Halophilicity of Thermolysin.Protein Engineering Studies

嗜热菌蛋白酶（Thermolysin，TLN）是一种嗜热的中性金属蛋白酶，我们曾报道TLN具有高度的嗜盐性，在饱和盐浓度存在下，其活性比无盐条件下提高30-60倍.结果表明，盐的活化与pH值、温度和有机溶剂的加入有很大的关系，表明盐的活化可能与TLN.2表面的静电相互作用有关.加入0.5- 1.5M NaCl可使TLN的温度稳定性提高2-3倍。对盐浓度的依赖性不同于对活化的依赖性，表明稳定性和活性可以通过独立的机制得到提高. TLN在普通缓冲液中的溶解度低至1 mg/ml。发现当盐的加入量高达70 mg/ml时，它显著增加。TLN为冷溶性。Trp 115位于S2亚位点。通过定点突变，发现在该位点上需要一个芳香族氨基酸才能产生活性. TLN的酪氨酰残基的硝化和胺化反应表明，TLN表面负电荷的引入降低了盐的活化程度。

Thermolysin (TLN) is a thermophilic neutral metalloprpteinase, and we have reported that TLN is highly halophilic and its activity enhances 30-60 times in the presence of saturated concentration of salts in comparison with that in the absence.In this study, the halophilicity was examined from the point of view of the enzyme structure and medium properties.1. The salt activation was shown to be dependent considerably on pH and temperature and the addition of organic solvent, suggesting that the activation might be related with electrostatic interaction on the surface of TLN.2. Temperature stability of TLN was enhanced 2-3 times by the addition of 0.5-1.5 M NaCl. The dependence on the salt concentration was different from that of the activation, suggesting that the stability and activity can be increased by indepenent mechanisms.3. The solubility of TLN is as low as 1 mg/ml in the common buffer. It was found that it increased remarkably by the addition of salts up to 70 mg/ml. TLN was revealed to be cold-soluble.4. Trp 115 is at the S2 suibsite. By the site-directed mutagenesis, an aromatic amino acid was shown to be needed strictly at this position for the activity.5. Nitration and amination of tyrosyl reasidues of TLN showed that introduction of negative charges on the surface of TLN decreased the degree of the salt activation.