高产GSH酵母突变株Y518谷胱甘肽合成酶结构分析.pdfVIP

258 2009, Vol. 30, No. 17 食品科学 ※生物工程 王雅楠，梅艳珍，郑丽雪，齐 斌，王立梅* (常熟理工学院发酵工程技术研究中心，江苏常熟 215500) 酿酒酵母突变株Saccharomyces cerevisiae Y518 比原始菌株谷胱甘肽产量有明显提高，有较好的耐酸性， 本研究通过克隆突变前后谷胱甘肽合成酶基因并进行结构比较，分析其突变后耐酸性提高的原因。结果表明： Pro54Leu 使突变残基扭转角度由－40.2 °变为－50.4 °，柔性增强，可能有利于酶催化过程中“帽子”结构域的 位移形成闭合构象而提高了与底物结合的稳定性；Leu54 侧链基团向外伸展，包裹暴露表面的—SH，避免因氧化 而影响酶分子活性；同时疏水性的增强可能使C 端羧基pKa 值下降从而使活性位点pKa 值降低，导致最适pH 值的 改变。 谷胱甘肽合成酶；酿酒酵母突变株Y518 ；亚硝基胍；耐酸性 Structural Analysis of Glutathione Synthase from High-yield Glutathione-producing Saccharomyces cerevisiae Y518 WANG Ya-nan ，MEI Yan-zhen ，ZHENG Li-xue ，QI Bin ，WANG Li-mei* (Fermentation Engineering Technology Center, Changshu Institute of Technology, Changshu 215500, China) Abstract ：The mutant strain of Saccharomyces cerevisiae Y518 yielded much more glutathione than the starting strain and presented an excellent acid resistance. The changes in glutathione synthase sequence and structure before and after mutation were analyzed using bioinformatics tools to account for the reason why the acid resistance was increased. The rigid residue Pro was replaced by flexible residue Leu (Pro54Leu) and its torsion angle changed from －40.2 ° to －50.4°, which may be conducive to the “lid ” domain shift in catalysis for the formation of closed conformation and thus the enzyme-substrate binding becomes more stable. The side chain of Leu54 stretch out to package —SH group exposed to protein surface might keep enzyme activity, and change the pKa value of protein C-terminal Tyr491 resulting in a decrease of the pKa of the active site and shifting the optimum pH to 3.5. Key words ：glutathione synthase ；Saccharomyces cerevisiae Y518 ；nitrosoguanidine ；acid resistance