Narinx E, Baise E, Gerday C
Laboratory of Biochemistry, Institute of Chemistry (B6), University of Liège, Belgium.
Protein Eng. 1997 Nov;10(11):1271-9. doi: 10.1093/protein/10.11.1271.
A subtilisin excreted by the Antarctic Bacillus TA39 has been purified to homogeneity and characterised. Two independent genes subt1 and subt2 are present but only subt1 is expressed significantly in the culture medium. The enzyme displays the usual characteristics of cold enzymes i.e. a high catalytic efficiency at low and moderate temperatures and an increased thermosensitivity originating from a 3D structure probably more flexible than its mesophilic counterpart. This is corroborated by the analysis of the computerized structure which shows a significant decrease in the number and strength of intramolecular weak bonds such as salt bridges and aromatic interactions. The affinity for calcium is also almost three orders of magnitude lower than that of mesophilic subtilisin and the interactions with the solvent are significantly higher thanks to a large increase in the number of Asp residues in the loops connecting secondary structures. The relation between flexibility and activity was investigated by site-directed mutagenesis tending mainly to increase the rigidity of the molecular edifice through the incorporation of additional salt bridge, disulfide bridge, aromatic interaction and by increasing the affinity of the enzyme for calcium. An important stabilization of the molecular structure was achieved through a modification of a calcium ligand T85D. The thermostability of the mutated product expressed in a mesophilic Bacillus reaches that of mesophilic subtilisin. Most important is the fact that this mutation further enhances the specific activity by a factor close to 2 when compared to the wild type enzyme so that the overall activity of the mutated cold enzyme is about 20 times higher than that of mesophilic subtilisin, illustrating the fact that thermostability is not systematically inversely related to specific activity. This opens new perspectives in the use of cold enzymes in biotechnology.
南极芽孢杆菌TA39分泌的一种枯草杆菌蛋白酶已被纯化至同质并进行了表征。存在两个独立的基因subt1和subt2，但只有subt1在培养基中大量表达。该酶具有冷适应酶的常见特性，即在低温和中温下具有较高的催化效率，并且由于其三维结构可能比嗜温同类酶更灵活而导致热敏感性增加。计算机辅助结构分析证实了这一点，该分析表明分子内弱键（如盐桥和芳香族相互作用）的数量和强度显著降低。其对钙的亲和力也比嗜温枯草杆菌蛋白酶低近三个数量级，并且由于二级结构连接环中Asp残基数量的大幅增加，与溶剂的相互作用显著增强。通过定点诱变研究了柔韧性与活性之间的关系，主要倾向于通过引入额外的盐桥、二硫键、芳香族相互作用以及增加酶对钙的亲和力来提高分子结构的刚性。通过对钙配体T85D的修饰实现了分子结构的重要稳定。在嗜温芽孢杆菌中表达的突变产物的热稳定性达到了嗜温枯草杆菌蛋白酶的水平。最重要的是，与野生型酶相比，这种突变使比活性进一步提高了近2倍，因此突变冷适应酶的总体活性比嗜温枯草杆菌蛋白酶高约20倍，这表明热稳定性并非总是与比活性呈负相关。这为在生物技术中使用冷适应酶开辟了新的前景。
