Einbu Aslak, Vårum Kjell M
Department of Biotechnology, Norwegian Biopolymer Laboratory, Norwegian University of Science and Technology, 7491 Trondheim, Norway.
Biomacromolecules. 2008 Jul;9(7):1870-5. doi: 10.1021/bm8001123. Epub 2008 Jun 10.
Proton NMR spectra of chitin dissolved in concentrated and deuterated hydrochloric acid (DCl) were found to be a simple and powerful method for identifying chitin from samples of biological origin. During the first hour after dissolving chitin in concentrated DCl (25 degrees C), insignificant de-N-acetylation occurred, meaning that the fraction of acetylated units (FA) of chitin could be determined. FA of demineralized shrimp shell samples treated with 1 M NaOH at 95 degrees C for 1-24 h were determined and were found to decrease linearly with time from 0.96 to 0.91 during the treatment with NaOH. Extrapolation to zero time suggested that chitin from shrimp shells has a FA of 0.96, that is, contains a small but significant fraction of de-N-acetylated units. Proton NMR spectra of chitin ( FA = 0.96) dissolved in concentrated DCl were obtained as a function of time until the samples were almost quantitatively hydrolyzed to the monomer glucosamine (GlcN). The initial phase of the reaction involves mainly depolymerization of the chitin chains, resulting in that almost 90% (molar fraction) of the chitin is converted to the monomer N-acetyl-glucosamine (GlcNAc).Thus, effective conversion of chitin to GlcNAc in concentrated acid is reported for the first time. GlcNAc is then further de-N-acetylated to GlcN. A new theoretical model was developed to simulate the experimental data of the kinetics of hydrolysis of chitin in concentrated acid. The model uses three different rate constants; two for the hydrolysis of the glycosidic linkages following an N-acetylated or a de-N-acetylated sugar unit and one for the de-N-acetylation reaction. The three rate constants were estimated by fitting model data to experimental results. The rate of hydrolysis of a glycosidic linkage following an N-acetylated unit was found to be 54 times higher as compared to the rate of de-N-acetylation and 115 times higher than the rate of hydrolysis of a glycosidic linkage following a de-N-acetylated unit. Two chitin samples with different F A values (0.96 and 0.70) were incubated in concentrated DCl until the samples were converted to the maximum yield of GlcNAc and the oligomer composition analyzed, showing that the maximum yield of GlcNAc was much higher when prepared from the chitin with the highest F A value.
研究发现，溶解于浓氘代盐酸（DCl）中的几丁质的质子核磁共振谱是从生物源样品中鉴定几丁质的一种简单而有效的方法。在将几丁质溶解于浓DCl（25℃）后的第一个小时内，脱N - 乙酰化作用不明显，这意味着可以测定几丁质的乙酰化单元分数（FA）。测定了在95℃下用1 M NaOH处理1 - 24小时的脱矿虾壳样品的FA，发现其在NaOH处理过程中随时间从0.96线性下降至0.91。外推至零时间表明，虾壳中的几丁质的FA为0.96，即含有一小部分但显著比例的脱N - 乙酰化单元。获得了溶解于浓DCl中的几丁质（FA = 0.96）的质子核磁共振谱随时间的变化情况，直至样品几乎定量水解为单体葡糖胺（GlcN）。反应的初始阶段主要涉及几丁质链的解聚，结果是几乎90%（摩尔分数）的几丁质转化为单体N - 乙酰葡糖胺（GlcNAc）。因此，首次报道了在浓酸中几丁质有效转化为GlcNAc的情况。然后GlcNAc进一步脱N - 乙酰化为GlcN。开发了一个新的理论模型来模拟浓酸中几丁质水解动力学的实验数据。该模型使用三个不同的速率常数；两个用于N - 乙酰化或脱N - 乙酰化糖单元后的糖苷键水解，一个用于脱N - 乙酰化反应。通过将模型数据与实验结果拟合来估计这三个速率常数。发现N - 乙酰化单元后的糖苷键水解速率比脱N - 乙酰化速率高54倍，比脱N - 乙酰化单元后的糖苷键水解速率高115倍。将两个具有不同FA值（0.96和0.70）的几丁质样品在浓DCl中孵育，直至样品转化为GlcNAc的最大产率并分析低聚物组成，结果表明，由FA值最高的几丁质制备的GlcNAc的最大产率要高得多。
