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Assembly of mutations for improving thermostability of Escherichia coli AppA2 phytase

  • Moon-Soo Kim
  • Jeremy D. Weaver
  • Xin Gen LeiEmail author
Biotechnologically Relevant Enzymes and Proteins

Abstract

We previously identified a number of mutations in Escherichia coli AppA2 phytase for enhancing its thermostability. The objective of the present study was to determine if these mutations (K46E, K65E, G103S, D112N, D144N, S209G, V227A, and G344D) could be sequentially added to further improve the thermostability of AppA2. Compared with the wild-type enzyme, two variants (D144N/V227A and D144N/V227A/G344D) out of the eight resulting mutants showed 15% enhancement in thermostability (as measured by residual activity after being heated at 80°C for 10 min) and 4 to 5°C increases in the melting temperatures (Tm). Based on the structural predictions with a highly homologous AppA phytase, the substitution D144N introduces a side-chain–side-chain hydrogen bond, thereby stabilizing the loop region (Gln137–Asn144), and the V227A substitution might eliminate structural hindrance between Val222 and Val227 that face each other in the β-hairpin structure. In addition, overall catalytic efficiency (kcat/Km) of the two mutants was also improved (P < 0.05) compared to the wild type. However, no further improvement in thermostability was observed by adding other mutations to D144N/V227A/G344D, which might result from unfavorable electrostatic interactions or structural perturbation. In conclusion, our results underscore the potential as well as difficulty of predicting synergistic effects of multiple mutations on thermostability within phytase.

Keywords

Enzyme Phytase Protein engineering Structure Thermostability 

Notes

Acknowledgment

This research was supported in part by a Cornell Biotechnology Program grant (to X.L.).

References

  1. Augspurger NR, Webel DM, Lei XG, Baker DH (2003) Efficacy of an E. coli phytase expressed in yeast for releasing phytate-bound phosphorus in young chicks and pigs. J Anim Sci 81:474–483CrossRefGoogle Scholar
  2. Chen KQ, Robinson AC, Van Dam ME, Martinez P, Economou C, Arnold FH (1991) Enzyme engineering for nonaqueous solvents. II. Additive effects of mutations on the stability and activity of subtilisin E in polar organic media. Biotechnol Prog 7:125–129CrossRefGoogle Scholar
  3. Dill KA (1997) Additivity principles in biochemistry. J Biol Chem 272:701–704CrossRefGoogle Scholar
  4. Green SM, Shortle D (1993) Patterns of nonadditivity between pairs of stability mutations in Staphylococcal nuclease. Biochemistry 32:10131–10139CrossRefGoogle Scholar
  5. Han Y, Lei XG (1999) Role of glycosylation in the functional expression of an Aspergillus niger phytase (phyA) in Pichia pastoris. Arch Biochem Biophys 364:83–90CrossRefGoogle Scholar
  6. Han YM, Wilson DB, Lei XG (1999) Expression of an Aspergillus niger phytase gene (phyA) in Saccharomyces cerevisiae. Appl Environ Microbiol 65:1915–1918CrossRefGoogle Scholar
  7. Hoseki J, Okamoto A, Takada N, Suenaga A, Futatsugi N, Konagaya A, Taiji M, Yano T, Kuramitsu S, Kagamiyama H (2003) Increased rigidity of domain structures enhances the stability of a mutant enzyme created by directed evolution. Biochemistry 42:14469–14475CrossRefGoogle Scholar
  8. Jermutus L, Tessier M, Pasamontes L, van Loon A, Lehmann M (2001) Structure-based chimeric enzymes as an alternative to directed enzyme evolution: phytase as a test case. J Biotechnol 85:15–24CrossRefGoogle Scholar
  9. Kim M-S, Lei XG (2008) Enhancing thermostability of Escherichia coli phytase AppA2 by error-prone PCR. Appl Microbiol Biotechnol 79(1):69–75CrossRefGoogle Scholar
  10. 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 69:4866–4874CrossRefGoogle Scholar
  11. Kim T, Mullaney EJ, Porres JM, Roneker KR, Crowe S, Rice S, Ko T, Ullah AHJ, Daly CB, Welch R, Lei XG (2006) Shifting the pH profile of Aspergillus niger PhyA phytase to match the stomach pH enhances its effectiveness as an animal feed additive. Appl Environ Microbiol 72:4397–4403CrossRefGoogle Scholar
  12. Kumar S, Tsai CJ, Nussinov R (2000) Factors enhancing protein thermostability. Protein Eng 13:179–191CrossRefGoogle Scholar
  13. Lee S, Kim T, Stahl CH, Lei XG (2005) Expression of Escherichia coli AppA2 phytase in four yeast systems. Biotechnol Lett 27:327–334CrossRefGoogle Scholar
  14. Lehmann M, Kostrewa D, Wyss M, Brugger R, D’Arcy A, Pasamontes L, van Loon A (2000a) From DNA sequence to improved functionality: using protein sequence comparisons to rapidly design a thermostable consensus phytase. Protein Eng 13:49–57CrossRefGoogle Scholar
  15. Lehmann M, Lopez-Ulibarri R, Loch C, Viarouge C, Wyss M, van Loon AP (2000b) Exchanging the active site between phytases for altering the functional properties of the enzyme. Protein Sci 9:1866–1872CrossRefGoogle Scholar
  16. LiCata VJ, Ackers GK (1995) Long-range, small magnitude nonadditivity of mutational effects in proteins. Biochemistry 34:3133–3139CrossRefGoogle Scholar
  17. Lim D, Golovan S, Forsberg CW, Jia Z (2000) Crystal structures of Escherichia coli phytase and its complex with phytate. Nat Struct Biol 7:108–113CrossRefGoogle Scholar
  18. Macedo-Ribeiro S, Martins BM, Pereira PJ, Buse G, Huber R, Soulimane T (2001) New insights into the thermostability of bacterial ferredoxins: high-resolution crystal structure of the seven-iron ferredoxin from Thermus thermophilus. J Biol Inorg Chem 6:663–674CrossRefGoogle Scholar
  19. Oue S, Okamoto A, Yano T, Kagamiyama H (1999) Redesigning the substrate specificity of an enzyme by cumulative effects of the mutations of non-active site residues. J Biol Chem 274:2344–2349CrossRefGoogle Scholar
  20. Ragone R (2001) Hydrogen-bonding classes in proteins and their contribution to the unfolding reaction. Protein Sci 10:2075–2082CrossRefGoogle Scholar
  21. Rodriguez E, Porres JM, Han Y, Lei XG (1999) Different sensitivity of recombinant Aspergillus niger phytase (r-PhyA) and Escherichia coli pH 2.5 acid phosphatase (r-AppA) to trypsin and pepsin in vitro. Arch Biochem Biophys 365:262–267CrossRefGoogle Scholar
  22. Rodriguez E, Wood ZA, Karplus PA, Lei XG (2000) Site-directed mutagenesis improves catalytic efficiency and thermostability of Escherichia coli pH 2.5 acid phosphatase/phytase expressed in Pichia pastoris. Arch Biochem Biophys 382:105–112CrossRefGoogle Scholar
  23. Schreiber G, Fersht AR (1995) Energetics of protein–protein interactions: analysis of the barnase–barstar interface by single mutations and double mutant cycles. J Mol Biol 248:478–486PubMedGoogle Scholar
  24. Shih P, Kirsch JF (1995) Design and structural analysis of an engineered thermostable chicken lysozyme. Protein Sci 4:2063–2072CrossRefGoogle Scholar
  25. Skinner MM, Terwilliger TC (1996) Potential use of additivity of mutational effects in simplifying protein engineering. Proc Natl Acad Sci USA 93:10753–10757CrossRefGoogle Scholar
  26. Spiller B, Gershenson A, Arnold FH, Stevens RC (1999) A structural view of evolutionary divergence. Proc Natl Acad Sci USA 96:12305–12310CrossRefGoogle Scholar
  27. Vieille C, Zeikus JG (1996) Thermozymes: identifying molecular determinants of protein structural and functional stability. Trends Biotechnol 14:183–190CrossRefGoogle Scholar
  28. Vieille C, Zeikus GJ (2001) Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol Mol Biol Rev 65:1–43CrossRefGoogle Scholar
  29. Voigt CA, Mayo SL, Arnold FH, Wang ZG (2001) Computational method to reduce the search space for directed protein evolution. Proc Natl Acad Sci USA 98:3778–3783CrossRefGoogle Scholar
  30. Wells JA (1990) Additivity of mutational effects in proteins. Biochemistry 29:8509–8517CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Moon-Soo Kim
    • 1
    • 2
  • Jeremy D. Weaver
    • 3
  • Xin Gen Lei
    • 1
    • 3
    Email author
  1. 1.Department of Animal Science and Graduate field of Food ScienceCornell UniversityIthacaUSA
  2. 2.Department of Medicinal Chemistry and Molecular PharmacologyPurdue UniversityWest LafayetteUSA
  3. 3.Department of Animal ScienceCornell UniversityIthacaUSA

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