Pitts J E, Uusitalo J M, Mantafounis D, Nugent P G, Quinn D D, Orprayoon P, Penttilä M E
Department of Crystallography, Birkbeck College, London, UK.
J Biotechnol. 1993 Mar;28(1):69-83. doi: 10.1016/0168-1656(93)90126-8.
The production of chymosin mutants designed to have altered pH optima using the cellulolytic filamentous fungus Trichoderma reesei is described. The strong promoter of the gene encoding the major cellulase, cellobiohydrolase I (CBHI) has been used for the expression and secretion of active calf chymosin. Structural analysis of the hydrogen bonding network around the two active site aspartates 32 and 215 in chymosin have suggested that residues Thr 218 and Asp 303 may influence the rate and pH optima for catalysis. The chymosin mutants Thr218Ala and the double mutant Thr218Ala/Asp303Ala have been made by site-directed mutagenesis and expressed in T. reesei. Enzyme kinetics of the active enzyme T218A indicate a pH optimum of 4.2 compared to 3.8 for native chymosin B using a synthetic octa-peptide substrate, confirming the previous analysis undertaken in E. coli. The double mutant T218A/D303A exhibits a similar optimum of 4.4 to that reported for the D303A, indicating that the combination of these changes is not additive. The application of protein engineering in the rational design of specific modifications to tailor the properties of enzymes offers a new approach to the development of industrial processes.
本文描述了利用纤维素分解丝状真菌里氏木霉生产pH最适值改变的凝乳酶突变体的过程。编码主要纤维素酶纤维二糖水解酶I（CBHI）的基因的强启动子已用于活性小牛凝乳酶的表达和分泌。对凝乳酶中两个活性位点天冬氨酸32和215周围氢键网络的结构分析表明，苏氨酸218和天冬氨酸303残基可能影响催化速率和pH最适值。通过定点诱变制备了凝乳酶突变体苏氨酸218丙氨酸和双突变体苏氨酸218丙氨酸/天冬氨酸303丙氨酸，并在里氏木霉中表达。使用合成八肽底物时，活性酶T218A的酶动力学表明其pH最适值为4.2，而天然凝乳酶B的pH最适值为3.8，这证实了之前在大肠杆菌中进行的分析。双突变体T218A/D303A的最适pH值与D303A报道的相似，为4.4，表明这些变化的组合不是累加的。蛋白质工程在合理设计特定修饰以调整酶的性质方面的应用为工业过程的开发提供了一种新方法。
