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Acid stabilization of Bacillus licheniformis alpha amylase through introduction of mutations

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Abstract

This paper provided further understanding of the relationships between acid resistance and structural features of different mutants in Bacillus licheniformis alpha amylase (BLA) due to the changes of two crucial positions Leu134 and Ser320. In order to investigate effect of the two positions on the acid stability, we described the detailed characterization of wild-type and the single mutants L134R and S320A as well as the double mutant L134R/S320A. The highest k cat/Km with pH 4.5, approximately 14 times that of wild type, was observed in L134R/S320A. The k cat/Km corresponding to L134R and S320A were at an intermediate values between those for wild type and L134R/S320A. In addition, compared with wild type, which had a rapid decline of the activity, L134R/S320A could maintain its activity strongly in low pH. Meanwhile, lower tolerance of L134R and S320A in acidic conditions than that of L134R/S320A was determined. Surprisingly, the acid-resistant capability of L134R/S320A was significantly enhanced by directed evolution. These results, combined with three-dimensional structure analysis, show that the electrostatic effects play a significant role in determining the stability of BLA at two crucial positions, 134 and 320.

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References

  1. Bernstein FC, Koetzle TF, Williams GJ, Meyer EF Jr, Brice MD, Rodgers JR, Kennard O, Shimanouchi T, Tasumi M (1977) The protein databank. A computer-based archival file for macromolecular structures. Eur J Biochem 80:319–324

  2. Bessler C, Schmitt J, Maurer KH, Schmid R (2003) Directed evolution of a bacterial α-amylase: toward enhanced pH-performance and higher specific activity. Protein Sci 12:2141–2149

  3. Cai H, Chen ZJ, Du LX, Lu FP (2005) Expression and secretion of an acid-stable α-amylase gene in Bacillus subtilis by sacB promoter and signal peptide. Biotechnol Lett 27:1731–1736

  4. Crabb WD, Shetty JK (1999) Commodity scale production of sugars from starches. Curr Opin Microbiol 2:252–256

  5. Davies GJ, Henrissat H (1995) Structures and mechanisms of glycosyl hydrolases. Structure 3:853–859

  6. Declerck N, Joyet P, Trosset JY, Garnier J, Gaillardin C (1995) Hyperthermostable mutants of Bacillus licheniformis α-amylase: multiple amino acid replacements and molecular modelling. Protein Eng 8:1029–1037

  7. Declerck N, Machius M, Chambert R, Wiegand G, Huber R, Gaillardin C (1997) Hyperthermostable mutants of Bacillus licheniformis α-amylase: thermodynamic studies and structural interpretation. Protein Eng 10:541–549

  8. Declerck N, Machius M, Wiegand G, Huber R, Gaillardin C (2000) Probing Structural determinants specifying high thermostability in Bacillus licheniformis α-amylase. J Mol Biol 301:1041–1057

  9. Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18:2714–2723

  10. Klein C, Hollender J, Bender H, Schulz GE (1992) Catalytic center of cyclodextrin glycosyltransferase derived from X-ray structure analysis combined with site-directed mutagenesis. Biochem 31:8740–8746

  11. Knegtel RM, Strokopytov B, Penninga D, Faber OG, Rozeboom HJ, Kalk KH, Dijkhuizen L, Dijkstra BW (1995) Crystallographic studies of the interaction of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 with natural substrates and products. J Biol Chem 270:29256–29264

  12. Kyte J (1995) Mechanism in protein chemistry. Garland, New York

  13. Lee S, Oneda H, Minoda M, Tanaka A, Inouye K (2006) Comparison of starch hydrolysis activity and thermal stability of two Bacillus licheniformis α-amylases and insights into engineering-amylase variants active under acidic conditions. J Biol Chem 139:997–1005

  14. Liu YH, Lu FP, Li Y, Yin XB, Wang Y, Gao C (2008) Characterisation of mutagenised acid-resistant alpha-amylase expressed in Bacillus subtilis WB600. Appl Microbiol Biotechnol 78:85–94

  15. Lowry OH, Rosbrough NL, Farr AL (1951) Protein measurement with folin phenol reagent. J Biol Chem 193:265–275

  16. MacGregor EA (1993) Relationships between structure and activity in the α-amylase family of starch-metabolising enzymes. Starch 7:232–237

  17. Machius M, Declerck N, Huber R, Wiegand G (2003) Kinetic stabilization of Bacillus licheniformis α-amylase through introduction of hydrophobic residues at the surface. J Biol Chem 278:11546–11553

  18. Matsumura M, Yasumura S, Aiba S (1986) Cumulative effect of intragenic amino-acid replacements on the thermostability of a protein. Nature 323:356–358

  19. Matsumura M, Signor G, Matthews BW (1989) Substantial increase of protein stability by multiple disulphide bonds. Nature 342:291–293

  20. Matthews BW (1993) Structural and genetic analysis of protein stability. Annu Rev Biochem 62:139–160

  21. McCarter JD, Withers SG (1994) Mechanisms of enzymatic glycoside hydrolysis. Curr Opin Struct Biol 4:885–892

  22. McCarter JD, Withers SG (1996) Unequivocal identification of Asp-214 as the catalytic nucleophile of Saccharomyces cerevisiae alpha glucosidase using 5-fluoroglycosyl fluorides. J Biol Chem 271:6889–6894

  23. Nielsen JE, Borchert TV (2000) Protein engineering of bacterial α-amylase. Biochim Biophys Acta 1543:253–274

  24. Nielsen JE, Beier L, Otzen D, Borchert TV, Frantzen HB, Andersen KV, Svendsen A (1999) Electrostatics in the active site of an α-amylase. Eur J Biochem 264:816–824

  25. Nielsen JE, Borchert TV, Vriend G (2001) The determinants of α-amylase pH-activity profiles. Protein Eng 14:505–512

  26. Pantoliano MW, Whitlow M, Wood JF, Dodd SW, Hardman KD, Rollence ML, Bryan PN (1989) Large increases in general stability for subtilisin BPN’ through incremental changes in the free energy of unfolding. Biochemistry 28:7205–7213

  27. Peitsch MC (1996) ProMod and Swiss-Model: internet-based tools for automated comparative protein modelling. Biochem Soc Trans 24:274–279

  28. Peitsch MC, Wells TN, Stampf DR, Sussman JL (1995) The Swiss-3DImage collection and PDB-browser on the world-wide web. Trends Biochem Sci 20:82–84

  29. Peitsch MC, Herzyk P, Wells TN, Hubbard RE (1996) Automated modelling of the transmembrane region of G-protein coupled receptor by Swiss-model. Receptors Channels 4:161–164

  30. Qian M, Haser R, Buisson G, Duee E, Payan F (1994) The active center of a mammalian α-amylase. Structure of the complex of a pancreatic α-amylase with a carbohydrate inhibitor refined to 2.2-Å resolution. Biochemistry 33:6284–6294

  31. Serrano L, Day AG, Fersht AR (1993) Step-wise mutation of barnase to binase. A procedure for engineering increased stability of proteins and an experimental analysis of the evolution of protein stability. J Mol Biol 233:305–312

  32. Shaw A, Bott R (1996) Engineering enzymes for stability. Curr Opin Biotechnol 6:546–550

  33. Shaw A, Bott R, Day AG (1999) Protein engineering of α-amylase for low pH performance. Curr Opin Biotechnol 10:349–352

  34. Sinnot ML (1990) Catalytic mechanisms of enzymic glycosyl transfer. Chem Rev 90:1171–1202

  35. Strokopytov B, Penninga D, Rozeboom HJ, Kalk KH, Dijkhuizen L, Dijkstra BW (1995) X-ray structure of cyclodextrin glycosyltransferase complexed with acarbose. Implications for the catalytic mechanism of glycosidases. Biochem 34:2234–2240

  36. Svensson B (1994) Protein engineering in the α-amylase family: catalytic mechanism, substrate specificity and stability. Plant Mol Biol 25:141–157

  37. Takagi T, Toda H, Isemura T (1971) Bacterial and mold amylases. In: Boyer PD (ed) The enzymes, 3rd edn. Academic, New York, pp 235–271

  38. Uitdehaag JC, Mosi R, Kalk KH, van der Veen BA, Dijkhuizen L, Withers SG, Dijkstra BW (1999) X-ray structures along the reaction pathway of cyclodextrin glycosyltransferase elucidate catalysis in the α-amylase family. Nat Struct Biol 6:432–436

  39. Vihinen M, Mantsala P (1989) Microbial amylolytic enzymes. Crit Rev Biochem Mol Biol 24:329–418

  40. Violet M, Meunier JC (1989) Kinetic study of the irreversible thermal denaturation of Bacillus licheniformis α-amylase. Biochem J 263:665–670

  41. Wind RD, Uitdehaag JC, Buitelaar RM, Dijkstra BW, Dijkhuizen L (1998) Engineering of cyclodextrin product specificity and pH optima of the thermostable cyclodextrin glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1. J Biol Chem 273:5771–5779

  42. Yasbin RE, Wilson GA, Young FE (1975) Transformation and transfection in lysogenic strains of Bacillus subtilis: evidence for selective induction of prophage. J Bacteriol 121:296–304

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Acknowledgments

The authors would like to thank Dr. Sui-Lam Wong for the supply of plasmid pWB980. Dr. Sui-Lam Wong is a senior medical scholar from University of Calgary. This work was supported by Key Program of Tianjin Science and Technology Development Plan (06YFGPSH03500).

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Correspondence to Fu-ping Lu.

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Liu, Y., Lu, F., Li, Y. et al. Acid stabilization of Bacillus licheniformis alpha amylase through introduction of mutations. Appl Microbiol Biotechnol 80, 795–803 (2008). https://doi.org/10.1007/s00253-008-1580-5

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Keywords

  • Bacillus licheniformis alpha amylase
  • Acid stability
  • Kinetics
  • Electrostatic field
  • Protein structure