Improved production of cyclodextrin glycosyltransferase from Bacillus stearothermophilus NO2 in Escherichia coli via directed evolution

  • Xiumei Tao
  • Lingqia Su
  • Lei Wang
  • Xixi Chen
  • Jing WuEmail author
Biotechnologically relevant enzymes and proteins


Cyclodextrin glycosyltransferases (CGTases) are widely used in starch deep processing, so reducing their cost by improving their production is of significant industrial interest. The CGTase from Bacillus stearothermophilus NO2 possesses excellent catalytic properties but suffers from low production in E. coli. In this study, directed evolution was used to create three point mutants (I631T, I641T and K647E) that were produced in E. coli with shake-flask yields 1.7-, 2.1-, and 2.2-fold higher than that of wild-type, respectively. The wild-type and K647E were then produced in a 3-L fermenter. The CGTase activity of the K647E (1904 U mL-1) was 2.0-fold higher than that of the wild-type. The K647E fermentation supernatant could be used directly to prepare 2-O-α-d-glucopyranosyl-l-ascorbic acid, reducing the costs associated with its production. Structural modeling of the three mutants suggested that hydrophilicity, hydrogen bonding, and negative charge may be responsible for their improved production. Since K647 is conserved in the CGTase family, the corresponding residues in the CGTases from Bacillus circulans 251, Paenibacillus macerans, and Anaerobranca gottschalkii were changed to glutamic acid. Productions of the resulting K647E mutants were 2.0-, 1.5-, and 1.0-fold higher than those of their respective wild-types. Electrostatic protein surface analysis suggested that mutations occurring at low negative surface charge may increase CGTase production.


Cyclodextrin glycosyltransferase High-efficient production Directed evolution Negative charge High-cell-density fermentation CGTase family 


Funding information

This study was financially supported by the National Natural Science Foundation of China (31730067, 31771916), the Science and Technology Project of Jiangsu Province - Modern Agriculture (BE2018305), the Natural Science Foundation of Jiangsu Province (BK20180082), National first-class discipline program of Light Industry Technology and Engineering (LITE2018-03), and the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX19_1832).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interests.

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2019_10249_MOESM1_ESM.pdf (182 kb)
ESM 1 (PDF 182 kb)


  1. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Carvajal-Vergara X, Sevilla A, D'Souza SL, Ang Y-S, Schaniel C, Lee D-F, Yang L, Kaplan AD, Adler ED, Rozov R, Ge Y, Cohen N, Edelmann LJ, Chang B, Waghray A, Su J, Pardo S, Lichtenbelt KD, Tartaglia M, Gelb BD, Lemischka IR (2010) Patient-specific induced pluripotent stem-cell-derived models of LEOPARD syndrome. Nature 465(7299):808–812. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Cheng J, Wu D, Chen S, Chen J, Wu J (2011) High-level extracellular production of alpha-cyclodextrin glycosyltransferase with recombinant Escherichia coli BL21 (DE3). J Agric Food Chem 59(8):3797–3802. CrossRefPubMedGoogle Scholar
  4. Chiti F, Stefani M, Taddei N, Ramponi G, Dobson CM (2003) Rationalization of the effects of mutations on peptide and protein aggregation rates. Nature 424(6950):805–808. CrossRefPubMedGoogle Scholar
  5. Deng C, Li J, Shin H-D, Du G, Chen J, Liu L (2018) Efficient expression of cyclodextrin glycosyltransferase from Geobacillus stearothermophilus in Escherichia coli by promoter engineering and downstream box evolution. J Biotechnol 266:77–83. CrossRefPubMedGoogle Scholar
  6. Deng C, Lv X, Li J, Liu Y, Du G, Amaro RL, Liu L (2019) Synthetic repetitive extragenic palindromic (REP) sequence as an efficient mRNA stabilizer for protein production and metabolic engineering in prokaryotic cells. Biotechnol Bioeng 116(1):5–18. CrossRefPubMedGoogle Scholar
  7. Fernandez-Escamilla AM, Rousseau F, Schymkowitz J, Serrano L (2004) Prediction of sequence-dependent and mutational effects on the aggregation of peptides and proteins. Nat Biotechnol 22(10):1302–1306. CrossRefPubMedGoogle Scholar
  8. Guo Y, Chen J, Jia X, Lin X (2016) Effects of chaperone co-expression on heterologous solubility expression of thermophilic cyclodextrin glucosetransferase. Microbiology China 43(3):518-526. doi:10.13344/j.microbiol.china.150453Google Scholar
  9. Han R, Li J, H-d S, Chen RR, Du G, Liu L, Chen J (2014) Recent advances in discovery, heterologous expression, and molecular engineering of cyclodextrin glycosyltransferase for versatile applications. Biotechnol Adv 32(2):415–428. CrossRefPubMedGoogle Scholar
  10. Jiang S, Li C, Zhang W, Cai Y, Yang Y, Yang S, Jiang W (2007) Directed evolution and structural analysis of N-carbamoyl-D-amino acid amidohydrolase provide insights into recombinant protein solubility in Escherichia coli. Biochem J 402:429–437. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Kramer RM, Shende VR, Motl N, Pace CN, Scholtz JM (2012) Toward a molecular understanding of protein solubility: increased negative surface charge correlates with increased solubility. Biophys J 102(8):1907–1915. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157(1):105–132. CrossRefPubMedGoogle Scholar
  13. Leemhuis H, Kelly RM, Dijkhuizen L (2010) Engineering of cyclodextrin glucanotransferases and the impact for biotechnological applications. Appl Microbiol Biotechnol 85(4):823–835. CrossRefPubMedGoogle Scholar
  14. Li P, Guan H, Li J, Lin ZL (2009) Heterologous expression, purification, and characterization of cytochrome P450sca-2 and mutants with improved solubility in Escherichia coli. Protein Expr Purif 65(2):196–203. CrossRefPubMedGoogle Scholar
  15. Li Y, Liu J, Wang Y, Liu B, Xie X, Jia R, Li C, Li Z (2017) A two-stage temperature control strategy enhances extracellular secretion of recombinant alpha-cyclodextrin glucosyltransferase in Escherichia coli. AMB Express 7(1):165. CrossRefPubMedPubMedCentralGoogle Scholar
  16. Li Z, Li B, Gu Z, Du G, Wu J, Chen J (2010) Extracellular expression and biochemical characterization of alpha-cyclodextrin glycosyltransferase from Paenibacillus macerans. Carbohydr Res 345(7):886–892. CrossRefPubMedGoogle Scholar
  17. Lombard V, Ramulu HG, Drula E, Coutinho PM, Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 42(D1):D490–D495. CrossRefPubMedGoogle Scholar
  18. McLoughlin SY, Jackson C, Liu JW, Ollis D (2005) Increased expression of a bacterial phosphotriesterase in Escherichia coli through directed evolution. Protein Expr Purif 41(2):433–440. CrossRefPubMedGoogle Scholar
  19. Melzer S, Sonnendecker C, Follner C, Zimmermann W (2015) Stepwise error-prone PCR and DNA shuffling changed the pH activity range and product specificity of the cyclodextrin glucanotransferase from an alkaliphilic Bacillus sp. Febs Open Bio 5:528–534. CrossRefPubMedPubMedCentralGoogle Scholar
  20. Mo H-M, Xu Y, Yu X-W (2018) Improved soluble expression and catalytic activity of a thermostable esterase using a high-throughput screening system based on a split-GFP assembly. J Agric Food Chem 66(48):12756–12764. CrossRefPubMedGoogle Scholar
  21. Mosavi LK, Peng ZY (2003) Structure-based substitutions for increased solubility of a designed protein. Protein Eng 16(10):739–745. CrossRefPubMedGoogle Scholar
  22. Overton TW (2014) Recombinant protein production in bacterial hosts. Drug Discov Today 19(5):590–601. CrossRefPubMedGoogle Scholar
  23. Qi QS, Zimmermann W (2005) Cyclodextrin glucanotransferase: from gene to applications. Appl Microbiol Biotechnol 66(5):475–485. CrossRefPubMedGoogle Scholar
  24. Reetz MT, Zheng HB (2011) Manipulating the expression rate and enantioselectivity of an epoxide hydrolase by using directed evolution. ChemBioChem 12(10):1529–1535. CrossRefPubMedGoogle Scholar
  25. Robert X, Gouet P (2014) Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res 42(W1):W320–W324. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Song B, Yue Y, Xie T, Qian S, Chao Y (2014) Mutation of Tyrosine167Histidine at Remote Substrate Binding Subsite-6 in alpha-Cyclodextrin Glycosyltransferase Enhancing alpha-Cyclodextrin Specificity by Directed Evolution. Mol Biotechnol 56(3):232–239. CrossRefPubMedGoogle Scholar
  27. Sonnendecker C, Wei R, Kurze E, Wang J, Oeser T, Zimmermann W (2017) Efficient extracellular recombinant production and purification of a Bacillus cyclodextrin glucanotransferase in Escherichia coli. Microb Cell Factories 16(1):87. CrossRefGoogle Scholar
  28. Sonnendecker C, Zimmermann W (2019a) Change of the product specificity of a cyclodextrin glucanotransferase by semi-rational mutagenesis to synthesize large-ring cyclodextrins. Catalysts 9(3):242. CrossRefGoogle Scholar
  29. Sonnendecker C, Zimmermann W (2019b) Domain shuffling of cyclodextrin glucanotransferases for tailored product specificity and thermal stability. Febs Open Bio 9(2):384–395. CrossRefPubMedPubMedCentralGoogle Scholar
  30. Su L, Ma Y, Wu J (2015) Extracellular expression of natural cytosolic arginine deiminase from Pseudomonas putida and its application in the production of L-citrulline. Bioresour Technol 196:176–183. CrossRefPubMedGoogle Scholar
  31. Sun LH, Petrounia IP, Yagasaki M, Bandara G, Arnold FH (2001) Expression and stabilization of galactose oxidase in Escherichia coli by directed evolution. Protein Eng 14(9):699–704. CrossRefPubMedGoogle Scholar
  32. Suominen I, Karp M, Lähde M, Kopio A, Glumoff T, Meyer P, Mäntsälä P (1987) Extracellular production of cloned α-amylase by Escherichia coli. Gene 61(2):165–176. CrossRefPubMedGoogle Scholar
  33. Tao X, Wang T, Su L, Wu J (2018) Enhanced 2-O-alpha-d-glucopyranosyl-l-ascorbic acid synthesis through iterative saturation mutagenesis of acceptor subsite residues in Bacillus stearothermophilus NO2 cyclodextrin glycosyltransferase. J Agric Food Chem 66(34):9052–9060. CrossRefPubMedGoogle Scholar
  34. Tesfai BT, Wu D, Chen S, Chen J, Wu J (2013) Effect of organic solvents on the yield and specificity of cyclodextrins by recombinant cyclodextrin glucanotransferase (CGTase) from Anaerobranca gottschalkii. J Incl Phenom Macr 77(1-4):147–153. CrossRefGoogle Scholar
  35. van der Veen BA, Leemhuis H, Kralj S, Uitdehaag JCM, Dijkstra BW, Dijkhuizen L (2001) Hydrophobic amino acid residues in the acceptor binding site are main determinants for reaction mechanism and specificity of cyclodextrin-glycosyltransferase. J Biol Chem 276(48):44557–44562. CrossRefPubMedGoogle Scholar
  36. Wang L, Chen S, Wu J (2018) Cyclodextrin enhanced the soluble expression of Bacillus clarkii γ-CGTase in Escherichia coli. BMC Biotechnol 18(1):72. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Willemot K, Cornelis P (1983) Growth defects of Escherichia coli cells which contain the gene of an α-amylase from Bacillus coagulans on a multicopy plasmid. Microbiology 129(2):311–319. CrossRefGoogle Scholar
  38. Yang Y, Wang L, Chen S, Wu J (2014) Optimization of beta-cyclodextrin production by recombinant beta-cyclodextrin glycosyltransferase. Biotechnol Bull(8):175-181.
  39. Zhang YB, Howitt J, McCorkle S, Lawrence P, Springer K, Freimuth P (2004) Protein aggregation during overexpression limited by peptide extensions with large net negative charge. Protein Expr Purif 36(2):207–216. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Xiumei Tao
    • 1
    • 2
    • 3
  • Lingqia Su
    • 1
    • 2
    • 3
  • Lei Wang
    • 1
    • 2
    • 3
  • Xixi Chen
    • 2
  • Jing Wu
    • 1
    • 2
    • 3
    Email author
  1. 1.State Key Laboratory of Food Science and TechnologyJiangnan UniversityWuxiChina
  2. 2.School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of EducationJiangnan UniversityWuxiChina
  3. 3.International Joint Laboratory on Food SafetyJiangnan UniversityWuxiChina

Personalised recommendations