Cold adaptation of thermostable enzymes by surface loops' mutagenesis

高活性的亲冷酶提供了潜在的好处，通过大量的能源节约大规模的化学过程。预计芽孢杆菌结核杆菌90尿酸酶的Trp279和Pro287位于界面环II的铰链上。W279L+P287G突变体的最适温度从50℃降至30℃，但其热稳定性未受影响。在这里，我们以2.1埃的分辨率确定了它的晶体结构。残基284 ~ 289缺失，环内其他残基温度因子较高。回路的柔韧性可能是低温适应成功的原因。对枯草芽孢杆菌α -淀粉酶表面环的7个残基进行了饱和诱变研究。通过淀粉降解试验筛选得到5个残基的亲心突变体。在保持热稳定性的情况下，活性增加了120-300%。

The high activity of psychrophilic enzymes offers potential benefits through substantial energy savings in large-scale chemical processes. Trp279 and Pro287 of Bacillus sp. TB-90 uricase were predicted to be located at hinges of the interface loop II. The optimum temperature of the W279L+P287G mutant decreased from 50 degree to 30 degree without loss of its thermal stability. Here, we determined its crystal structure at a 2.1 angstrom resolution. The residues 284-289 were missing and the other residues in the loop showed higher temperature factors. The flexibility of the loop may be responsible for the successful cold adaptation. The saturation mutagenesis studies on seven residues in the surface loops of alpha-amylase from Bacillus subtilis were also performed. Psychorphilic mutants of five residues were obtained by the screening using the starch degradation assay. They showed 120-300% increases of the activities with having kept thermal stability.