Expression,purification and immobilization of the intracellular invertase INVA,from <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> on crystalline cellulose and Nylon-6 |
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Authors: | 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 |
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Institution: | (1) Departamento de Biotecnología y Bioingeniería, CINVESTAV-IPN. Av. Instituto Politécnico, Nacional 2508 Col. San Pedro Zacatenco, CP 07360 Mexico D.F., Mexico |
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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. |
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Keywords: | Enzyme immobilization Invertase Nylon-6 Sucrose Zymomonas mobilis |
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