摘 要

我们从深海海洋细菌Flammeovirgasp中鉴定并克隆编码新的褐藻胶裂解酶FsAlgA的基因NJ-04。并将其进行重组表达和亲和纯化。实验结果表明它在pH 7.0和50℃下表现出最高的活性（3343.68U / mg）。值得注意的是，在50℃温育30分钟后，FsAlgA保留了80％以上的活性，在中高温下表现出极好的热稳定性。另外，它具有广泛的底物特异性，并且显示出对polyM（聚β-D-甘露糖酸钠）和polyG（聚α-L-古洛糖醛酸酯）的活性。此外，FsAlgA对海藻酸钠（0.48mM）和polyG（0.94mM）的Km值比对polyM（1.42mM）的低。TLC和ESI-MS分析表明，FsAlgA主要释放DP为2~5的寡糖。因此，它可能是生产具有低DP的藻酸盐寡糖的有效工具。

关键词：深海细菌 Flammeovirgasp NJ-04 热稳定性 藻酸盐裂解酶 寡糖

Expression and Characterization of a New Thermal Stable Endo-type Alginate Lyase from Deep-Sea Bacterium Flammeovirga sp. NJ-04

ABSTRACT

A gene, encoding a new alginate lyase FsAlgA, was identified and cloned from deep

sea marine bacterium Flammeovirga sp. NJ-04. The recombinant alginate lyase was

characterized followed by being purified on Ni-NTA Sepharose. It exhibited the

highest activity (3343.68 U/mg) at pH 7.0 and 50 °C. Notably, the FsAlgA retained

more than 80% activity after being incubated at 50 °C for 30 min, which exhibited

an excellent thermostability in medium-high temperatures. Additionally, it possessed

broader substrate specificity and showed activities towards both polyM (poly

β-D-mannuronate) and polyG (poly α-L-guluronate). Furthermore, theKm values of

FsAlgA toward sodiumalginate (0.48 mM) and polyG (0.94 mM) werelower than

those toward polyM (1.42 mM).The TLC and ESI-MS analysis suggested that

FsAlgA mainly released oligosaccharides with DP of 2~5 in an endolytic manner.

Therefore, it may be a potent tool to produce alginate oligosaccharides with low DP.

Keywords: deep-sea bacterium; Flammeovirga sp. NJ-04; thermostable; alginatelyase; oligosaccharides

目 录

摘 要.........................................................................................................................................Ⅰ

ABSTRACT....................................................................................................................................Ⅱ

1. 引言............................................................................................................................1
2. 实验材料与方法.....................................................................................................2

2.1实验材料...........................................................................................................................2

2.2实验方法...........................................................................................................................2

2.2.1序列分析................................................................................................................

2.2.2 FsAlgA
的表达和纯化.........................................................................................

2.2.3底物特异性和酶动力学........................................................................................

2.2.4 FsAlgA的生化表征..............................................................................................

2.2.5行动模式和降级产品分析...................................................................................

1. 结果与讨论...............................................................................................................

3.1基因FsAlgA的序列分析................................................................................................

3.2 FsAlgA的表达与纯化.....................................................................................................

3.3 FsAlgA的生化表征.........................................................................................................

3.5降解产物的TLC和ESI-MS分析..................................................................................

1. 结论与展望...............................................................................................................

4.1结论...................................................................................................................................

4.2展望...................................................................................................................................

参考文献......................................................................................................................................

致谢................................................................................................................................................

第一章 引言

藻酸盐，一种亲水胶体，是最丰富的多糖棕色藻类^[1]。它由两种糖醛酸组成，以β-D-甘露糖醛酸盐（M）和α-L-古洛糖酸盐（G）作为单体单元。这些单位有三种个不同的嵌段，聚β-D-甘露聚糖（polyM），聚α-L-古洛糖醛酸（polyG）和异源聚合物（polyMG）2。由于其有利的特性，如化学品稳定性，高粘度和凝胶形成性能，藻酸盐可广泛应用于食品工业中的添加剂3-5。

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