Chitinase and uses thereof

專利類型

發明

專利國別 (專利申請國家)

美國

專利申請案號

12/013,518

專利證號

US 7,670,820 B2

專利獲證名稱

Chitinase and uses thereof

專利所屬機關 (申請機關)

中央研究院

獲證日期

2010/02/01

技術說明

木瓜幾丁質酶基因(CpCHI)全長1002 bp，預測有275個胺基酸，包括其氮端含有一個訊號胜肽約28個胺基酸，而其成熟蛋白 (無訊號胜肽)預測之分子量為26.2 kDa、pI值為6.32。根據其胺基酸序列與植物中第四類的幾丁質酶最相近，因而推測CpCHI屬於第四類的植物幾丁質酶。將此基因轉殖到大腸菌中大量表現，可以觀察到所產生的幾丁質酶具有活性，以乙二醇幾丁質為基質進行水解，其最適反應pH 值為5.0到9.0，最適反應溫度為30 C，此重組基丁質酶極為穩定，在30 C貯存三周仍維持64%的活性；進行抗菌實驗可觀察到CpCHI對Rhizoctonia solani, Sclerotium rolfsii, Phytophthora capsici, and Alternaria brassicicola有抗真菌活性表現。將15 μM重組機丁質酶塗佈於甘藍、青椒、番茄的離葉上，可抑制Sclerotium rolfsii所引起的病徵達98%。 A chitinase cDNA clone (CpCHI, 1002 bp) is isolated from papaya fruit CpCHI which encodes a 275-amino-acid protein containing a 28 amino-acid signal peptide in the N-terminal end. The predicted molecular weight of the mature protein is 26.2 kDa, and its pI value is 6.32. Based on its amino acid sequence homology with other plant chitinases, it is classified as class IV chitinase. An active recombinant CpCHI enzyme is overexpressed in Escherichia coli. The purified recombinant papaya chitinase shows an optimal reaction temperature at 30 ºC and broad optimal pH ranging from pH 5.0 to 9.0. The recombinant enzyme is quite stable, retaining more than 64% activity for 3 weeks at 30 ºC. It appears to reduce the fungal activity of Rhizoctonia solani, Sclerotium rolfsii, Phytophthora capsici, and Alternaria brassicicola.

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