Enzymatic sequencing of partially acetylated chitosan oligomers
Enzymatic sequencing of partially acetylated chitosan oligomers
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DOI：
10.1016/j.carres.2014.04.006
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发表时间：
2014-06-17
影响因子：
3.1
通讯作者：
Kolkenbrock, Stephan
中科院分区：
文献类型：
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作者：
Hamer, Stefanie Nicole;Moerschbacher, Bruno Maria;Kolkenbrock, Stephan
壳寡糖具有多种生物活性，在医药和农业等领域具有潜在的应用价值。这些功能被认为取决于它们的聚合度和乙酰化程度，并可能取决于乙酰化的特定模式。具有完全确定的结构的壳聚糖低聚物很难生产，并且它们的完整分析是苛刻的。分析通常使用MS或NMR进行，需要使用昂贵的基础设施，并且仅在相当小的低聚物的情况下产生明确的结果。我们在这里描述了一种简单且具有成本效益的方法，用于对吉格量的壳寡糖进行测序，该方法基于两种重组葡糖苷酶的顺序作用，即来自枯草芽孢杆菌168的外切-β-N-乙酰氨基己糖苷酶（GlcNA-case）和来自科达卡热球菌KOD 1的外切-β-D-氨基葡萄糖苷酶（GlcNase）。从非还原末端开始，GlcNAcase和GlcNase分别特异性去除N-乙酰基葡糖胺（A）和葡糖胺（D）单元。通过以交替的方式依次添加和去除这些酶，然后使用高效薄层色谱法分析产物，可以揭示壳寡糖的序列。重要的是，这两种酶在相同的条件下工作，因此在步骤之间不需要缓冲液交换，并且可以使用简单的超滤装置方便地去除酶。作为原理证明，该方法用于使用来自霍乱弧菌的重组几丁质脱乙酰酶对几丁质五聚体的酶促脱乙酰化的产物进行测序，所述几丁质脱乙酰酶特异性地从底物的非还原端旁边的第二单元去除乙酰基，产生具有序列ADAM的单脱乙酰化五聚体。(C)2014爱思唯尔有限公司版权所有。
Chitosan oligosaccharides have diverse biological activities with potentially valuable applications, for example, in the fields of medicine and agriculture. These functionalities are thought to depend on their degree of polymerization and acetylation, and possibly on specific patterns of acetylation. Chitosan oligomers with fully defined architecture are difficult to produce, and their complete analysis is demanding. Analysis is typically done using MS or NMR, requiring access to expensive infrastructure, and yielding unequivocal results only in the case of rather small oligomers. We here describe a simple and cost-efficient method for the sequencing of jig amounts of chitosan oligosaccharides which is based on the sequential action of two recombinant glycosidases, namely an exo-beta-N-acetylhexosaminidase (GlcNA-case) from Bacillus subtilis 168 and an exo-beta-D-glucosaminidase (GlcNase) from Thermococcus kodakarensis KOD1. Starting from the non-reducing end, GlcNAcase and GlcNase specifically remove N-acetyl glucosamine (A) and glucosamine (D) units, respectively. By the sequential addition and removal of these enzymes in an alternating way followed by analysis of the products using high-performance thin-layer chromatography, the sequence of chitosan oligosaccharides can be revealed. Importantly, both enzymes work under identical conditions so that no buffer exchange is required between steps, and the enzyme can be removed conveniently using simple ultra-filtration devices. As proof-of-principle, the method was used to sequence the product of enzymatic deacetylation of chitin pentamer using a recombinant chitin deacetylase from Vibrio cholerae which specifically removes the acetyl group from the second unit next to the non-reducing end of the substrate, yielding mono-deacetylated pentamer with the sequence ADAM. (C) 2014 Elsevier Ltd. All rights reserved.