Novel Use for the Osmolyte Trimethylamine N-oxide: Retaining the Psychrophilic Characters of Cold-Adapted Protease Deseasin MCP-01 and Simultaneously Improving its Thermostability
Novel Use for the Osmolyte Trimethylamine N-oxide: Retaining the Psychrophilic Characters of Cold-Adapted Protease Deseasin MCP-01 and Simultaneously Improving its Thermostability
复制标题
渗透剂三甲胺 N-氧化物的新用途：保留冷适应蛋白酶 deseasin MCP-01 的嗜冷特性，同时提高其热稳定性
DOI：
10.1007/s10126-009-9185-2
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发表时间：
2009-12-01
影响因子：
3
通讯作者：
Zhang, Yu-Zhong
中科院分区：
文献类型：
--
作者：
He, Hai-Lun;Chen, Xiu-Lan;Zhang, Yu-Zhong
低温酶的热稳定性差是其应用的主要障碍。一个简单可靠的方法来提高低温酶的稳定性和活性仍然很少见。作为一种蛋白质稳定剂，氧化三甲胺（TMAO）对冷适应酶或蛋白质的影响还未见报道。本文研究了TMAO对低温蛋白酶Deseasin MCP-01的结构、活性和稳定性的影响。Deseasin MCP-01是一种新型的枯草杆菌酶，来源于深海耐冷菌Pseudoalteromonas sp.SM9913。荧光光谱和圆二色性光谱表明TMAO对MCP-01的结构没有影响，保持了MCP-01的构象柔性。1摩尔TMAO使MCP-01的活性提高了174%，其催化效率（k（cat）/K（m））在0A ℃下提高了290%。在1 M TMAO的存在下，MCP-01的热稳定性（t（1/2））在60 ℃（类似于40 ℃）下增加了2至5倍。CD结构分析表明，当在40 ℃孵育1 h时，1 M TMAO可使MCP-01的结构热稳定性接近其嗜温对应物枯草杆菌蛋白酶Carlsberg的结构热稳定性。此外，IM TMAO使MCP-01的熔融温度（T（m））增加了10.5A摄氏度。这些结果表明，TMAO可以作为一个完美的稳定剂，以保持冷适应酶的嗜冷特性，同时提高其热稳定性。
The low thermostability of cold-adapted enzymes is a main barrier for their application. A simple and reliable method to improve both the stability and the activity of cold-adapted enzymes is still rare. As a protein stabilizer, the effect of trimethylamine N-oxide (TMAO) on a cold-adapted enzyme or protein has not been reported. In this study, effects of TMAO on the structure, activity, and stability of a cold-adapted protease, deseasin MCP-01, were studied. Deseasin MCP-01 is a new type of subtilase from deep-sea psychrotolerant bacterium Pseudoalteromonas sp. SM9913. Fluorescence and CD spectra showed that TMAO did not perturb the structure of MCP-01 and therefore kept the conformational flexibility of MCP-01. One molar TMAO improved the activity of MCP-01 by 174% and its catalytic efficiency (k (cat) /K (m)) by 290% at 0A degrees C. In the presence of 1 M TMAO, the thermostability (t (1/2)) of MCP-01 increased by two- to fivefold at 60 similar to 40A degrees C. Structural analysis with CD showed that 1 M TMAO could keep the structural thermostability of MCP-01 close to that of its mesophilic counterpart subtilisin Carlsberg when incubated at 40A degrees C for 1 h. Moreover, 1 M TMAO increased the melting temperature (T (m)) of MCP-01 by 10.5A degrees C. These results suggest that TMAO can be used as a perfect stabilizing agent to retain the psychrophilic characters of a cold-adapted enzyme and simultaneously improve its thermostability.