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

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 CBDCex 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 degrees 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. Km and Vmax 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 degrees C were maintained, subsequent to immobilization on Nylon-6 as well as with immobilization on crystalline cellulose. The kinetic parameters relating to Vmax increased up to 5.7-fold, following immobilization, whereas Km 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.

Exo‐inulinase production from Aspergillus fumigatus NFCCI 2426: purification, characterization, and immobilization for continuous fructose production

“…The increase in K m after immobilization was probably due to the mass transfer resistance to the substrate into the polymer matrix, and effect of the increasing degree of polymerization of substrate caused increase in this value (Abdel-Naby et al, 1999;Curcio et al, 2014;Kusano et al, 1989;Mazutti et al, 2007). Higher V max observed for the immobilized enzyme was rather unusual; however, such increase has been observed in case of immobilized inulinase (Calixto-Romo et al, 2008;Mohamed et al, 2014;Risso et al, 2010). According to Ahmed et al (2019), increased reaction velocities are observed probably due to the configurational modifications on enzyme after interacting with the matrix and molecular flocking caused by immobilization.…”

Catalytical Properties of Free and Immobilized Aspergillus niger Tannase

“…Typically, immobilized enzymes have lower V max values than their free counterparts [37], and this phenomenon is associated with conformational changes of the enzyme during immobilization [18]. Higher V max values after immobilization have been reported for inulinase [38], invertase [39], and β -galactosidase [40]. However, this is an unusual behavior and the mechanistic basis for this positive feature requires further investigation [37].…”

Immobilization of lipase on the modified magnetic diatomite earth for effective methyl esterification of isoamyl alcohol to synthesize banana flavor

“…The FTIR spectra of DE, mDE, and mDE-APTES consist of the remarkable peaks for asymmetric stretching vibration of Si-O-Si (1090 cm -1 ), symmetric stretching vibration of Si-O (798 cm -1 ), and bending vibration of Si-O-Si (468 cm -1 ) (Fig. 1b) (de los Ángeles Calixto-Romo et al 2008;Janićijević et al 2015). The band of 3600 cm -1 is also due to stretching vibration frequency of SiO-H.…”