Stabilization of multimeric enzymes via immobilization and post-immobilization techniques

Fernández‐Lafuente, Roberto; Rodríguez, Verónica; Mateo, César; Penzol, Guadalupe; Hernández-Jústiz, Odette; Irazoqui, Gabriela; Villarino, Andréa; Ovsejevi, Karen; Batista, Francisco; Guisán, José M.

doi:10.1016/s1381-1177(99)00028-4

Cited by 122 publications

(97 citation statements)

References 11 publications

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“…The free enzyme, a dimeric protein, exhibited a characteristic decrease in the enzyme stability when decreasing the enzyme concentration, suggesting that the dissociation of the subunits may be the first step in its inactivation (Fernandez-Lafuente et al, 1999). This enzyme seemed to be adsorbed on anionic exchanger matrices via both subunits, resulting in an enzyme preparation with much higher stability than the one of the soluble enzyme and with its stability fully independent from the protein concentration (Fig.…”

Section: Immobilization Of Some Industrial Enzymes On the Optimized Pmentioning

confidence: 98%

Reversible enzyme immobilization via a very strong and nondistorting ionic adsorption on support-polyethylenimine composites

Mateo

Abián

Fernández‐Lafuente

et al. 2000

Biotechnol. Bioeng.

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241

155

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Section: Immobilization Of Some Industrial Enzymes On the Optimized Pmentioning

confidence: 98%

Reversible enzyme immobilization via a very strong and nondistorting ionic adsorption on support-polyethylenimine composites

Mateo

Abián

Fernández‐Lafuente

et al. 2000

Biotechnol. Bioeng.

Self Cite

241

155

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“…The monomeric nature of our enzyme excludes the possi- bility of multimeric stabilization. 30 Fern´andez-Lafuente et al [7][8][9][10] studied different mechanisms of protein immobilization on GAactivated supports. They studied the effect of support activation, immobilization conditions, and techniques on the stability of immobilized enzymes.…”

Section: Effect Of Ph Of the Reaction Mediummentioning

confidence: 99%

“…These strategies included formation of multipoint immobilization, multimeric units' enzymes immobilization, oriented immobilization, and finally, generation of more convenient microenvironment. [7][8][9][10] Recently, nanomaterials have been presented as a novel paradigm for enzyme immobilization and stabilization. 11 Moreover, it is believed that the possible disadvantages, such as low retained activity as a consequence of the immobilization procedure, are largely compensated by the possibility of the reuse of the enzyme.…”

mentioning

confidence: 99%

Covalent Immobilization of β‐Galactosidase onto Amino‐Functionalized Polyvinyl Chloride Microspheres: Enzyme Immobilization and Characterization

et al. 2013

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ABSTRACT:In this study, β-galactosidase (Aspergillus oryzae) was covalently immobilized onto aminated polyvinyl chloride (PVC) via glutaraldehyde coupling. Effect of immobilization conditions was investigated. The maximum obtained immobilization yield is 27 units g −1 while the maximum expressed activity is 12 μmol minThe optimum pH of the free enzyme was observed at 5.2, whereas the optimum pH of the immobilized one was schiffed to 4.4. The optimum temperature for both the free and immobilized forms was recognized at 60• C. A change in the temperature profile of the immobilized enzyme below 60• C was observed when response to the elevation temperature was less than that to the free counterpart. The K m and V max values of the free and immobilized β-galactosidase were found to be 52.03 and 123.1 mM, and 6.12 and 9.98 μ mol/min, respectively. Lactose hydrolysis, 500 mL of 100 mM concentration, was studied using 400 units of either free or immobilized enzymes at pH 6.0 and 60• C. Almost 80% hydrolysis percentage has been obtained after 6 h hydrolysis time with immobilized enzyme compared with 63% hydrolysis percentage obtained after 2 h for the free counterpart. Investigation of the operational stability at 40• C shows that the immobilized enzyme kept its activity for 5 h over 10 successive lactose hydrolysis cycles, which provides potentials for practical application in milk and whey hydrolysis. C

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“…This treatment releases any protein molecule that is not covalently bound to support [35,42]. The knowledge about the stability of the enzyme-support complex gives us an idea about whether or not postimmobilization stabilization is needed.…”

Section: Covalent Binding Analysis Of L-n-carbamoylase On Eupergit R Cmentioning

confidence: 99%

Enhanced stability of newly isolated trimeric l‐methionine‐N‐carbamoylase from Brevibacillus reuszeri HSN1 by covalent immobilization

Nandanwar

Vohra

Hoondal

2013

Biotech and App Biochem

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Newly isolated and partially purified trimeric l-methionine-N-carbamoylase from Brevibacillus reuszeri HSN1 was immobilized by covalent coupling to a well-known support material, Eupergit® C. Approximately 80% enzyme activity yield was achieved with ≈61% binding of a soluble protein from a solution containing 5 mg/mL protein. The immobilized preparation was found to be quite unstable due to a poor multisubunit covalent interaction of trimeric enzyme. Additional cross-linking with polyaldehyde-dextran was done to sustain the biotechnological application of immobilized enzyme. The temperature and pH optima of immobilized enzyme were increased by 10°C and 0.5 unit, respectively. The enzyme was significantly stabilized and retained ≈93% enzyme activity when incubated at 60°C for 60 Min, whereas free enzyme lost ≈50% activity. It was recycled nine times with ≈100% conversion efficiency when batch experiments were carried out at 35°C, pH 7.5, for the 180 Min cycle, using 5% N-carbamoyl-l-methionine as the substrate. The half-life of the immobilized preparation was determined as 23 cycles and is significant. Approximately 50% of enzyme activity was retained even after 5 months of storage at 4°C, whereas free enzyme lost complete enzyme activity. Hence, we could enhance the stability of l-methionine-N-carbamoylase to make it a potential biocatalyst for biotechnological production of α-amino acids.

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Stabilization of multimeric enzymes via immobilization and post-immobilization techniques

Cited by 122 publications

References 11 publications

Reversible enzyme immobilization via a very strong and nondistorting ionic adsorption on support-polyethylenimine composites

Reversible enzyme immobilization via a very strong and nondistorting ionic adsorption on support-polyethylenimine composites

Covalent Immobilization of β‐Galactosidase onto Amino‐Functionalized Polyvinyl Chloride Microspheres: Enzyme Immobilization and Characterization

Enhanced stability of newly isolated trimeric l‐methionine‐N‐carbamoylase from Brevibacillus reuszeri HSN1 by covalent immobilization

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