Characterization of Halomonas sp Strain H11 α-Glucosidase Activated by Monovalent Cations and Its Application for Efficient Synthesis of α-D-Glucosylglycerol
Characterization of Halomonas sp Strain H11 α-Glucosidase Activated by Monovalent Cations and Its Application for Efficient Synthesis of α-D-Glucosylglycerol
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DOI：
10.1128/aem.07514-11
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发表时间：
2012-03-01
影响因子：
4.4
通讯作者：
Kudo, Toshiaki
中科院分区：
文献类型：
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作者：
Ojima, Teruyo;Saburi, Wataru;Kudo, Toshiaki
从Halomonas sp.菌株H11中分离到一种具有以下独特性质的α -葡萄糖苷酶（HaG）：(i)高转糖基化活性，（ii）被单价阳离子激活，（iii）底物特异性非常窄。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）测定，纯化的HaG分子量为58 kDa。HaG对麦芽糖、蔗糖和对硝基苯α - d -葡萄糖苷（pNPG）具有较高的水解活性，但对其他双糖或麦芽糖寡糖的水解活性几乎没有高于三糖。HaG在30℃、pH 6.5条件下对麦芽糖的活性最佳。K+、Rb+、Cs+、NH4+等一价阳离子使酶活性提高到原活性的2 ~ 9倍。这些离子使活性- ph曲线向碱性一侧偏移。在10 mM NH4+存在下，最适温度升至40℃，但温度稳定性不受影响。一价阳离子显著提高了麦芽糖和pNPG的表观k -m和k（cat）值。令人惊讶的是，在它们的存在下，pNPG的k(cat)/ k -m增加了372至969倍。HaG在转糖基化过程中利用一些醇作为受体底物，有效地合成了- d -葡萄糖甘油。在产生少量副产物方面，该生产水平的效率优于先前报道的黑曲霉α -葡萄糖苷酶。序列分析表明，HaG属于糖苷水解酶家族13。其氨基酸序列与油菜黄单胞菌WU-9701 α -葡萄糖苷酶、油菜黄单胞菌pv的同源性分别为60%、58%、57%和56%。raphani 756C寡聚-1,6-葡萄糖苷酶、stutzeri假单胞菌DSM 4166寡聚-1,6-葡萄糖苷酶和农杆菌F2 α -葡萄糖苷酶。
An alpha-glucosidase (HaG) with the following unique properties was isolated from Halomonas sp. strain H11: (i) high transglucosylation activity, (ii) activation by monovalent cations, and (iii) very narrow substrate specificity. The molecular mass of the purified HaG was estimated to be 58 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). HaG showed high hydrolytic activities toward maltose, sucrose, and p-nitrophenyl alpha-D-glucoside (pNPG) but to almost no other disaccharides or malto-oligosaccharides higher than trisaccharides. HaG showed optimum activity to maltose at 30 degrees C and pH 6.5. Monovalent cations such as K+, Rb+, Cs+, and NH4+ increased the enzymatic activity to 2- to 9-fold of the original activity. These ions shifted the activity-pH profile to the alkaline side. The optimum temperature rose to 40 degrees C in the presence of 10 mM NH4+, although temperature stability was not affected. The apparent K-m and k(cat) values for maltose and pNPG were significantly improved by monovalent cations. Surprisingly, k(cat)/K-m for pNPG increased 372- to 969-fold in their presence. HaG used some alcohols as acceptor substrates in transglucosylation and was useful for efficient synthesis of alpha-D-glucosylglycerol. The efficiency of the production level was superior to that of the previously reported enzyme Aspergillus niger alpha-glucosidase in terms of small amounts of by-products. Sequence analysis of HaG revealed that it was classified in glycoside hydrolase family 13. Its amino acid sequence showed high identities, 60%, 58%, 57%, and 56%, to Xanthomonas campestris WU-9701 alpha-glucosidase, Xanthomonas campestris pv. raphani 756C oligo-1,6-glucosidase, Pseudomonas stutzeri DSM 4166 oligo-1,6-glucosidase, and Agrobacterium tumefaciens F2 alpha-glucosidase, respectively.