Norwegian Biopolymer Laboratory (NOBIPOL), Department of Biotechnology, Norwegian University of Science and Technology, 7491 Trondheim, Norway.
Biomacromolecules. 2010 Sep 13;11(9):2487-97. doi: 10.1021/bm1006745.
We have studied the degradation of well-characterized soluble heteropolymeric chitosans by a novel family 46 chitosanase, ScCsn46A from Streptomyces coelicolor A3(2), to obtain insight into the enzyme's mode of action and to determine its potential for production of different chitooligosaccharides. The degradation of both a fully deacetylated chitosan and a 32% acetylated chitosan showed a continuum of oligomeric products and a rapid disappearance of the polymeric fraction, which is diagnostic for a nonprocessive endomode of action. The kinetics of the degradation of the 32% acetylated chitosan demonstrated an initial rapid phase and a slower second phase, in addition to a third and even slower kinetic phase. The first phase reflects the cleavage of the glycosidic linkage between two deacetylated units (D-D), the primary products being fully deacetylated dimers, trimers, and tetramers, as well as longer oligomers with increasing degrees of acetylation. In the subsequent slower kinetic phases, oligomers with a higher degree of acetylated units (A) appear, including oligomers with A's at the reducing or nonreducing end, which indicate that there are no absolute preferences for D in subsites -1 and +1. After maximum degradation of the chitosan, the dimers DA and DD were the dominant products. The degradation of chitosans with varying degrees of acetylation to a maximum degree of scission showed that ScCsn46A could degrade all chitosan substrates extensively, although to decreasing degrees of scission with increasing F(A). The potential use of ScCsn46A to prepare fully deacetylated oligomers and more highly acetylated oligomers from chitosan substrates with varying degrees of acetylation is discussed.
我们研究了一种新型的 46 家族壳聚糖酶 ScCsn46A 对具有良好特征的可溶性杂合壳聚糖的降解，以深入了解酶的作用模式，并确定其生产不同壳寡糖的潜力。完全脱乙酰壳聚糖和 32%乙酰化壳聚糖的降解都表现出寡聚物产物的连续谱和聚合物部分的快速消失，这是内切酶作用的非连续模式的特征。32%乙酰化壳聚糖降解的动力学表现出初始快速相和较慢的第二相，以及第三甚至更慢的动力学相。第一相反映了两个脱乙酰单元（D-D）之间糖苷键的裂解，主要产物是完全脱乙酰的二聚体、三聚体和四聚体，以及具有增加乙酰化度的更长寡聚物。在随后较慢的动力学相中，出现了具有更高乙酰化单元（A）的寡聚物，包括在还原端或非还原端具有 A 的寡聚物，这表明在亚位点-1 和 +1 中没有对 D 的绝对偏好。在壳聚糖的最大降解后，DA 和 DD 二聚体是主要产物。不同乙酰化程度的壳聚糖降解到最大程度的断裂表明 ScCsn46A 可以广泛降解所有壳聚糖底物，尽管随着 F(A)的增加，断裂程度逐渐降低。讨论了 ScCsn46A 从具有不同乙酰化程度的壳聚糖底物制备完全脱乙酰寡聚物和更高度乙酰化寡聚物的潜在用途。
