball-milled chitin.
从副腐败梭菌M-21中克隆出一个β-N-乙酰氨基葡萄糖苷酶基因（nag3A），并将其克隆到大肠杆菌中。nag3A基因由一个1239 bp的开放阅读框组成，编码413个氨基酸，推导分子量为45531 Da。Nag3A是一种单结构域酶，含有一个3型糖苷水解酶催化结构域。从重组大肠杆菌中纯化并鉴定了Nag3A。该酶能水解几丁寡糖，如二-N-乙酰壳二糖、三-N-乙酰壳三糖、四-N-乙酰壳四糖、五-N-乙酰壳五糖、六-N-乙酰壳六糖、球磨甲壳素，以及合成底物，如4-甲基伞形酮基-N-乙酰-β-D-氨基葡萄糖苷[4-MU-(GlcNAc)]，但对对硝基苯基-β-D-葡萄糖苷、对硝基苯基-β-D-木糖苷或对硝基苯基-β-D-半乳糖胺完全没有活性。该酶在50℃和pH 7.0时活性最佳，对4-MU-(GlcNAc)的表观K(m)和V(max)值分别为7.9 μM和21.8 μmol min(-1) mg蛋白(-1)。SDS-PAGE、酶谱和免疫分析表明，该酶是由球磨甲壳素诱导产生的。
