Cloning, sequence analysis, and expression of gene alyPI encoding an alginate lyase from marine bacterium Pseudoalteromonas sp CY24
Cloning, sequence analysis, and expression of gene alyPI encoding an alginate lyase from marine bacterium Pseudoalteromonas sp CY24
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DOI：
10.1139/w09-051
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发表时间：
2009-09-01
影响因子：
2.8
通讯作者：
Yu, Wengong
中科院分区：
文献类型：
--
作者：
Duan, Gaofei;Han, Feng;Yu, Wengong
利用PCR技术克隆了海洋细菌Pseudoalteromonassp.CY24的海藻酸裂解酶基因（alyPI）。alyPI基因全长1575 bp，开放阅读框编码57.4 kDa的蛋白质，含524个氨基酸残基和23个氨基酸的信号肽。在大肠杆菌中表达的AlyPI蛋白的His-标签序列融合在C-末端和纯化的电泳同质性使用Ni-琼脂糖亲和层析。AlyPI在0.1 mol/L NaCl存在下，在40 ℃和pH 7.0下最具活性，并且在pH 6.0-10.6的宽范围内稳定。Na+、K+、Mn ~（2+）、Ca ~（2+）、Fe ~（3+）等离子的存在均能提高酶的活性。在AlyPI中发现了褐藻酸裂解酶共有区YFKAGXYXQ，其被认为是类似于30 kDa的几种褐藻酸裂解酶的C末端的显著特征，AlyPI属于类似于60 kDa的组。另一个9个氨基酸的共有区，YXRSELREM，只发现在G-特异性的海藻酸裂解酶先前存在于AlyPI，它可以降解海藻酸钠，M块，和G块，似乎是一个广泛的底物特异性的海藻酸裂解酶。
The alginate lyase encoding gene (alyPI) of marine bacterium Pseudoalteromonas sp. CY24 was cloned using a battery of PCR techniques. Gene alyPI was composed of a 1575 bp open reading frame encoding a protein of 57.4 kDa containing 524 amino acid residues with a signal peptide of 23 amino acids. The AlyPI protein was expressed in Escherichia coli with a His-tag sequence fused at the C-terminal end and purified to electrophoretic homogeneity using Ni-sepharose affinity chromatography. AlyPI was most active at 40 degrees C and pH 7.0 in the presencce of 0.1 mol/L NaCl and stable over a broad range of pH, 6.0-10.6. The presence of Na+, K+, Mn2+, Ca2+, and Fe3+, can enhance the enzyme activity. The alginate lyase consensus region YFKAGXYXQ, regarded as a striking feature at the C termini of several alginate lyase of similar to 30 kDa, was found in AlyPI, which belongs to the similar to 60 kDa group. Another nine amino acid consensus region, YXRSELREM, only found in G-specific alginate lyases previously existed in AlyPI, which could degrade sodium alginate, M blocks, and G blocks and appeared to be a broad substrate-specific alginate lyase.