Expression, purification and immobilization of the intracellular invertase INVA, from Zymomonas mobilis on crystalline cellulose and Nylon-6

  • María de los Ángeles Calixto-Romo
  • José Alejandro Santiago-Hernández
  • Vanessa Vallejo-Becerra
  • Lorena Amaya-Delgado
  • María del Carmen Montes-Horcasitas
  • María Eugenia Hidalgo-Lara
Original Paper

Abstract

This paper presents two immobilization methods for the intracellular invertase (INVA), from Zymomonas mobilis. In the first method, a chimeric protein containing the invertase INVA, fused through its C-terminus to CBD Cex from Cellulomonas fimi was expressed in Escherichia coli strain BL21 (DE3). INVA was purified and immobilized on crystalline cellulose (Avicel) by means of affinity, in a single step. No changes were detected in optimal pH and temperature when INVA-CBD was immobilized on Avicel, where values of 5.5 and 30 °C, respectively, were registered. The kinetic parameters of the INVA-CBD fusion protein were determined in both its free form and when immobilized on Avicel. K m and V max were affected with immobilization, since both showed an increase of up to threefold. Additionally, we found that subsequent to immobilization, the INVA-CBD fusion protein was 39% more susceptible to substrate inhibition than INVA-CBD in its free form. The second method of immobilization was achieved by the expression of a 6xHis-tagged invertase purified on Ni-NTA resin, which was then immobilized on Nylon-6 by covalent binding. An optimal pH of 5.5 and a temperature of 30 °C were maintained, subsequent to immobilization on Nylon-6 as well as with immobilization on crystalline cellulose. The kinetic parameters relating to V max increased up to 5.7-fold, following immobilization, whereas K m increased up to 1.7-fold. The two methods were compared showing that when invertase was immobilized on Nylon-6, its activity was 1.9 times that when immobilized on cellulose for substrate concentrations ranging from 30 to 390 mM of sucrose.

Keywords

Enzyme immobilization Invertase Nylon-6 Sucrose Zymomonas mobilis 

Notes

Acknowledgments

This research was funded by Centro de Investigación y de Estudios Avanzados (CINVESTAV-IPN), México. MACR. gratefully acknowledges the scholarship from Consejo Nacional de Ciencia y Tecnología (Conacyt), México.

References

  1. 1.
    Ahmad S, Anwar A, Saleemuddin M (2001) Immobilization and stabilization of invertase on cajanus cajan lectin support. Bioresour Technol 79:121–127PubMedCrossRefGoogle Scholar
  2. 2.
    Alberto F, Bignon C, Sulzenbacher G, Henrissat B, Czjzek M (2004) The three-dimensional structure of invertase (β-fructosidase) from Thermotoga maritima reveals a bimodular arrangement and an evolutionary relationship between retaining and inverting glycosidases. J Biol Chem 279:18903–18910PubMedCrossRefGoogle Scholar
  3. 3.
    Amaya-Delgado L, Hidalgo-Lara ME, Montes-Horcasitas MC (2006) Hydrolysis of sucrose by invertase immobilized on nylon-6 microbeads. Food Chem 99:299–304CrossRefGoogle Scholar
  4. 4.
    Arslan F, Tümtürk H, Çaykara T, Sen M, Güven O (2000) The effect of gel composition on the adsorption of invertase on poly(acrylamide/maleic acid) hidrogels. Food Chem 70:33–38CrossRefGoogle Scholar
  5. 5.
    Bayramo IG, Akgöl S, Bulut A, Denizli A, Arica MY (2003) Covalent immobilization of invertase onto a reactive film composed of 2-hydroxyethyl mathacrylate and glycidyl methacrylate: properties and applications in a continuous flow system. Biochem Eng J 14:117–126CrossRefGoogle Scholar
  6. 6.
    Bray MR, Johnson PE, Gilkes NR, Kilburn LP, Warren RAJ (1996) Probing the role of tryptophan residues in a cellulose-binding domain by chemical modification. Protein Sci 5:2311–2318PubMedCrossRefGoogle Scholar
  7. 7.
    Kennedy JF, Phillips GO, Williams PA (1990) Cellulose sources and explotation industrial utilization, biotechnology, and physico-chemical properties. Ellis Horwood Limited Press, New YorkGoogle Scholar
  8. 8.
    Danisman T, Tan S, Kacar Y, Ergene A (2004) Covalent immobilization of invertase on microporous pHEMA-GMA membrane. Food Chem 85:461–466CrossRefGoogle Scholar
  9. 9.
    Erginer R, Toppare L, Alkan S, Bakir U (2000) Immobilization of invertase in functionalized copolymer matrices. React Funct Polym 45:227–233CrossRefGoogle Scholar
  10. 10.
    Gilkes NR, Kilburn DG, Miller RC, Warren RAJ (1993) Visualization of the adsortion of a bacterial endo-β-1, 4-glucanase and its isolated cellulose-binding domain to crystalline cellulose. Int J Biol Macromol 15:347–351PubMedCrossRefGoogle Scholar
  11. 11.
    Gunasekaran P, Karunakaran T, Cami B, Mukundan AG, Preziosi L, Baratti J (1990) Cloning and sequencing of the sacA gene: charaterization of a sucrase from Zymomonas mobilis. J Bacteriol 172:6727–6735PubMedGoogle Scholar
  12. 12.
    Henrissat B (1991) A classification of glycosyl hydrolases based on amino acids similarities sequences. Biochem J 280:309–316PubMedGoogle Scholar
  13. 13.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  14. 14.
    Lineweaver H, Burk D (1934) The determination of enzyme dissociation constants. J Am Chem Soc 56:658–666CrossRefGoogle Scholar
  15. 15.
    Lowry OH, Rosehbrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  16. 16.
    Mansfeld J, Forster M, Schellenberger A, Dautzenberg H (1991) Immobilization of invertase by encapsulation in polyelectrolyte complex. Enzyme Microbial Technol 13:240–244CrossRefGoogle Scholar
  17. 17.
    Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugars. Anal Chem 31:426–428CrossRefGoogle Scholar
  18. 18.
    Nakane K, Takashi O, Ogata N, Kurokawa Y (2000) Entrap-immobilization of invertase on composite gel fiber of cellulose acetate and zirconium alkoxides by sol-gel process. J Appl Polym Sci 81:2084–2088CrossRefGoogle Scholar
  19. 19.
    Ong E, Gilkes NR, Warren RAJ, Miller RC, Kilburn DG (1989) Enzyme immobilization using the cellulose-binding domain of a Cellulomonas fimi exoglucanase. Biotechnol 7:604–607CrossRefGoogle Scholar
  20. 20.
    Ong E, Greenwood JM, Gilkes NR, Kilburn DG, Miller RC, Warren RAJ (1989) The cellulose binding domains of cellulases: tools for biotechnology. Trends Biotechnol 7:239–243CrossRefGoogle Scholar
  21. 21.
    Salleh AB (1982) Activation of nylon by alkaline gluteraldehyde solution for enzyme immobilization. Biotechnol Letters 4:769–774CrossRefGoogle Scholar
  22. 22.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, vol 2. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  23. 23.
    Santiago-Hernández JA, Vásquez-Bahena JM, Calixto-Romo MA, Xoconostle-Cázares B, Ortega-López J, Ruíz-Medrano R, Montes-Horcasitas MC, Hidalgo-Lara ME (2006) Direct immobilization of a recombinant invertase to Avicel by E. coli overexpression of a fusion protein containing the extracellular invertase from Zymomonas mobilis and the carbohydrate-binding domain CBDCex from Cellulomonas fimi. Enzyme Microb Technol 40:172–176Google Scholar
  24. 24.
    Segura-Ceniceros EP, Dabek KR, Ilyiná AD (2006) Invertase immovilization on nylon-6 activated by hydrochloric acid in the presence of glutaraldehyde as cross-linker. Mosc Univ Chem Bull 47:143–148Google Scholar
  25. 25.
    Simionescu C, Popa M, Dumitru S (1987) Bioactive polymers XXX, immobilization of invertase of the diazonium salt of 4-aminobenzoylcellulose. Biotechnol Bioeng 28:198–203Google Scholar
  26. 26.
    Tümtürk H, Arslan F, Disli A, Tufan Y (2000) Immobilization of invertase attached to a granular dimer acid-co-alkyl polyamine. Food Chem 69:5–9CrossRefGoogle Scholar
  27. 27.
    Vallejo-Becerra V, Marín-Zamora ME, Vásquez-Bahena JM, Rojas-Melgarejo F, Hidalgo-Lara ME, García-Ruíz PA (2008) Immobilization of recombinant invertase (re-INVB) from Zymomonas mobilis on D-sorbitol cinnamic ester for production of invert sugar. J Agric Food Chem 56:1392–1397PubMedCrossRefGoogle Scholar
  28. 28.
    Vallejo-Becerra V, Vásquez-Bahena JM, Santiago-Hernández JA, Hidalgo-Lara ME (2008) Immobilization of the recombinant invertase INVB from Zymomonas mobilis on Nylon-6 (companion manuscript)Google Scholar
  29. 29.
    Vásquez-Bahena JM, Montes-Horcasitas MC, Ortega-López J, Magaña-Plaza I, Flores-Clotera LB (2004) Multiple steady states in a continuous stirred tank reactor: an experimental case study for hydrolysis of sucrose by invertase. Process Biochem 39:2179–2182CrossRefGoogle Scholar
  30. 30.
    Vásquez-Bahena JM, Vega-Estrada J, Santiago-Hernández JA, Ortega-López J, Flores-Cotera LB, Montes-Horcasitas MC, Hidalgo-Lara ME (2006) Expression and improved production of the soluble extracellular invertase from Zymomonas mobilis in Escherichia coli. Enzyme Microb Technol 40:172–176CrossRefGoogle Scholar
  31. 31.
    Vigants A, Zikmanis P, Bekers M (1996) Sucrose medium osmolarity as a regulator of anabolic and catabolic parameters in Zymomonas culture. Acta Biotechnol 16:321–7327CrossRefGoogle Scholar
  32. 32.
    Yanase H, Fukushi H, Ueda N, Maeda Y, Toyoda A, Tonomura K (1991) Cloning, sequencing, and characterization of the intracellular invertase gene from Zymomonas mobilis. Agric Biol Chem 55:1383–1390PubMedGoogle Scholar

Copyright information

© Society for Industrial Microbiology 2008

Authors and Affiliations

  • María de los Ángeles Calixto-Romo
    • 1
  • José Alejandro Santiago-Hernández
    • 1
  • Vanessa Vallejo-Becerra
    • 1
  • Lorena Amaya-Delgado
    • 1
  • María del Carmen Montes-Horcasitas
    • 1
  • María Eugenia Hidalgo-Lara
    • 1
  1. 1.Departamento de Biotecnología y BioingenieríaCINVESTAV-IPN. Av. Instituto PolitécnicoMexico D.F.Mexico

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