Skip to main content
SpringerLink
Log in

Characterization of Recombinant Enzymes

  • Chapter
  • First Online:
Recombinant Enzymes - From Basic Science to Commercialization

  • 1359 Accesses

Abstract

Commercial enzymes can be obtained from natural sources or they can be produced recombinantly. A full enzyme characterization involved many methodologies but the spectrum of methods and the extent of characterization are related to the end use of the enzyme. Understanding the biochemical properties and mechanisms of enzymes are essential for research and development as well as for enzyme commercialization. These include among others the establishment of the optimum pH and stability of enzyme to different pH, the effects of temperature for the enzyme and the meaning of the apparent temperature optimum, the meaning of substrate specificity and enzyme specificity constant, the effects of small molecules including substrates, inhibitors and ions on enzyme activity, and last but not least the factors that are important for enzyme catalysis and enzyme mechanisms.

An erratum to this chapter can be found at http://dx.doi.org/10.1007/978-3-319-12397-4_13

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Denslow N, Wingfield PT, Rose K (1995) Overview of the characterization of recombinant proteins. Curr Protoc Protein Sci Unit 7.1.1–7.1.13

    Google Scholar 

  2. Lehninger AL (1982) Principles of biochemistry. Worth Publishers, USA

    Google Scholar 

  3. Allison RD (1996) Kinetic assay methods. Curr Protoc Protein Sci (Suppl. 5), Unit 3.5.1–3.5.11

    Google Scholar 

  4. Roberts SM, Davies GJ (2012) The crystallization and structural analysis of cellulases (and other glycoside hydrolases): strategies and tactics. Method Enzymol 510:141–168

    Article  CAS  Google Scholar 

  5. Johnston N, Dettmar PW, Bishwokarma B, Lively MO, Koufman JA (2007) Activity/stability of human pepsin: implications for reflux attributed laryngeal disease. Laryngoscope 117:1036–1039

    Article  PubMed  Google Scholar 

  6. Nielsen JK, McCammon, JA (2003) Calculating pK a values in enzyme active sites. Protein Sci 12:1894–1901

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  7. Mehler EL, Guarnieri F (1999) A self-consistent, microenvironment modulated screened coulomb potential approximation to calculate pH-dependent electrostatic effects in proteins. Biophys J 77:3–22

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  8. McIntosh, LP, Hand G, Johnson PE, Joshi MD, Korner M, Plesniak LA, Ziser L, Wakarchuk WW, Withers SG (1996) The pK a of the general acid/base carboxyl group of a glycosidase cycles during catalysis: a 13C-NMR study of Bacillus circulans xylanase. Biochemistry 35:9958–9966

    Article  CAS  PubMed  Google Scholar 

  9. Daniel RM, Danson MJ, Eisenthal R, Lee CK, Peterson ME (2008) The effect of temperature on enzyme activity: new insights and their implications. Extremophiles 12:51–59

    Article  CAS  PubMed  Google Scholar 

  10. Daniel RM, Danson MJ, Eisenthal R (2001) The temperature optima of enzymes: a new perspective on an old phenomenon. Trends Biochem Sci 26:223–225

    Article  CAS  PubMed  Google Scholar 

  11. Daniel RM, Danson MJ (2010) A new understanding of how temperature affects the catalytic activity of enzymes. Trends Biochem Sci 35:584–91

    Article  CAS  PubMed  Google Scholar 

  12. Hedstrom L (2001) Enzyme specificity and selectivity. Encyclopedia of life sciences. Nature Publishing Group, pp 1–7

    Google Scholar 

  13. Fersht AR (1985). Enzyme structure and mechanism, 2nd edn. WH Freeman, New York

    Google Scholar 

  14. Miller BG, Wolfenden R (2002) Catalytic proficiency: the unusual case of OMP decarboxylase. Annu Rev Biochem 71:847–885

    Article  CAS  PubMed  Google Scholar 

  15. Eisenthal R, Danson MJ, Hough DW (2007) Catalytic efficiency and k cat/K m: a useful comparator? Trends Biotechnol. 25:247–249

    Article  CAS  PubMed  Google Scholar 

  16. Aharoni A, Gaidukov L, Kheronsky O, McQ Gould S, Roodveldt C, Tawfik DS (2005) The ‘evolvability’ of promiscuous protein functions. Nat Genet 17:73–76

    Google Scholar 

  17. Tokuriki N, Tawfik DS (2009) Stability effects of mutations and protein evolvability. Curr Opin Struct Biol 19:596–604

    Article  CAS  PubMed  Google Scholar 

  18. Kheronsky O, Tawfik DS (2010) Enzyme promiscuity: a mechanistic and evolutionary perspective. Annu Rev Biochem. 79:471–505

    Article  Google Scholar 

  19. Medyantseva EP, Vertlib MG, Budnikov GK (1998) Metals ions as enzyme effectors. Russ Chem Rev 67:225–232

    Article  Google Scholar 

  20. Andreini C, Bertini I, Cavallaro G, Holliday GL, Thornton JM (2008) Metal ions in biological catalysis: from enzyme databases to general principles. J Biol Inorg Chem 13:1205–1218

    Article  CAS  PubMed  Google Scholar 

  21. Berg JM, Tymoczko JL, Stryer L (2002) Biochemistry, 5th edn. Freeman, New York

    Google Scholar 

  22. Shramm VL (2011) Enzymatic transition states, transition state analogs, dynamics, thermodynamics and lifetimes. Ann Rev Biochem 80:703–732

    Article  Google Scholar 

  23. Gluza K, Kafarski P (2013) Transition state analogues of enzymatic reactions as potential drugs. Intech, pp 325–372. http://dx.doi.org/10.5772/52504

    Google Scholar 

  24. Rempel BP, Withers SG (2008) Covalent inhibitors of glycosidases and their applications in biochemistry and biology. Glycobiology 18:570–586

    Article  CAS  PubMed  Google Scholar 

  25. Fersht A (1999) Structure and mechanism in protein science. Freeman, New York, pp 273–288

    Google Scholar 

  26. Vodovozova EL (2007) Photoaffinity labeling and its application in structural biology. Biochem Mosc 72:1–20

    Article  CAS  Google Scholar 

  27. Liessem B, Glombitza GJ, Knoll F, Lehmann J, Kellermann J, Lottspeich F, Sandhoff K. (1995) Photoaffinity-labeling of human lysosomal betahexosaminidase B: identification of Glu-355 at the substrate binding site. J Biol Chem. 270:23693–23699

    Article  CAS  PubMed  Google Scholar 

  28. Hou YM, Vocadlo DJ, Leung A, Withers SG, Mahuran D (2001) Characterization of the Glu and Asp residues in the active site of human beta-hexosaminidase B. Biochemistry 40:2201–2209

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  29. Fernandes MJG, Yew S, Leclerc D, Henrissat B, Vorgias CE, Gravel RA, Hechtman P, Kaplan F (1997) Identification of candidate active site residues in lysosomal beta-hexosaminidase A. J Biol Chem 272:814–820

    Article  CAS  PubMed  Google Scholar 

  30. Mark BL, Mahuran DJ, Cherney MM, Zhao DL, Knapp S, James MNG (2003) Crystal structure of human beta-hexosaminidase B: understanding the molecular basis of Sandhoff and Tay-Sachs disease. J Mol Biol 327:1093–1109

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  31. Lemieux MJ, Mark BL, Cherney MM, Withers SG, Mahuran DJ, James MNG (2006) Crystallographic structure of human beta-hexosaminidase A: interpretation of Tay-Sachs mutations and loss of G(M2) ganglioside hydrolysis. J Mol Biol 359:913–929

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Legler G (1990) Glycoside hydrolases: mechanistic information from studies with reversible and irreversible inhibitors. Adv Carbohydr Chem Biochem 48:319–384

    Article  CAS  PubMed  Google Scholar 

  33. Withers SG, Aebersold R (1995) Approaches to labeling and identification of active-site residues in glycosidases. Protein Sci 4:361–372

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  34. Radzicka A, Wolfenden R (1995) A proficient enzyme. Science 267:90–93

    Article  CAS  PubMed  Google Scholar 

  35. Gao J, Ma S, Major DT, Nam K, Pu J, Truhlar DG (2006) Mechanisms and free energies of enzymatic reactions. Chem Rev 106:3188–3209

    Article  CAS  PubMed  Google Scholar 

  36. Goodey NM, Benkovic SJ (2009) Understanding enzyme mechanism through protein chimeragenesis. In: Kohrer C, RajBhandary UL (eds) Protein engineering. Springer, Heidelberg, pp 1–28

    Google Scholar 

  37. Kraut DA, Carroll KS, Herschlag D (2003) Challenges in enzyme mechanism and energetics. Annu Rev Biochem 72:517–571

    Article  CAS  PubMed  Google Scholar 

  38. Walsh C (1979) Enzymatic reaction mechanisms. Freeman, San Francisco

    Google Scholar 

  39. CAZypedia. http://www.cazypedia.org/index.php/Glycoside_Hydrolases

  40. CAZy. http://www.cazy.org/Glycoside-Hydrolases.html

Download references

Author information

Authors and Affiliations

  1. Bioprocess & Molecular Engineering Research Unit, Department of Biotechnology Engineering, International Islamic University Malaysia, P.O. Box 10, 50728, Kuala Lumpur, Malaysia

    Farah Fadwa Ben Belgasem & Hamzah Mohd. Salleh

Authors
  1. Farah Fadwa Ben Belgasem
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hamzah Mohd. Salleh
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hamzah Mohd. Salleh .

Editor information

Editors and Affiliations

  1. Department of Biotechnology Engineering Kulliyyah of Engineering, International Islamic University Malaysia, Kuala Lumpur, Malaysia

    Azura Amid

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer International Publishing Switzerland

About this chapter

Cite this chapter

Belgasem, F., Salleh, H. (2015). Characterization of Recombinant Enzymes. In: Amid, A. (eds) Recombinant Enzymes - From Basic Science to Commercialization. Springer, Cham. https://doi.org/10.1007/978-3-319-12397-4_4

Download citation

Publish with us

Policies and ethics

Search

Navigation