Xie Ting, Song Binghong, Yue Yang, Chao Yapeng, Qian Shijun
State Key Laboratories of Transducer Technology, National Engineering Lab for Industrial Enzymes, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China; CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
State Key Laboratories of Transducer Technology, National Engineering Lab for Industrial Enzymes, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
J Biotechnol. 2014 Jan 20;170:10-6. doi: 10.1016/j.jbiotec.2013.10.032. Epub 2013 Nov 15.
Central tyrosine 195 plays an important role in the active site of cyclodextrin glycosyltransferase (CGTase) that is highly conservative among various CGTases. However, a detailed functional understanding of this subsite is lacking. In this study, we applied site-directed saturation mutagenesis to investigate the effect of tyrosine 195 on the hydrolytic activity and cyclization specificity of an α-CGTase. A total of 17 mutant CGTases were obtained and heterologously expressed in E. coli. The mutant Y195F α-CGTase showed similar characteristics with wild-type α-CGTase. The other mutant α-CGTases showed considerably lower activity for starch-degradation and cyclodextrin (CD) formation. Interestingly, we found that the main product of mutant Y195R α-CGTase was γ-CDs (50%), not α-CDs (35%). The mutant Y195I α-CGTase drastically altered the CD specificity of α-CGTase, which showed a switch toward the synthesis of both β- and γ-CDs with percentages of 34% and 38%, respectively. Other mutant CGTases retained the α-CD as the main product but with lower percentages than wild-type α-CGTase. Mutant Y195F, Y195I, and Y195R CGTases showed an optimal temperature of 50°C and pH 6.5. The mutants Y195I and Y195R also showed better thermostability. These findings suggested that aromatic amino acids Tyr or Phe at the 195 position were important for the amylolytic activity and cyclization specificity of α-CGTase. The mutants Y195I CGTase and Y195R CGTase have potential applications for γ-CD production in the future.
位于环糊精糖基转移酶（CGTase）活性位点中心的酪氨酸195在各种CGTase中高度保守，发挥着重要作用。然而，对该亚位点的详细功能了解尚缺。在本研究中，我们应用定点饱和诱变来研究酪氨酸195对α-CGTase水解活性和环化特异性的影响。共获得17种突变型CGTase，并在大肠杆菌中进行了异源表达。突变型Y195F α-CGTase表现出与野生型α-CGTase相似的特性。其他突变型α-CGTase对淀粉降解和环糊精（CD）形成的活性则显著较低。有趣的是，我们发现突变型Y195R α-CGTase的主要产物是γ-CD（50%），而非α-CD（35%）。突变型Y195I α-CGTase极大地改变了α-CGTase的CD特异性，转而合成β-CD和γ-CD，其比例分别为34%和38%。其他突变型CGTase仍以α-CD为主要产物，但比例低于野生型α-CGTase。突变型Y195F、Y195I和Y195R CGTase的最适温度为50°C，最适pH为6.5。突变型Y195I和Y195R还表现出更好的热稳定性。这些发现表明，195位的芳香族氨基酸Tyr或Phe对α-CGTase的淀粉分解活性和环化特异性很重要。突变型Y195I CGTase和Y195R CGTase未来在γ-CD生产方面具有潜在应用价值。
