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A Paenibacillus sp. dextranase mutant pool with improved thermostability and activity

Abstract

Random mutagenesis was used to create a library of chimeric dextranase (dex1) genes. A plate-screening protocol was developed with improved thermostability as a selection criterion. The mutant library was screened for active dextranase variants by observing clearing zones on dextran-blue agar plates at 50°C after exposure to 68°C for 2 h, a temperature regime at which wild-type activity was abolished. A number of potentially improved variants were identified by this strategy, five of which were further characterised. DNA sequencing revealed ten nucleotide substitutions, ranging from one to four per variant. Thermal inactivation studies showed reduced (2.9-fold) thermostability for one variant and similar thermostability for a second variant, but confirmed improved thermostability for three mutants with 2.3- (28.9 min) to 6.9-fold (86.6 min) increases in half-lives at 62°C compared to that of the wild-type enzyme (12.6 min). Using a 10-min assay, apparent temperature optima of the variants were similar to that of the wild type (T opt 60°C). However, one of these variants had increased enzyme activity. Therefore, the first-generation dextranase mutant pool obtained in this study has sufficient molecular diversity for further improvements in both thermostability and activity through recombination (gene shuffling).

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References

  1. Aharoni A, Griffiths AD, Tawfik DS (2005) High-throughput screens and selections of enzyme-encoding genes. Curr Opin Chem Biol 9:210–216

  2. Akashi H (2001) Gene expression and molecular evolution. Curr Opin Genet Dev 11:660–666

  3. Bloom JD, Meyer MM, Meinhold P, Otey CR, MacMillan D, Arnold FH (2005) Evolving strategies for enzyme engineering. Curr Opin Struct Biol 15:447–452

  4. Cadwell RC, Joyce GF (1992) Randomization of genes by PCR mutagenesis. PCR Methods Appl 2:28–33

  5. Chen FM, Zhao YM, Wu H, Deng ZH, Wang QT, Zhou W, Liu Q, Dong GY, Li K, Wu ZF, Jin Y (2006) Enhancement of peridontal tissue regeneration by locally controlled delivery of insulin-like growth factor-I from dextran-co-gelatin microspheres. J Control Release 114:209–222

  6. Eggleston G, Monge A (2005) Optimization of sugarcane factory application of commercial dextranases. Process Biochem 40:1881–1894

  7. Eijsink VGH, Gåseidnes S, Borchert TV, Van den Burg B (2005) Directed evolution of enzyme stability. Biomol Eng 22:21–30

  8. Finnegan PM, Brumbley SM, O’Shea MG, Nevalainen KMH, Bergquist PL (2004a) Paenibacillus isolates possess diverse dextran-degrading enzymes. J Appl Microbiol 97:477–485

  9. Finnegan PM, Brumbley SM, O’Shea MG, Nevalainen H, Bergquist PL (2004b) Isolation and characterization of genes encoding thermoactive and thermostable dextranases from two thermotolerant soil bacteria. Curr Microbiol 49:327–333

  10. Finnegan PM, Brumbley SM, O’Shea MG, Nevalainen H, Bergquist PL (2005) Diverse dextranase genes from Paenibacillus species. Arch Microbiol 183:140–147

  11. Flores H, Ellington AD (2002) Increasing the thermal stability of an oligomeric protein, beta-glucuronidase. J Mol Biol 315:325–337

  12. Giver L, Gershenson A, Freskgard PO, Arnold FH (1998) Directed evolution of a thermostable esterase. Proc Natl Acad Sci U S A 95:12809–12813

  13. Hayacibara MF, Koo H, Vacca Smith AM, Kopec LK, Scott-Anne K, Cury JA, Bowen WH (2004) The influence of mutanase and dextranase on the production and structure of glucans synthesized by streptococcal glucosyltransferases. Carbohydr Res 339:2127–2137

  14. Hibbert EG, Dalby PA (2005) Directed evolution strategies for improved enzymatic performance. Microb Cell Fact 4:29. DOI https://doi.org/10.1186/1475-2859-4-29

  15. Hibbert EG, Baganz F, Hailes HC, Ward JM, Lye GJ, Woodley JM, Dalby PA (2005) Directed evolution of biocatalytic processes. Biomol Eng 22:11–19

  16. Höcker B (2005) Directed evolution of (βα)8-barrel enzymes. Biomol Eng 22:31–38

  17. Holmberg RC, Henry AA, Romesberg FE (2005) Directed evolution of novel polymerases. Biomol Eng 22:39–49

  18. Ingelman B (1948) Enzymatic beakdown of dextran. Acta Chem Scand 2:803–812

  19. Johannes TW, Zhao H (2006) Directed evolution of enzymes and biosynthetic pathways. Curr Opin Microbiol 9:261–267

  20. Khalikova E, Susi P, Korpela T (2005) Microbial dextran-hydrolyzing enzymes: fundamentals and applications. Microbiol Mol Biol Rev 69:306–325

  21. Kim YW, Choi JH, Kim JW, Park C, Kim JW, Cha H, Lee SB, Oh BH, Moon TW, Park KH (2003) Directed evolution of Thermus maltogenic amylase toward enhanced thermal resistance. Appl Environ Microbiol 69:4866–4874

  22. Kubic C, Sikora B, Bielecki S (2004) Immobilization of dextransucrase and its use with soluble dextranase for glucooligosaccharides synthesis. Enzyme Microb Technol 34:555–560

  23. Kumar S, Halpert JR (2005) Use of directed evolution of mammalian cytochromes P450 for investigating the molecular basis of enzyme function and generating novel biocatalysts. Biochem Biophys Res Commun 338:456–464

  24. Larsson AM, Andersson R, Ståhlberg J, Kenne L, Jones TA (2003) Dextranase from Penicillium minioluteum: reaction course, crystal structure, and product complex. Structure 11:1111–1121

  25. Lever M (1972) A new reaction for colorimetric determination of carbohydrates. Anal Biochem 47:273–279

  26. Marotta M, Martino A, De Rosa A, Farina E, Carteni M, De Rosa M (2002) Degradation of dental plaque glucans and prevention of glucan formation using commercial enzymes. Process Biochem 38:101–108

  27. Morisaki H, Igarashi T, Yamamoto A, Goto N (2002) Analysis of a dextran-binding domain of the dextranase of Streptococcus mutans. Lett Appl Microbiol 35:223–227

  28. Morley KL, Kazlauskas RJ (2005) Improving enzyme properties: when are closer mutations better? Trends Biotechnol 23:231–237

  29. Otten LG, Quax WJ (2005) Directed evolution: selecting today’s biocatalysts. Biomol Eng 22:1–9

  30. Schauder B, Blöcker H, Frank R, McCarthy JEG (1987) Inducible expression vectors incorporating the Escherichia coli atpE translational initiation region. Gene 52:279–283

  31. Schmidt-Dannert C, Arnold FH (1999) Directed evolution of enzymes. Trends Biotechnol 17:135–136

  32. Zaccolo M, Williams DM, Brown DM, Gherardi E (1996) An approach to random mutagenesis of DNA using mixtures of triphosphate derivatives of nucleoside analogues. J Mol Biol 255:589–603

  33. Zhou YH, Zhang X, Ebright RH (1991) Random mutagenesis of gene-sized DNA molecules by use of PCR with Taq DNA polymerase. Nucleic Acids Res 19:6052

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Acknowledgements

We thank Dr. Anwar Sunna and Prof. Roy Daniel for valuable discussions. This work was funded with the aid of a Linkage Grant from the Australian Research Council.

Author information

Correspondence to Peter L. Bergquist.

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Hild, E., Brumbley, S.M., O’Shea, M.G. et al. A Paenibacillus sp. dextranase mutant pool with improved thermostability and activity. Appl Microbiol Biotechnol 75, 1071–1078 (2007). https://doi.org/10.1007/s00253-007-0936-6

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Keywords

  • Dextranase
  • Thermostability
  • Activity
  • Directed evolution
  • Paenibacillus sp.
  • Random mutagenesis