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

Santiago-Hernández, J.A.; Vásquez-Bahena, J.M.; Calixto-Romo, María de los Ángeles; Xoconostle-Cázares, G.B.; Ortega‐López, Jaime; Ruíz-Medrano, Roberto; Montes-Horcasitas, M.C.; Hidalgo-Lara, María Eugenia

doi:10.1016/j.enzmictec.2005.10.052

Cited by 19 publications

(23 citation statements)

References 28 publications

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“…The optimum pH for the hydrolysis of sucrose by immobilized re-INVB on Nylon-6 was pH 5.5; the same as that observed for the free enzyme. The optimum pH was shown to be similar to that reported for other immobilized invertases [5,16,17]. Other reports [15,[18][19][20] show optimal pH for immobilized invertases to be in the range of 3.0-6.1.…”

Section: Behavior Of Immobilized Re-invb On Nylon-6supporting

confidence: 75%

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Immobilization of the recombinant invertase INVB from Zymomonas mobilis on Nylon-6

Vallejo-Becerra

Vásquez-Bahena

Santiago-Hernández

et al. 2008

J Ind Microbiol Biotechnol

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The recombinant invertase INVB (re-INVB) from Zymomonas mobilis was immobilized on microbeads of Nylon-6, by means of covalent bonding. The enzyme was strongly and successfully bound to the support. The activity of the free and immobilized enzyme was determined, using 10% (w/v) sucrose, at a temperature ranging between 15 and 60 degrees C and a pH ranging between 3.5 and 7. The optimal pH and temperature for the immobilized enzyme were 5.5 and 25 degrees C, respectively. Immobilization of re-INVB on Nylon-6 showed no significant change in the optimal pH, but a difference in the optimal temperature was evident, as that for the free enzyme was shown to be 40 degrees C. The values for kinetic parameters were determined as: 984 and 98 mM for Kappm of immobilized and free re-INVB, respectively. Kappcat values for immobilized and free enzymes were 6.1x10(2) and 1.2x10(4) s(-1), respectively, and immobilized re-INVB showed Vappmax of 158.73 micromol h min(-1) mg(-1). Immobilization of re-INVB on Nylon-6 enhanced the thermostability of the enzyme by 50% at 30 degrees C and 70% at 40 degrees C, when compared to the free enzyme. The immobilization system reported here may have future biotechnological applications, owing to the simplicity of the immobilization technique, the strong binding of re-INVB to the support and the effective thermostability of the enzyme.

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Section: Behavior Of Immobilized Re-invb On Nylon-6supporting

confidence: 75%

“…2). These results indicate that immobilization on Nylon-6 happens more quickly than on other supports [15], and is similar to that for the fusion protein INVB-CBD cex , when immobilized on crystalline cellulose [16].…”

Section: Expression Of the Invb Recombinant Proteinsupporting

confidence: 65%

Immobilization of the recombinant invertase INVB from Zymomonas mobilis on Nylon-6

Vallejo-Becerra

Vásquez-Bahena

Santiago-Hernández

et al. 2008

J Ind Microbiol Biotechnol

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“…INVA-CBD was puriWed and immobilized on Avicel as described [23]. Immobilization was conducted by gently mixing 5 g of Avicel with 8.5 mL of bacterial lysate [E. coli BL21(DE3)/invA-pET38b(+) cells] (20 mg of protein/mL) at 4 °C for 18 h on an orbitron Bockel ScientiWc rotor.…”

Section: Puriwcation and Immobilization Of Inva-cbd On Avicelmentioning

confidence: 99%

“…Unfortunately, the Avicelimmobilized INVB-CBD lost about 60% of its catalytic activity compared to the free fusion protein, probably due to unspeciWc interaction of INVB-CBD with the support, although the immobilized enzyme was more thermo stable than free INVB-CBD and showed an improvement in the aYnity for the substrate [23]. Also, we have reported the successful immobilization of the extracellular invertase from S. cerevisiae [3] and the extracellular recombinant invertase INVB from Z. mobilis on Nylon-6 [27].…”

Section: Introductionmentioning

confidence: 95%

“…We have previously reported the puriWcation and immobilization of Z. mobilis INVB-CBD fusion protein to crystalline cellulose (Avicel) [23]. Unfortunately, the Avicelimmobilized INVB-CBD lost about 60% of its catalytic activity compared to the free fusion protein, probably due to unspeciWc interaction of INVB-CBD with the support, although the immobilized enzyme was more thermo stable than free INVB-CBD and showed an improvement in the aYnity for the substrate [23].…”

Section: Introductionmentioning

confidence: 96%

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

Calixto-Romo¹,

Santiago-Hernández²,

Vallejo-Becerra³

et al. 2008

J Ind Microbiol Biotechnol

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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.

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Design and characterization of a chimeric alginate lyase for immobilization to produce well‐defined oligosaccharides

Zhang

Lyu

Liu

et al. 2022

Food Bioengineering

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Recently, a novel two-domain alginate lyase (AlyAL01) was cloned from Algibacter sp. and successfully overexpressed in Escherichia coli BL21(DE3). Biochemical analysis showed that the N-terminal carbohydrate-binding module (CBM) of AlyAL01 had no effect on the enzyme activity and product distributions. Therefore, the N-terminal CBM was substituted with CBM3 to confer the designed chimeric protein with the ability to specifically bind to regenerated amorphous cellulose. As anticipated, the designed chimeric protein (CBM3-L1) exhibited a similar enzyme activity. Moreover, it was found that the CBM3-L1 combined the purification and immobilization in one step with high immobilized efficiency of 65.8%. The immobilized enzyme exhibited good storage stability and moderate reusability. The immobilized enzyme could keep 85% activity when incubated at 4°C for 60 days and 70% activity when incubated at 25°C for 30 days. Furthermore, the immobilized CBM3-L1 kept about 50% of its initial activity after being reused five times. Finally, immobilized CBM3-L1 successively produced well-defined alginate oligosaccharides (AOS) with DPs of 2-6 by controlling reaction time. In sum, our current study provided a feasible strategy for well-defined AOS production.K E Y W O R D S alginate lyase, alginate oligosaccharides, cellulose-binding domain, immobilized enzyme

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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

Cited by 19 publications

References 28 publications

Immobilization of the recombinant invertase INVB from Zymomonas mobilis on Nylon-6

Immobilization of the recombinant invertase INVB from Zymomonas mobilis on Nylon-6

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

Design and characterization of a chimeric alginate lyase for immobilization to produce well‐defined oligosaccharides

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