A major disadvantage of cyclodextrin production is the limited cyclodextrin product specificity of cyclodextrin glycosyltransferase (CGTase). Here, we described mutations of Asp372 and Tyr89 at subsite −3 in the CGTase from Paenibacillus macerans strain JFB05-01. The results showed that Asp372 and Tyr89 played important roles in cyclodextrin product specificity of CGTase. The replacement of Asp372 by lysine and Tyr89 by aspartic acid, asparagine, lysine, and arginine resulted in a shift in specificity towards the production of α-cyclodextrin, which was most apparent for the mutants D372K and Y89R. Furthermore, the changes in cyclodextrin product specificity for the single mutants D372K and Y89R could be combined in the double mutant D372K/Y89R, which displayed a 1.5-fold increase in the production of α-cyclodextrin, with a concomitant 43% decrease in the production of β-cyclodextrin when compared to the wild-type CGTase. Thus, the D372K and Y89R single and double mutants were much more suitable for the industrial production of α-cyclodextrin than the wild-type enzyme. The enhanced α-cyclodextrin specificity of these mutants might be a result of stabilizing the bent conformation of the intermediate in the cyclization reaction.
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This work was supported financially by the Program for Innovative Research Team of Jiangnan University, the Natural Science Foundation of Jiangsu Province (BK2007019), the Major State Basic Research Development Program of China (973 Program; 2007CB714036), the National Outstanding Youth Foundation of China (20625619), Research Program of the State Key Laboratory of Food Science and Technology (No. SKLF-MB-200802), the National High-tech Research and Development Program of China (863 Program; 2006AA10Z335), and the Graduate Student Creative Research Program of Jiangsu Province in 2008 (CX08B_127Z).
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Li, Z., Zhang, J., Wang, M. et al. Mutations at subsite −3 in cyclodextrin glycosyltransferase from Paenibacillus macerans enhancing α-cyclodextrin specificity. Appl Microbiol Biotechnol 83, 483–490 (2009). https://doi.org/10.1007/s00253-009-1865-3
- Cyclodextrin glycosyltransferase
- Product specificity
- Paenibacillus macerans
- Subsite −3