Norwegian Biopolymer Laboratory (NOBIPOL), Department of Biotechnology, Norwegian University of Science and Technology, Trondheim, Norway.
Biomacromolecules. 2012 Jun 11;13(6):1733-41. doi: 10.1021/bm201521h. Epub 2012 May 14.
Chitooligosaccharides (CHOS) are oligomers composed of glucosamine and N-acetylglucosamine with several interesting bioactivities that can be produced from enzymatic cleavage of chitosans. By controlling the degree of acetylation of the substrate chitosan, the enzyme, and the extent of enzyme degradation, CHOS preparations with limited variation in length and sequence can be produced. We here report on the degradation of chitosans with a novel family 75 chitosanase, SaCsn75A from Streptomyces avermitilis . By characterizing the CHOS preparations, we have obtained insight into the mode of action and subsite specificities of the enzyme. The degradation of a fully deacetylated and a 31% acetylated chitosan revealed that the enzyme degrade these substrates according to a nonprocessive, endo mode of action. With the 31% acetylated chitosan as substrate, the kinetics of the degradation showed an initial rapid phase, followed by a second slower phase. In the initial faster phase, an acetylated unit (A) is productively bound in subsite -1, whereas deacetylated units (D) are bound in the -2 subsite and the +1 subsite. In the slower second phase, D-units bind productively in the -1 subsite, probably with both acetylated and deacetylated units in the -2 subsite, but still with an absolute preference for deacetylated units in the +1 subsite. CHOS produced in the initial phase are composed of deacetylated units with an acetylated reducing end. In the slower second phase, higher amounts of low DP fully deacetylated oligomers (dimer and trimer) are produced, while the higher DP oligomers are dominated by compounds with acetylated reducing ends containing increasing amounts of internal acetylated units. The degradation of chitosans with varying degrees of acetylation to maximum extents of degradation showed that increasingly longer oligomers are produced with increasing degree of acetylation, and that the longer oligomers contain sequences of consecutive acetylated units interspaced by single deacetylated units. The catalytic properties of SaCsn75A differ from the properties of a previously characterized family 46 chitosanase from S. coelicolor (ScCsn46A).
壳寡糖（CHOS）是由氨基葡萄糖和 N-乙酰氨基葡萄糖组成的低聚物，具有多种有趣的生物活性，可通过壳聚糖的酶切产生。通过控制底物壳聚糖、酶和酶降解程度的乙酰化程度，可以生产出长度和序列有限变化的 CHOS 制剂。我们在这里报道了一种新型家族 75 壳聚糖酶 SaCsn75A 对壳聚糖的降解。通过对 CHOS 制剂进行表征，我们深入了解了酶的作用模式和亚基特异性。完全脱乙酰化和 31%乙酰化壳聚糖的降解表明，该酶按照非连续的内切作用模式进行降解。以 31%乙酰化壳聚糖为底物，降解的动力学表现出初始快速相，随后是第二较慢的相。在初始较快的阶段，一个乙酰化单元（A）在-1 亚基中有效地结合，而脱乙酰化单元（D）在-2 亚基和+1 亚基中结合。在较慢的第二阶段，D-单元在-1 亚基中有效地结合，可能在-2 亚基中同时含有乙酰化和脱乙酰化单元，但在+1 亚基中仍然绝对优先于脱乙酰化单元。在初始阶段产生的 CHOS 由脱乙酰化单元组成，具有乙酰化的还原端。在较慢的第二阶段，产生更多数量的低 DP 完全脱乙酰化低聚物（二聚体和三聚体），而更高 DP 的低聚物主要由含有越来越多内部乙酰化单元的乙酰化还原端的化合物组成。具有不同乙酰化程度的壳聚糖降解至最大程度的降解表明，随着乙酰化程度的增加，产生的低聚物越来越长，并且较长的低聚物含有连续的乙酰化单元序列，由单个脱乙酰化单元间隔开。SaCsn75A 的催化性质与先前从 S. coelicolor 中鉴定的家族 46 壳聚糖酶（ScCsn46A）的性质不同。
