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