Chitinase from Bacillus thuringiensis subsp pakistani
Chitinase from Bacillus thuringiensis subsp pakistani
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DOI：
10.1007/s002530100630
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发表时间：
2001-08-01
影响因子：
5
通讯作者：
Panbangred, W
中科院分区：
文献类型：
--
作者：
Thamthiankul, S;Suan-Ngay, S;Panbangred, W
苏云金芽孢杆菌几丁质酶基因（chiA71）。巴基斯坦由一个开放阅读框组成，包含1905个核苷酸，编码635个氨基酸残基，估计分子质量为71 kDa。将成熟酶的氨基酸序列与其他微生物几丁质酶的氨基酸序列进行比较，发现了一个推定的催化结构域和一个与纤维连接蛋白III型模块和几丁质结合结构域相似的保守氨基酸区域。在SDS-PAGE上进行几丁质酶活性检测，具有几丁质酶活性的蛋白带分子量分别为66、60、47和32 kDa。各几丁质酶活性带的n端氨基酸序列相同（Asp-Ser-Pro-Lys-Gln），说明60-、47-和32-kDa几丁质酶是由66-kDa几丁质酶的c端加工而来。该酶是一种外几丁质酶，在胶体几丁质水解的早期产生n -乙酰氨基葡萄糖。含有最终活性为8、16、32和64 mU/ml几丁质酶的粗蛋白（2.3 ~ 18.4 mg/ml）对埃及伊蚊幼虫具有毒性，死亡率分别为7.5%、15.0%、51.3%和70.0%，而苏云金芽孢杆菌亚种的粗蛋白含量相同。缺乏几丁质酶的巴基斯坦突变体没有毒性。
The chitinase gene (chiA71) from Bacillus thuringiensis subsp. pakistani consists of an open reading frame of 1,905 nucleotides encoding 635 amino acid residues with an estimated molecular mass of 71 kDa. Comparison of the deduced amino acid sequence of the mature enzyme to other microbial chitinases shows a putative catalytic domain and a region with conserved amino acids similar to that of the type III module of fibronectin and a chitin-binding domain. By activity detection of chitinase on SDS-PAGE after renaturation, the molecular mass of protein bands with chitinase activity were 66, 60, 47, and 32 kDa. The N-terminal amino acid sequence of each chitinase activity band was the same (Asp-Ser-Pro-Lys-Gln), suggesting that the 60-, 47-, and 32-kDa chitinases were derived from the 66-kDa chitinase by processing step(s) at the C-terminus. The enzyme was identified as an exochitinase, since it generated N-acetylglucosamine from early stage of colloidal chitin hydrolysis. The crude protein (2.3-18.4 mg/ml), containing chitinase at final activities of 8, 16, 32, and 64 mU/ml, was toxic to Aedes aegypti larvae and caused mortalities of 7.5, 15.0, 51.3, and 70.0% respectively, but the same amount of crude protein from a B. thuringiensis subsp. pakistani mutant lacking chitinase was not toxic.