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

从十字花科粉蝶（Pieris raiscrucivcra Boisduval）蛹中提取几丁质酶，粗酶液经CM-SephadexC-50离子交换层析和SephadexG-150凝胶过滤层析纯化。然后得到活性组分Chi-A和Chi-B。酶的纯度分别提高到12.4倍和2.17倍，酶活回收率分别为42.4%和4.58%。SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析纯化的Chi-A的均一性和分子量。在SDS-PAGE上确认Chi-A的均一性为单一条带，估计分子量为48，000。以乙二醇甲壳素为底物时，纯化的Chi-A的最适pH为5.0。Chi-A在pH4.0 -8.0范围内稳定，在310 K时仍保持70%的活性。十字粉蝶（Pieris raiscrucivara Boisduval）蛹几丁质酶具有典型的米氏动力学。我们还发现Chi-A具有几丁质合成酶活性。N-乙酰氨基葡萄糖经Chi-A转糖基化后，可有效地生成大量的N-乙酰壳五糖。另一方面，通过Sephadex G-25凝胶过滤层析和CM-Sephadex C-50离子交换层析，从柑橘凤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308-328 K，转移酶活性的最适pH出现在6.5-7.0。每种酶都能与N-乙酰氨基葡萄糖反应，并产生不溶性存款，表明壳寡糖高度聚合。这些催化性质与同一目的短十字粉蝶（Pieris raiscrucivora Boisduval）腐皮几丁质转移酶的催化性质有所不同。

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.