Expression and improved production of the soluble extracellular invertase from Zymomonas mobilis in Escherichia coli

Vásquez-Bahena, J.M.; Vega-Estrada, Jesús; Santiago-Hernández, J.A.; Ortega‐López, Jaime; Flores-Cotera, Luis B.; Montes-Horcasitas, M.C.; Hidalgo-Lara, María Eugenia

doi:10.1016/j.enzmictec.2005.11.051

Cited by 20 publications

(20 citation statements)

References 19 publications

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“…The analysis of protein extracts from bacteria, harboring the construct invBpetK, showed a band of 51 kDa (Fig. 1, lines 7-10), which accords with the expected, theoretical, molecular weight of re-INVB [4].…”

Section: Expression Of the Invb Recombinant Proteinsupporting

confidence: 71%

“…The construction of the plasmid invbpetK has been described previously [4]. E. coli BL21(DE3) strain was provided by Invitrogene (US).…”

Section: Methodsmentioning

confidence: 99%

“…3b indicate the activity of immobilized and free enzyme at temperatures between 15 and 60°C. The optimal temperature for re-INVB immobilized on Nylon-6 was 25°C, while for the free enzyme it was 40°C [4]. A decrease in optimal temperature, upon enzyme immobilization has been reported for other enzymes.…”

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

confidence: 96%

“…Re-INVB was immobilized at pH 5.5, which is the optimum pH for the free enzyme [4]. To immobilize re-INVB, one gram of Nylon-6, previously activated as described above, was suspended in 5 ml of coupling buffer (50 mM sodium acetate, pH 5.5, 50 mM KCl, 10 mM MgCl 2 , 5 mM L-cysteine and 5% glycerol) containing 20 mg of purified re-INVB.…”

Section: Enzyme Immobilizationmentioning

confidence: 99%

“…The immobilization of enzymes in their active state may increase their potential application for industrial purposes [3]. Recombinant enzymes are active in their accurate-folded and soluble forms, but can be totally inactivated, if converted into insoluble aggregates [4]. Thus, for industrial purposes, it is paramount to find a method for immobilizing the enzyme invertase, in its active state [5].…”

Section: Introductionmentioning

confidence: 99%

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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: Expression Of the Invb Recombinant Proteinsupporting

confidence: 71%

“…The construction of the plasmid invbpetK has been described previously [4]. E. coli BL21(DE3) strain was provided by Invitrogene (US).…”

Section: Methodsmentioning

confidence: 99%

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

confidence: 96%

Section: Enzyme Immobilizationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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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Bacterial invertases: Occurrence, production, biochemical characterization, and significance of transfructosylation

Lincoln

2017

J. Basic Microbiol

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Invertase or β-D-fructofuranoside fructohydrolase (EC 3.2.1.26) was one of the foremost enzyme biocatalysts and established the primary concepts of most enzyme-kinetic principles. Invertases are glycoside hydrolases and occur mostly in microorganisms. Among microbial strains, for many decades yeast species have been extensively researched for invertase production, characterization, and applications in industries. Besides, limited literature is available on invertases from bacterial strains. The enzymic and molecular biological reports from bacterial invertases are scarce. In this minireview, occurrence, production, biochemical properties, and significance of transfructosylation of bacterial invertases are reported.

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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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Expression and improved production of the soluble extracellular invertase from Zymomonas mobilis in Escherichia coli

Cited by 20 publications

References 19 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

Bacterial invertases: Occurrence, production, biochemical characterization, and significance of transfructosylation

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

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