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


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.


Enzyme immobilization Invertase Nylon-6 Sucrose Zymomonas mobilis 



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.


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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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