Quantitative analysis of cellulose-reducing ends
Quantitative analysis of cellulose-reducing ends
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DOI：
10.1385/abab:113:1-3:213
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发表时间：
2004-03-01
影响因子：
3
通讯作者：
Penner, MH
中科院分区：
文献类型：
--
作者：
Kongruang, S;Han, MJ;Penner, MH
传统纤维素底物上的总还原末端和可及还原末端的定量方法已被评估，因为它们与酶催化的纤维素糖化相关。例如，可接近的还原末端的定量可能是外作用的、还原末端偏好的纤维二糖水解酶的底物浓度的最直接的量度。本申请评价了两种比色测定法（二硝基水杨酸[DNS]和二喹啉甲酸[BCA]测定法）和放射性同位素方法（（NaBH 4）-H-3，标记）。纤维素基质包括微晶纤维素、细菌纤维素和滤纸。发现每单位质量纤维素的还原末端数的估计值取决于测定系统（即DNS和BCA测定给出了显著不同的结果）。基于DNS的值比使用BCA测定获得的值高几倍，其中倍数差异是底物特异性的。硼氢化钠还原纤维素，使用冷或放射性标记的试剂在相对温和的条件下，被用来评估表面（溶剂可及）还原端的数量。结果表明，传统纤维素底物上30 - 40%的还原末端是溶剂不可及的;也就是说，它们被埋在纤维素结构的内部，因此不可用于外作用酶。
Methods for the quantification of total and accessible reducing ends on traditional cellulose substrates have been evaluated because of their relevance to enzyme-catalyzed cellulose saccharificaion. For example, quantification of accessible reducing ends is likely to be the most direct measure of substrate concentration for the exo-acting, reducing end-preferring cellobiohydrolases. Two colorimetric assays (dinitrosalicylic acid [DNS] and bicinchoninic acid [BCA] assay) and a radioisotope approach ((NaBH4)-H-3, labeling) were evaluated for this application. Cellulose substrates included microcrystalline celluloses, bacterial celluloses, and filter paper. Estimates of the number of reducing ends per unit mass cellulose were found to be dependent on the assay system (i.e. the DNS and BCA assays gave strikingly different results). DNS-based values were several-fold higher than those obtained using the BCA assay, with fold-differences being substrate specific. Sodium borohydride reduction of celluloses, using cold or radiolabeled reagent under relatively mild conditions, was used to assess the number of surface (solvent-accessible) reducing ends. The results indicate that 30-40% of the reducing ends on traditional cellulose substrates are not solvent accessible; that is, they are buried in the interior of cellulose structures and thus not available to exo-acting enzymes.