Fukamizo T, Honda Y, Goto S, Boucher I, Brzezinski R
Laboratory of Biophysical Chemistry, Faculty of Agriculture, Kinki University, Nara, Japan.
Biochem J. 1995 Oct 15;311 ( Pt 2)(Pt 2):377-83. doi: 10.1042/bj3110377.
Chitosanase was produced by the strain of Streptomyces lividans TK24 bearing the csn gene from Streptomyces sp. N174, and purified by S-Sepharose and Bio-Gel A column chromatography. Partially (25-35%) N-acetylated chitosan was digested by the purified chitosanase, and structures of the products were analysed by NMR spectroscopy. The chitosanase produced heterooligosaccharides consisting of D-GlcN and GlcNAc in addition to glucosamine oligosaccharides [(GlcN)n, n = 1, 2 and 3]. The reducing- and non-reducing-end residues of the heterooligosaccharide products were GlcNAc and GlcN respectively, indicating that the chitosanase can split the GlcNAc-GlcN linkage in addition to that of GlcN-GlcN. Time-dependent 1H-NMR spectra showing hydrolysis of (GlcN)6 by the chitosanase were obtained in order to determine the anomeric form of the reaction products. The chitosanase was found to produce only the alpha-form; therefore it is an inverting enzyme. Separation and quantification of (GlcN)n was achieved by HPLC, and the time course of the reaction catalysed by the chitosanase was studied using (GlcN)n (n = 4, 5 and 6) as the substrate. The chitosanase hydrolysed (GlcN)6 in an endo-splitting manner producing (GlcN)2, (GlcN)3 and (GlcN)4, and did not catalyse transglycosylation. Product distribution was (GlcN)3 >> (GlcN)2 > (GlcN)4. Cleavage to (GlcN)3 + (GlcN)3 predominated over that to (GlcN)2 + (GlcN)4. Time courses showed a decrease in rate of substrate degradation from (GlcN)6 to (GlcN)5 to (GlcN)4. It is most likely that the substrate-binding cleft of the chitosanase can accommodate at least six GlcN residues, and that the cleavage point is located at the midpoint of the binding cleft.
壳聚糖酶由携带来自链霉菌属N174的csn基因的淡紫灰链霉菌TK24菌株产生，并通过S-Sepharose和Bio-Gel A柱色谱法进行纯化。部分（25%-35%）N-乙酰化壳聚糖被纯化的壳聚糖酶消化，产物结构通过核磁共振光谱进行分析。该壳聚糖酶除了产生葡糖胺寡糖[（GlcN）n，n = 1、2和3]外，还产生由D-GlcN和GlcNAc组成的杂合寡糖。杂合寡糖产物的还原端和非还原端残基分别为GlcNAc和GlcN，这表明壳聚糖酶除了能裂解GlcN-GlcN键外，还能裂解GlcNAc-GlcN键。为了确定反应产物的异头物形式，获得了壳聚糖酶水解（GlcN）6的时间依赖性1H-NMR光谱。发现壳聚糖酶仅产生α-形式；因此它是一种转化酶。通过高效液相色谱法实现了（GlcN）n的分离和定量，并以（GlcN）n（n = 4、5和6）为底物研究了壳聚糖酶催化反应的时间进程。壳聚糖酶以内切方式水解（GlcN）6，产生（GlcN）2、（GlcN）3和（GlcN）4，且不催化转糖基化反应。产物分布为（GlcN）3 >> （GlcN）2 > （GlcN）4。裂解为（GlcN）3 + （GlcN）3比裂解为（GlcN）2 + （GlcN）4占优势。时间进程表明，底物降解速率从（GlcN）6到（GlcN）5再到（GlcN）4逐渐降低。最有可能的是，壳聚糖酶的底物结合裂隙可以容纳至少六个GlcN残基，并且裂解点位于结合裂隙的中点。
