Synthesis of Long-Chain Chitooligosaccharides by a Hypertransglycosylating Processive Endochitinase of Serratia proteamaculans 568
Synthesis of Long-Chain Chitooligosaccharides by a Hypertransglycosylating Processive Endochitinase of Serratia proteamaculans 568
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DOI：
10.1128/jb.06473-11
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发表时间：
2012-08-01
影响因子：
3.2
通讯作者：
Podile, Appa Rao
中科院分区：
文献类型：
--
作者：
Purushotham, Pallinti;Podile, Appa Rao
我们描述了来自革兰氏阴性原斑沙雷氏菌 568 的 407 个残基单域糖基水解酶家族 18 几丁质酶 (SpChiD) 的异源表达和表征，该酶具有前所未有的催化特性。 SpChiD 在 pH 6.0 和 40 摄氏度下具有最佳活性，在胶体几丁质上，其 K-m 为 83 mg ml(-1)，k(cat) 为 3.9 x 10(2) h(-1)，k(cat)/K-m 为 4.7 h mg(-1) ml(-1)。壳二糖的K-m、k(cat)和k(cat)/K-m分别为203μM、1.3×10(2)h(-1)和0.62h(-1)μM-1。壳寡糖 (CHOS) 和胶体甲壳素的水解活性表明 SpChiD 是一种内作用加工酶，具有将释放的壳二糖转化为主要最终产物 N-乙酰氨基葡萄糖的独特能力。 SpChiD 显示出与三聚体-六聚体 CHOS 底物的过度糖基化 (TG)，产生大量的长链 CHOS。 SpChiD 的 TG 活性取决于寡聚底物的长度和浓度以及酶浓度。积累的TG产物的长度和数量随着底物长度和浓度的增加而增加，并随着酶浓度的增加而减少。尽管不存在辅助结构域，但 SpChiD 仍能与不溶性和可溶性几丁质底物结合。序列比对和结构建模表明，SpChiD 将具有一个排列有芳香族残基的深底物结合凹槽，这是加工酶的特征。 SpChiD 显示了 18 族几丁质酶中罕见的特性组合，并且可能类似于人类壳三糖苷酶的特性。
We describe the heterologous expression and characterization of a 407-residue single-domain glycosyl hydrolase family 18 chitinase (SpChiD) from Gram-negative Serratia proteamaculans 568 that has unprecedented catalytic properties. SpChiD was optimally active at pH 6.0 and 40 degrees C, where it showed a K-m of 83 mg ml(-1), a k(cat) of 3.9 x 10(2) h(-1), and a k(cat)/K-m of 4.7 h mg(-1) ml(-1) on colloidal chitin. On chitobiose, the K-m, k(cat) and k(cat)/K-m were 203 mu M, 1.3 x 10(2) h(-1), and 0.62 h(-1) mu M-1, respectively. Hydrolytic activity on chitooligosaccharides (CHOS) and colloidal chitin indicated that SpChiD was an endo-acting processive enzyme, with the unique ability to convert released chitobiose to N-acetylglucosamine, the major end product. SpChiD showed hyper transglycosylation (TG) with trimer-hexamer CHOS substrates, generating considerable amounts of long-chain CHOS. The TG activity of SpChiD was dependent on both the length and concentration of the oligomeric substrate and also on the enzyme concentration. The length and amount of accumulated TG products increased with increases in the length of the substrate and its concentration and decreased with increases in the enzyme concentration. The SpChiD bound to insoluble and soluble chitin substrates despite the absence of accessory domains. Sequence alignments and structural modeling indicated that SpChiD would have a deep substrate-binding groove lined with aromatic residues, which is characteristic of processive enzymes. SpChiD shows a combination of properties that seems rare among family 18 chitinases and that may resemble the properties of human chitotriosidase.