Misset O, Gerritse G, Jaeger K E, Winkler U, Colson C, Schanck K, Lesuisse E, Dartois V, Blaauw M, Ransac S
Lehrstuhl Biologie der Mikroorganismen, Ruhr Universität, Bochum, Germany.
Protein Eng. 1994 Apr;7(4):523-9. doi: 10.1093/protein/7.4.523.
Within the BRIDGE T-project on lipases we investigate the structure-function relationships of the lipases from Bacillus subtilis and Pseudomonas aeruginosa. Construction of an overproducing Bacillus strain allowed the purification of > 100 mg lipase from 30 l culture supernatant. After testing a large variety of crystallization conditions, the Bacillus lipase gave crystals of reasonable quality in PEG-4000 (38-45%), Na2SO4 and octyl-beta-glucoside at 22 degrees C, pH 9.0. A 2.5 A dataset has been obtained which is complete from 15 to 2.5 A resolution. P.aeruginosa wild-type strain PAC1R was fermented using conditions of maximum lipase production. More than 90% of the lipase was cell bound and could be solubilized by treatment of the cells with Triton X-100. This permitted the purification of approximately 50 mg lipase. So far, no crystals of sufficient quality were obtained. Comparison of the model we built for the Pseudomonas lipase, on the basis of sequences and structures of various hydrolases which were found to possess a common folding pattern (alpha/beta hydrolase fold), with the X-ray structure of the P.glumae lipase revealed that it is possible to correctly build the structure of the core of a protein even in the absence of obvious sequence homology with a protein of known 3-D structure.
在BRIDGE T项目的脂肪酶研究中，我们探究了枯草芽孢杆菌和铜绿假单胞菌脂肪酶的结构-功能关系。构建一个高产芽孢杆菌菌株，使得从30升培养上清液中纯化出了超过100毫克的脂肪酶。在测试了多种结晶条件后，枯草芽孢杆菌脂肪酶在22℃、pH 9.0的PEG - 4000（38 - 45%）、硫酸钠和辛基-β-葡萄糖苷体系中得到了质量尚可的晶体。已获得一个从15到2.5埃分辨率完整的2.5埃数据集。使用最大脂肪酶产量的条件发酵铜绿假单胞菌野生型菌株PAC1R。超过90%的脂肪酶与细胞结合，可用Triton X - 100处理细胞使其溶解。这使得能够纯化出约50毫克的脂肪酶。到目前为止，尚未获得足够质量的晶体。我们基于各种水解酶的序列和结构构建了铜绿假单胞菌脂肪酶的模型，发现这些水解酶具有共同的折叠模式（α/β水解酶折叠），与谷壳假单胞菌脂肪酶的X射线结构比较表明，即使在与已知三维结构的蛋白质没有明显序列同源性的情况下，也有可能正确构建蛋白质核心的结构。
