Applied Microbiology and Biotechnology

, Volume 99, Issue 10, pp 4245–4253 | Cite as

Thermostability enhancement of an endo-1,4-β-galactanase from Talaromyces stipitatus by site-directed mutagenesis

  • Dorte M. Larsen
  • Christian Nyffenegger
  • Maria M. Swiniarska
  • Anders Thygesen
  • Mikael L. Strube
  • Anne S. Meyer
  • Jørn D. Mikkelsen
Biotechnologically relevant enzymes and proteins


Enzymatic conversion of pectinaceous biomasses such as potato and sugar beet pulp at high temperatures is advantageous as it gives rise to lower substrate viscosity, easier mixing, and increased substrate solubility and lowers the risk of contamination. Such high-temperature processing requires development of thermostable enzymes. Talaromyces stipitatus was found to secrete endo-1,4-β-galactanase when grown on sugar beet pectin as sole carbon source. The mature protein contained 353 AA and the MW was estimated to 36.5 kDa. It was subjected to codon optimization and produced in Pichia pastoris in 2 l scale yielding 5.3 g. The optimal reaction condition for the endo-1,4-β-galactanase was determined to be 46 °C at pH 4.5 at which the specific activity was estimated to be 6.93 μmol/min/mg enzyme with half-lives of 13 and 2 min at 55 and 60 °C, respectively. For enhancement of the half-life of TSGAL, nine single amino acid residues were selected for site-directed mutagenesis on the basis of semi-rational design. Of these nine mutants, G305A showed half-lives of 114 min at 55 °C and 15 min at 60 °C, respectively. This is 8.6-fold higher than that of the TSGAL at 55 °C, whereas the other mutants displayed moderate positive to negative changes in their half-lives.


Protein engineering Semi-rational design Multiple alignment GH53 Half-life 

Supplementary material

253_2014_6244_MOESM1_ESM.pdf (3.1 mb)
ESM 1(PDF 3157 kb)


  1. Blaber M, Zhang X, Matthews B (1993) Structural basis of amino acid alpha-helix propensity. Science 260:1637–1640CrossRefPubMedGoogle Scholar
  2. Bornscheuer UT, Huisman GW, Kazlauskas RJ, Lutz S, Moore JC, Robins K (2012) Engineering the third wave of biocatalysis. Nature 485:185–194CrossRefPubMedGoogle Scholar
  3. Boyce R, Chilana P, Rose TM (2009) iCODEHOP: a new interactive program for designing COnsensus-DEgenerate Hybrid Oligonucleotide Primers from multiply aligned protein sequences. Nucleic Acids Res 37:W222–W228CrossRefPubMedCentralPubMedGoogle Scholar
  4. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) The carbohydrate-active EnZymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res 37:D233–D238CrossRefPubMedCentralPubMedGoogle Scholar
  5. Cregg JM, Cereghino JL, Shi JY, Higgins DR (2000) Recombinant protein expression in Pichia pastoris. Mol Biotechnol 16:23–52CrossRefPubMedGoogle Scholar
  6. Daniel R, Dines M, Petach H (1996) The denaturation and degradation of stable enzymes at high temperatures. Biochem J 317:1–11PubMedCentralPubMedGoogle Scholar
  7. Davids T, Schmidt M, Boettcher D, Bornscheuer UT (2013) Strategies for the discovery and engineering of enzymes for biocatalysis. Curr Opin Chem Biol 17:215–220CrossRefPubMedGoogle Scholar
  8. De Maria L, Svendsen A, Borchert TV, Christensen LL, Larsen S, Ryttersgaard C (2013) Galactanase variants (Patent: US8367390 B2)Google Scholar
  9. Declerck N, Machius M, Wiegand G, Huber R, Gaillardin C (2000) Probing structural determinants specifying high thermostability in Bacillus licheniformis alpha-amylase. J Mol Biol 301:1041–1057CrossRefPubMedGoogle Scholar
  10. Dehouck Y, Grosfils A, Folch B, Gilis D, Bogaerts P, Rooman M (2009) Fast and accurate predictions of protein stability changes upon mutations using statistical potentials and neural networks: PoPMuSiC-2.0. Bioinformatics 25:2537–2543CrossRefPubMedGoogle Scholar
  11. Duret L, Gasteiger E, Perriere G (1996) LALNVIEW: a graphical viewer for pairwise sequence alignments. Comput Appl Biosci 12:507–510PubMedGoogle Scholar
  12. Eijsink VGH, Bjork A, Gaseidnes S, Sirevag R, Synstad B, van den Burg B, Vriend G (2004) Rational engineering of enzyme stability. J Biotechnol 113:105–120CrossRefPubMedGoogle Scholar
  13. Eijsink V, Gaseidnes S, Borchert T, van den Burg B (2005) Directed evolution of enzyme stability. Biomol Eng 22:21–30CrossRefPubMedGoogle Scholar
  14. Gasteiger E, Gattiker A, Hoogland C, Ivanyi I, Appel R, Bairoch A (2003) ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res 31:3784–3788CrossRefPubMedCentralPubMedGoogle Scholar
  15. Gilis D, Rooman M (2000) PoPMuSiC, an algorithm for predicting protein mutant stability changes. Application to prion proteins. Protein Eng 13:849–856CrossRefPubMedGoogle Scholar
  16. Goujon M, McWilliam H, Li W, Valentin F, Squizzato S, Paern J, Lopez R (2010) A new bioinformatics analysis tools framework at EMBL-EBI. Nucleic Acids Res 38:W695–W699CrossRefPubMedCentralPubMedGoogle Scholar
  17. Gruber K, Klintschar G, Hayn M, Schlacher A, Steiner W, Kratky C (1998) Thermophilic xylanase from Thermomyces lanuginosus: high-resolution X-ray structure and modeling studies. Biochemistry (N Y) 37:13475–13485CrossRefGoogle Scholar
  18. Guo H, Xiong J (2006) A specific and versatile genome walking technique. Gene 381:18–23CrossRefPubMedGoogle Scholar
  19. Holck J, Hjerno K, Lorentzen A, Vigsnaes LK, Hemmingsen L, Licht TR, Mikkelsen JD, Meyer AS (2011) Tailored enzymatic production of oligosaccharides from sugar beet pectin and evidence of differential effects of a single DP chain length difference on human faecal microbiota composition after in vitro fermentation. Process Biochem 46:1039–1049CrossRefGoogle Scholar
  20. Houbraken J, de Vries RP, Samson RA (2014) Modern taxonomy of biotechnologically important Aspergillus and Penicillium species. Adv Appl Microbiol 86:199–249CrossRefPubMedGoogle Scholar
  21. Kwasigroch JM, Gilis D, Dehouck Y, Rooman M (2002) PoPMuSiC, rationally designing point mutations in protein structures. Bioinformatics 18:1701–1702CrossRefPubMedGoogle Scholar
  22. Le Nours J, Ryttersgaard C, Lo Leggio L, Ostergaard P, Borchert T, Christensen L, Larsen S (2003) Structure of two fungal beta-1,4-galactanases: searching for the basis for temperature and pH optimum. Protein Sci 12:1195–1204CrossRefPubMedCentralPubMedGoogle Scholar
  23. Li D, Yang Y, Peng Y, Shen C (1998) Purification and characterization of extracellular glucoamylase from the thermophilic Thermomyces lanuginosus. Mycol Res 102:568–572CrossRefGoogle Scholar
  24. Lutz S (2010) Beyond directed evolution—semi-rational protein engineering and design. Curr Opin Biotechnol 21:734–743CrossRefPubMedCentralPubMedGoogle Scholar
  25. Lyu P, Wang P, Liff M, Kallenbach N (1991) Local effect of glycine substitution in a model helical peptide. J Am Chem Soc 113:3568–3572CrossRefGoogle Scholar
  26. Magrane M, UniProt Consortium (2011) UniProt Knowledgebase: a hub of integrated protein data. Database-the Journal of Biological Databases and CurationGoogle Scholar
  27. Margarit I, Campagnoli S, Frigerio F, Grandi G, Defilippis V, Fontana A (1992) Cumulative stabilizing effects of glycine to alanine substitutions in Bacillus subtilis neutral protease. Protein Eng 5:543–550CrossRefPubMedGoogle Scholar
  28. Matthews BW, Nicholson H, Becktel WJ (1987) Enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding. Proc Natl Acad Sci U S A 84:6663–6667CrossRefPubMedCentralPubMedGoogle Scholar
  29. Melgaard M, Svendsen I (1994) Different effects of N-glycosylation on the thermostability of highly homologous bacterial (1,3-1,4)-beta-glucanases secreted from yeast. Microbiology-Uk 140:159–166CrossRefGoogle Scholar
  30. Michalak M, Thomassen LV, Roytio H, Ouwehand AC, Meyer AS, Mikkelsen JD (2012) Expression and characterization of an endo-1,4-beta-galactanase from Emericella nidulans in Pichia pastoris for enzymatic design of potentially prebiotic oligosaccharides from potato galactans. Enzyme Microb Technol 50:121–129CrossRefPubMedGoogle Scholar
  31. Myers JK, Pace CN, Scholtz JM (1997) A direct comparison of helix propensity in proteins and peptides. Proc Natl Acad Sci U S A 94:2833–2837CrossRefPubMedCentralPubMedGoogle Scholar
  32. Naumoff DG (2011) Hierarchical classification of glycoside hydrolases. Biochemistry-Moscow 76:622–635CrossRefPubMedGoogle Scholar
  33. Novoradovsky A, Zhang V, Ghosh M, Hogrefe H, Sorge JA, Gaasterland T (2005) Computational Principles of Primer Design for Site Directed Mutagenesis. Technical Proceedings of the 2005 NSTI Nanotechnology Conference and Trade Show, Volume 1 : 532–533Google Scholar
  34. Otten H, Michalak M, Mikkelsen JD, Larsen S (2013) The binding of zinc ions to Emericella nidulans endo-beta-1,4-galactanase is essential for crystal formation. Acta Crystallographica Section F-Structural Biology and Crystallization Communications 69:850–854CrossRefGoogle Scholar
  35. Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785–786CrossRefPubMedGoogle Scholar
  36. R Core Team (2014) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL
  37. Rahimzadeh M, Khajeh K, Mirshahi M, Khayatian M, Schwarzenbacher R (2012) Probing the role of asparagine mutation in thermostability of Bacillus KR-8104 alpha-amylase. Int J Biol Macromol 50:1175–1182CrossRefPubMedGoogle Scholar
  38. Ravn HC, Meyer AS (2014) Chelating agents improve enzymatic solubilization of pectinaceous co-processing streams. Process Biochem 49:250–257CrossRefGoogle Scholar
  39. Ryttersgaard C, Lo Leggio L, Coutinho P, Henrissat B, Larsen S (2002) Aspergillus aculeatus beta-1,4-galactanase: substrate recognition and relations to other glycoside hydrolases in clan GH-A. Biochemistry (N Y) 41:15135–15143CrossRefGoogle Scholar
  40. Sakamoto T, Nishimura Y, Makino Y, Sunagawa Y, Harada N (2013) Biochemical characterization of a GH53 endo-beta-1,4-galactanase and a GH35 exo-beta-1,4-galactanase from Penicillium chrysogenum. Appl Microbiol Biotechnol 97:2895–2906CrossRefPubMedGoogle Scholar
  41. Schmid F (2011) Lessons about protein stability from in vitro selections. Chembiochem 12:1501–1507CrossRefPubMedGoogle Scholar
  42. Silva IR, Jers C, Otten H, Nyffenegger C, Larsen DM, Derkx PMF, Meyer AS, Mikkelsen JD, Larsen S (2014) Design of thermostable rhamnogalacturonan lyase mutants from Bacillus licheniformis by combination of targeted single point mutations. Appl Microbiol Biotechnol 98:4521–4531CrossRefPubMedGoogle Scholar
  43. Silva IR, Larsen DM, Jers C, Derkx P, Meyer AS, Mikkelsen JD (2013) Enhancing RGI lyase thermostability by targeted single point mutations. Appl Microbiol Biotechnol 97:9727–9735CrossRefPubMedGoogle Scholar
  44. Silva IR, Larsen DM, Meyer AS, Mikkelsen JD (2011) Identification, expression, and characterization of a novel bacterial RGI Lyase enzyme for the production of bio-functional fibers. Enzyme Microb Technol 49:160–166CrossRefPubMedGoogle Scholar
  45. Soeding J (2005) Protein homology detection by HMM-HMM comparison. Bioinformatics (Oxford) 21:951–960CrossRefGoogle Scholar
  46. Spek EJ, Olson CA, Shi ZS, Kallenbach NR (1999) Alanine is an intrinsic alpha-helix stabilizing amino acid. J Am Chem Soc 121:5571–5572CrossRefGoogle Scholar
  47. Stanke M, Keller O, Gunduz I, Hayes A, Waack S, Morgenstern B (2006) AUGUSTUS: ab initio prediction of alternative transcripts. Nucleic Acids Res 34:W435–W439CrossRefPubMedCentralPubMedGoogle Scholar
  48. Thomassen LV, Larsen DM, Mikkelsen JD, Meyer AS (2011a) Definition and characterization of enzymes for maximal biocatalytic solubilization of prebiotic polysaccharides from potato pulp. Enzyme Microb Technol 49:289–297CrossRefPubMedGoogle Scholar
  49. Thomassen LV, Vigsns LK, Licht TR, Mikkelsen JD, Meyer AS (2011b) Maximal release of highly bifidogenic soluble dietary fibers from industrial potato pulp by minimal enzymatic treatment. Appl Microbiol Biotechnol 90:873–884CrossRefPubMedGoogle Scholar
  50. Watanabe K, Masuda T, Ohashi H, Mihara H, Suzuki Y (1994) Multiple proline substitutions cumulatively thermostabilize bacillus-cereus Atcc7064 oligo-1,6-glucosidase. Irrefragable proof supporting the proline rule. Eur J Biochem 226:277–283CrossRefPubMedGoogle Scholar
  51. Yi Z-L, Pei X-Q, Wu Z-L (2011) Introduction of glycine and proline residues onto protein surface increases the thermostability of endoglucanase CelA from Clostridium thermocellum. Bioresour Technol 102:3636–3638CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Dorte M. Larsen
    • 1
  • Christian Nyffenegger
    • 1
  • Maria M. Swiniarska
    • 2
  • Anders Thygesen
    • 1
  • Mikael L. Strube
    • 1
  • Anne S. Meyer
    • 1
  • Jørn D. Mikkelsen
    • 1
  1. 1.Center for Bioprocess Engineering, Department of Chemical and Biochemical EngineeringTechnical University of DenmarkKongens LyngbyDenmark
  2. 2.Dako Denmark A/SGlostrupDenmark

Personalised recommendations