Halophilicity of Thermolysin.Protein Engineering Studies

热溶酶（Thermolysin， TLN）是一种嗜热中性金属蛋白酶，我们报道过TLN具有高度的嗜盐性，其活性在饱和盐浓度下比不存在饱和盐浓度时提高30-60倍。本研究从酶的结构和培养基性质两方面对其嗜盐性进行了考察。结果表明，盐的活化与pH、温度和有机溶剂的加入有很大关系，表明盐的活化可能与TLN.2表面的静电相互作用有关。添加0.5 ~ 1.5 M 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，表明其稳定性和活性的提高可以通过独立的机制实现。TLN在普通缓冲液中的溶解度低至1mg /ml。当盐的添加量达到70 mg/ml时，其含量显著增加。TLN是冷溶的。Trp 115位于S2亚位。通过定点诱变，表明在这个位置上严格需要一个芳香氨基酸才能发挥活性。对TLN酪氨酸残基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.