Characterisation of a chitinase from Pseudoalteromonas sp. DL-6, a marine psychrophilic bacterium.
Characterisation of a chitinase from Pseudoalteromonas sp. DL-6, a marine psychrophilic bacterium.
复制标题
DOI：
10.1016/j.ijbiomac.2014.07.033
复制
发表时间：
2014-09
影响因子：
8.2
通讯作者：
Xiaohui Wang;Yong Zhao;Haidong Tan;N. Chi;Qingfang Zhang;Yuguang Du;H. Yin
中科院分区：
文献类型：
--
作者：
Xiaohui Wang;Yong Zhao;Haidong Tan;N. Chi;Qingfang Zhang;Yuguang Du;H. Yin
本研究中，我们分离到一种新的嗜冷细菌，假交替单胞菌。DL-6来自海洋沉积物，其在4 °C下在含几丁质的平板上生长良好。从该菌基因组DNA中克隆了一个内切型几丁质酶基因chiA，并在大肠杆菌BL 21（DE 3）中异源表达。即使在4 °C下，ChiA也显示出非常高的催化活性，并且在pH 8.0和20 °C下对几丁质底物表现出最大活性。动力学研究表明，ChiA对4-甲基伞形酮基-β-d-N，N′，N″-三乙酰壳三糖[4-MU（GlcNAc）3]的催化效率高于对4-甲基伞形酮基-β-d-N，N′-二乙酰壳二糖苷[4-MU（GlcNAc）2]的催化效率。电喷雾电离质谱（ESI-MS）分析表明，粉末甲壳素的水解产物后，ChiA消化由一系列的甲壳素低聚物具有不同程度的聚合。以（GlcNAc）2- 6为底物研究了ChiA的作用方式，结果表明ChiA是一种非加工性内切型几丁质酶。
In this study, we isolated a new psychrophilic bacterium,Pseudoalteromonassp. DL-6 from marine sediments, which grew well on chitin-containing plates at 4 °C. One endo-type chitinase gene,chiA, was cloned from the genomic DNA of this bacterium and heterologously expressed inEscherichia coliBL21 (DE3). ChiA showed very high catalytic activity, even at 4 °C, and exhibited maximal activity on a chitinous substrate at pH 8.0 and 20 °C. Kinetic studies indicated that ChiA has a greater catalytic efficiency on 4-methylumbelliferyl-β-d-N,N′,N″-triacetylchitotriose[4-MU(GlcNAc)3] than on 4-methylumbelliferyl-β-d-N,N′-diacetylchitobioside[4-MU(GlcNAc)2]. Electrospray ionisation mass spectrometry (ESI-MS) analysis showed that the hydrolysis products of powdered chitin after ChiA digestion consisted of a series of chitin oligomers with different degrees of polymerisation. The ChiA mode of action was also examined using (GlcNAc)2–6as a substrate, and the results suggested that ChiA is a non-processive endo-type chitinase.