Sulová Z, Lednická M, Farkas V
Institute of Chemistry, Slovak Academy of Sciences, Bratislava.
Anal Biochem. 1995 Jul 20;229(1):80-5. doi: 10.1006/abio.1995.1381.
One of the key enzymes involved in the breakdown of reserve xyloglucan in seeds of some dicotyledonous plants during germination is the specific endo-beta-(1,4)-glucanase. The enzyme operates predominantly by a transglycosylic mechanism, i.e., by random splitting the beta-(1,4)-linked polyglucose backbone of xyloglucan molecules and rejoining the newly created reducing ends by beta-(1,4) glycosidic bonds to nonreducing ends of other xyloglucan molecules or xyloglucan subunit oligosaccharides. For this reason, the enzyme is regarded primarily as xyloglucan-endotransglycosylase (XET). Since almost no net formation of reducing ends occurs in the course of transglycosylation, the conventional reductometric methods used for the assessment of glycanase activities are not applicable for detection and determination of XET activity. The described colorimetric assay is based on the property of xyloglucan-derived subunit oligosaccharides (DP 5-10) to stimulate selectively the breakdown of xyloglucan by endotransglycosylation while serving as additional glycosyl acceptors. The depolymerization of xyloglucan in the course of reaction is followed colorimetrically by measuring the disappearance of the blue--green-colored iodine:xyloglucan complex. The transglycosylase activity is calculated as the difference of activities measured in the presence of stimulating xyloglucan-derived oligosaccharides and in their absence. The advantages of the described colorimetric method include its low cost, simplicity, speed, and the possibility to analyze multiple samples simultaneously.
在一些双子叶植物种子萌发过程中，参与储备木葡聚糖分解的关键酶之一是特异性内切-β-(1,4)-葡聚糖酶。该酶主要通过转糖基机制起作用，即随机切断木葡聚糖分子的β-(1,4)-连接的聚葡萄糖主链，并通过β-(1,4)糖苷键将新产生的还原端重新连接到其他木葡聚糖分子或木葡聚糖亚基寡糖的非还原端。因此，该酶主要被视为木葡聚糖内转糖基酶（XET）。由于在转糖基化过程中几乎不发生还原端的净形成，用于评估聚糖酶活性的传统还原法不适用于检测和测定XET活性。所描述的比色测定法基于木葡聚糖衍生的亚基寡糖（聚合度5-10）的特性，即通过内转糖基化选择性地刺激木葡聚糖的分解，同时作为额外的糖基受体。在反应过程中，木葡聚糖的解聚通过比色法测量蓝绿色碘：木葡聚糖复合物的消失来跟踪。转糖基酶活性通过在存在刺激木葡聚糖衍生寡糖和不存在刺激木葡聚糖衍生寡糖的情况下测量的活性差异来计算。所描述的比色法的优点包括成本低、操作简单、速度快以及能够同时分析多个样品。
