Studied on low molecular weight chitinase RS-9K

几丁质酶是几丁质水解酶。由于几丁质是真菌细胞壁的主要成分之一，几丁质酶被认为在植物防御系统中起着重要作用。为了揭示几丁质酶的生理功能，本论文以水稻种子中的几丁质酶为研究对象，通过离子交换层析、凝胶过滤和反相层析等方法从水稻种子中分离纯化了一种新型的几丁质酶。分离的几丁质酶的分子量为8，902，通过TOF质谱测定，揭示了最小的酶之一。几丁质酶RS-9 K是水稻种子中含有的一种微量组分。从水稻基因组DNA中克隆了几丁质酶9 K DNA，并通过几种表达系统进行了表达。杆状病毒表达系统、pET大肠杆菌表达系统和pMAL大肠杆菌表达系统均不成功。利用pGEX表达系统获得了重组几丁质酶RS-9 K，但该酶无活性。重组几丁质酶RA-9 K的还原、再氧化等方法在某些试验中也难以再活化。最后，通过酵母（毕赤酵母）进行表达，并获得重组酶的活性形式。

Chitinase is the chitin hydrolytic enzyme. Since chitin is one of the major components of the cell wall of fungi, chitinase is thought to play the important role in the defense system of plants. To reveal the physiological role of chitinase, I studied chitinase of rice plant.Novel chitinase was purified from rice seeds by ion-exchange chromatography, gel filtration and reversed phase chromatography. The molecular weight of the chitinase isolated was 8,902 determined by TOF mass spectrometry, revealing one of the smallest enzymes. I was designated as chitinase RS-9K.Chitinase RS-9K was included as a small amount component in rice seed. Chitinase 9K DNA was cloned from rice genomic DNA and expressed by several expression systems. Baculovirus expression system, pET expression system in E.coli And pMAL expression system in E.coli weren't successful. By using pGEX expression system, recombinant chitinase RS-9K was obtained whereas it was not active. It was also difficult to reactivate recombinant chitinase RA-9K in some trials by means of reduction and reoxidation. Finally, the expression by yeast (pichia) was performed and the Active form of the recombinant enzyme was obtained.