Characterization of chitinase genes from an alkaliphilic actinomycete, Nocardiopsis prasina OPC-131
Characterization of chitinase genes from an alkaliphilic actinomycete, Nocardiopsis prasina OPC-131
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DOI：
10.1128/aem.69.2.894-900.2003
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发表时间：
2003-02-01
影响因子：
4.4
通讯作者：
Inamori, Y
中科院分区：
文献类型：
--
作者：
Tsujibo, H;Kubota, T;Inamori, Y
一种嗜碱放线菌，Nocardiopsis prasina OPC-131，在几丁质存在下分泌几丁质酶，ChiA，Chilli和ChiB Delta。对编码ChiA和Chilli的基因进行了克隆和序列测定。ChiA的开放阅读框（ORF）编码336个氨基酸，计算分子量为35，257 Da，仅由催化结构域组成，与家族18几丁质酶有很高的同源性。ChiB ORF编码296个氨基酸的蛋白质，计算分子量为31，500DaXiB是由几丁质结合结构域3型（ChtBD 3型）和催化结构域组成的模块化酶。ChiB的催化结构域与Streptomyces family 19几丁质酶具有很高的相似性。ChiBDelta是缺乏ChtBD 3型的ChiB的截短形式。构建了编码ChiA、ChiB和ChiBDelta的表达质粒，以研究这些重组蛋白的生化特性。这些酶显示出与天然酶相似的最适pH和温度。结果表明，ChiB的ChtBD 3型在几丁质的高效水解和抗真菌活性中起着重要作用。此外，还发现了N. prasina OPC-131表明家族19几丁质酶广泛分布于除链霉菌属以外的放线菌中。
An alkaliphilic actinomycete, Nocardiopsis prasina OPC-131, secretes chitinases, ChiA, Chilli, and ChiBDelta, in the presence of chitin. The genes encoding ChiA and Chilli were cloned and sequenced. The open reading frame (ORF) of chiA encoded a protein of 336 amino acids with a calculated molecular mass of 35,257 Da. ChiA consisted of only a catalytic domain and showed a significant homology with family 18 chitinases. The chiB ORF encoded a protein of 296 amino acids with a calculated molecular mass of 31,500 Da. ChiB is a modular enzyme consisting of a chitin-binding domain type 3 (ChtBD type 3) and a catalytic domain. The catalytic domain of ChiB showed significant similarity to Streptomyces family 19 chitinases. ChiBDelta was the truncated form of ChiB lacking ChtBD type 3. Expression plasmids coding for ChiA, ChiB, and ChiBDelta were constructed to investigate the biochemical properties of these recombinant proteins. These enzymes showed pHs and temperature optima similar to those of native enzymes. ChiB showed more efficient hydrolysis of chitin and stronger antifungal activity than ChiBDelta, indicating that the ChtBD type 3 of ChiB plays an important role in the efficient hydrolysis of chitin and in antifungal activity. Furthermore, the finding of family 19 chitinase in N. prasina OPC-131 suggests that family 19 chitinases are distributed widely in actinomycetes other than the genus Streptomyces.