Construction of System for Formation of Chitin Oligomer Having Highly Physiological Activity by Enzymatic Hydrolysis Reaction

通过CM-Sephadex C-50离子交换层析和Sephadex G-150凝胶过滤层析，从菜粉蝶蛹粗提物中分离纯化出几丁质酶。得到了活性部位CHI-A和CHI-B。酶纯度分别提高了12.4倍和2.17倍，酶活力回收率分别为42.4%和4.58%。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法测定CHI-A分离物的均一性和相对分子质量。经SDS-PAGE分析，CHI-A为单一条带，分子量约为48,000。以乙二醇甲壳质为底物时，纯化的CHI-A的最适pH为5.0。CHI-A在pH 4.0-8.0范围内稳定，在310K时仍保持其70%的活性。菜粉蝶几丁质酶活性符合典型的Michaelis-Menten动力学规律。我们还发现CHI-A具有甲壳素合成酶活性。N-乙酰氨基葡萄糖在CHI-A作用下发生糖基化反应，有效地生成了大量的N-乙酰基壳五糖。另外，通过Sephadex G-25凝胶过滤层析和CM-Sephadex C-50离子交换层析，从黄粉蝶蛹粗提物中分离纯化了3种N-乙酰氨基葡萄糖转移酶。这些酶的热稳定性为308~328K，最适pH为6.5~7.0。每种酶都能与N-乙酰氨基葡萄糖反应，并产生不溶沉淀，表明甲壳低聚糖高度聚合。这些催化性质与菜粉蝶同属几丁质转移酶的性质略有不同。

Chitinase from the extract of pupae of Pieris rapae crucivcra Boisduval was purified through the successive steps of CM-Sephadex C-50 ion exchange chromatography and gel filtration chromatography with Sephadex G-150 from crude enzyme extract. Then the active fractions named Chi-A and Chi-B were obtained. The purity of the enzyme increased up to 12.4- and 2.17-fold and the recovery of the enzyme activity were 42.4 and 4.58%, for the fraction Chi-A and.Chi-B, respectively. The homogeneity and molecular weight of isolated Chi-A were evaluated by SDS PAGE. The homogeneity of Chi-A was confirmed as a single band on SDS-PAGE and the molecular weight was estimated to be 48,000. The purified Chi-A had an optimal pH of 5.0 for the hydrolysis reaction when glycol chitin was used as a substrate. Chi-A was stable in the pH range of 4.0-8.0 and retained its 70% activity at 310K. Chitinase from pupae of Pieris rapae crucivara Boisduval exhibited typical Michaelis-Menten type kinetics. We also found that Chi-A revealed a chitin synfase activity. A large amount of N-acetylchitopentaose was effectively formed by the transglycosylation from N-acetylglucosamine with Chi-A.On the other hand, three kinds of N-acetylglucosamine transferase were purified from the crude extract of pupae of Papilio xuthus Linne through Sephadex G-25 gel filtration chromatography and CM-Sephadex C-50 ion exchange chromatography. Thermal stability of these enzymes was 308-328 K and optimal pH for transferase activity appeared at 6.5-7.0. Each enzyme could react with N-acetylglucosamine, and produced insoluble deposit suggesting highly polymerized chitooligosaccharides. These catalytic properties somewhat differ from those of chitintransferase fron pupae of Pieris rapae crucivora Boisduval belonging to the same order.