Halophilicity of Thermolysin.Protein Engineering Studies

Thermolysin(TLN)是一种嗜热的中性金属蛋白水解酶，我们曾报道TLN是高度嗜盐的，在饱和盐浓度下其活性是无盐时的30-60倍。本研究从酶结构和介质性质的角度考察了TLN的嗜盐性。盐的活化与pH、温度和有机溶剂的加入有很大的依赖关系，表明盐的活化可能与TLN表面的静电相互作用有关。添加0.5~1.5M的氯化钠可使TLN的温度稳定性提高2~3倍。其对盐浓度的依赖性与活性对盐浓度的依赖程度不同，表明其稳定性和活性的提高可以通过独立的机制来实现。TLN在普通缓冲液中的溶解度低至1 mg/ml。结果表明，当盐浓度达到70 mg/ml时，TLN的溶出度显着增加，TLN为冷溶型。Trp 115位于S2 SuibSite。通过定点突变，证明在该位置的活性完全需要芳香族氨基酸。TLN酪氨酰还原产物的硝化和胺化反应表明，在TLN表面引入负电荷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.