Preparation, optimization, and structures of cross‐linked enzyme aggregates (CLEAs)

Schoevaart, Rob; Wolbers, M.W.; Golubovic, M.; Ottens, Marcel; Kieboom, A. P. G.; Rantwijk, Fred van; Wielen, Luuk A.M. van der; Sheldon, Roger A.

doi:10.1002/bit.20184

Cited by 449 publications

(356 citation statements)

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“…A β-gal imobilizada por CLEA foi reportada em várias obras da literatura (Schoevaart et al, 2004;Gaur et al, 2006;Sheldon, 2010;Li et al, 2015). Contudo, não foi avaliada a β-gal de K. lactis ou a adição de uma proteína espaçadora durante a imobilização, visando a diminuição da resistência à transferência de massa e o entrecruzamento excessivo.…”

Section: Ufscar -São Carlos -Sp 16 a 19 De Julho De 2017unclassified

Imobilização De Beta-Galactosidase Utilizando a Técnica Clea (Cross-Linked Enzyme Aggregates)

Ulrich¹,

Mafra²,

Tardioli³

et al. 2017

Blucher Chemical Engineering Proceedings

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RESUMO -Realizou-se a otimização da imobilização por agregados enzimáticos reticulados (Cross-Linked Enzyme Aggregates CLEAs) de β-galactosidase (EC 3.2.1.23) de Kluyveromyces lactis. A metodologia de preparo dos CLEAs consiste na precipitação da proteína e entrecruzamento com grupos amino reativos livres em sua superfície, via reação com um reagente bifuncional. Dessa forma, foram avaliados os rendimentos de imobilização de derivados preparados com diferentes agentes precipitantes (polietilenoglicol 400, álcool terc-butílico, acetona, dimetoxietano, etanol e sulfato de amônio), diferentes concentrações de agente entrecruzante (0, 0,3, 0,6 e 1 mM) e presença de proteína espaçadora soja nas proporções mgSoja/mgEnzima 2 e 4 e mmolGutaraldeído/gProteína Total 2, 2,5 e 3. O derivado que apresentou rendimento de imobilização máximo de 74,6% foi preparado utilizando álcool terc-butílico, glutaraldeído na razão 2 mmolG/gProteína Total e razão entre proteína espaçadora soja e enzima, respectivamente, 0,4 mg/0,2 mg. INTRODUÇÃOOs chamados agregados enzimáticos reticulados (Cross-linked Enzymes AggregatesCLEAs) são obtidos por meio da preparação e imobilização enzimáticas em um único passo. A metodologia consiste na precipitação da proteína e entrecruzamento com grupos amino reativos livres de lisina em sua superfície, via reação com um reagente bifuncional, tal como glutaraldeído (Sheldon, 2010). A metodologia CLEA, demonstrada pela Figura 1, é simples, amplamente aplicável, não requer uma enzima altamente purificada ou uma matriz sólida (Sheldon et al., 2013). O método pode, ainda, estabilizar as estruturas quaternárias de enzimas multiméricas (Wilson et al., 2004).Figura 1 -Representação da preparação dos CLEAs. A enzima solúvel é agregada pela ação de um precipitante e, em seguida, ocorre o entrecruzamento dos agregados enzimáticos pela ação de um agente intercruzante.

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Section: Ufscar -São Carlos -Sp 16 a 19 De Julho De 2017unclassified

Imobilização De Beta-Galactosidase Utilizando a Técnica Clea (Cross-Linked Enzyme Aggregates)

Ulrich¹,

Mafra²,

Tardioli³

et al. 2017

Blucher Chemical Engineering Proceedings

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“…On the other hand, glycosylated enzymes ss-Galactosidase formed "well dispersible suspensions" which showed no difference in activities when assayed by two different fast and slow assays [42]. The issue of cluster size and nature of enzyme molecule had been studied at a greater length by the same group [49]. Majumder et al, (2008) [24] showed that extent of crosslinking also affected cluster size and same enzyme can give CLEA clusters of different morphology and size.…”

Section: Crosslinked Enzyme Aggregates (Clea)mentioning

confidence: 99%

Use of high activity enzyme preparations in neat organic solvents for organic synthesis

Gupta¹,

Mukherjee²,

Malhotra³

2013

Univers Org Chem

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The use of enzymes in nearly anhydrous organic solvents generally results in low initial rates/percentage conversions. The current review focuses on some biocatalyst designs like immobilization on nanomaterials, enzyme precipitated and rinsed with organic solvents (EPROS), crosslinked enzyme crystals (CLEC), crosslinked enzyme aggregates (CLEA), protein coated microcrystals (PCMC) and crosslinked protein coated microcrystals (CLPCMC) which show much better catalytic efficiency in such media as compared to other kinds of biocatalyst preparations. The basic methodology and principle behind these efficient designs are described. The relatively recent results on catalytic promiscuity as seen in low water media have also been covered. It is hoped that this will further encourage wider applications of enzymes in neat organic solvents in organic synthesis.

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“…[10][11][12][13][14][15] In spite of these attempts, a standard process for GOx immobilization has not yet been developed. Furthermore, GOx immobilization research to date has mostly focused on only increasing the amount of immobilized GOx without serious consideration of how to improve the electron transfer between the immobilized GOx molecules and supporter materials.…”

Section: Introductionmentioning

confidence: 99%

A new biocatalyst employing pyrenecarboxaldehyde as an anodic catalyst for enhancing the performance and stability of an enzymatic biofuel cell

2017

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A new enzyme catalyst consisting of pyrenecarboxaldehyde (PCA) and glucose oxidase (GOx) immobilized on polyethyleneimine (PEI) and a carbon nanotube supporter (CNT/PEI/[PCA/GOx]) is suggested, and the performance and stability of an enzymatic biofuel cell (EBC) using the new catalyst are evaluated. Using PCA, the amount of immobilized GOx increases (3.3 U mg − 1 ) and the electron transfer rate constant of the CNT/PEI/[PCA/GOx] is promoted (11.51 s − 1 ). Also, the catalyst induces excellent EBC performance (maximum power density (MPD) of 2.1 mW cm − 2 ), long-lasting stability (maintenance of 93% of the initial MPD after 4 weeks) and superior catalytic activity (flavin adenine dinucleotide redox reaction rate of 0.62 mA cm − 2 and Michaelis-Menten constant of 0.99 mM). These characteristics are ascribed to effects of (i) electron collection due to hydrophobic interactions, (ii) electron transfer pathways due to π-conjugated bonds and (iii) enzyme stabilization due to π-hydrogen bonds that are newly induced by the PCA/GOx composite. The existence of such positive interactions is properly verified using X-ray photoelectron spectroscopy and enzyme activity measurements. NPG Asia Materials (2017) 9, e386; doi:10.1038/am.2017.75; published online 2 June 2017 INTRODUCTIONThe demand for clean electrical energy is increasing as a result of weather changes caused by the greenhouse effect and depletion of fossil fuels. 1 To meet this demand, related research and development efforts are being eagerly pursued. As one of the efforts, enzymatic biofuel cells (EBCs) that convert bioenergy into electrical energy are being considered. 2,3 In particular, EBC systems employing glucose oxidase (GOx) as a biocatalyst have emerged because of their advantageous properties, such as biocompatibility, excellent selectivity toward specific substrates and strong activity near neutral pH and room temperature. 2 In addition, because EBC systems use human body-friendly fuels, such as glucose, glycerol, water and oxygen, for electricity generation, they can be embedded as power generators for the operation of artificial internal organs, such as artificial hearts and brains, insulin pumps or bone stimulators. [4][5][6][7][8] However, in spite of such promising facets of EBC systems, the commercialization of EBCs using GOx has been limited because of their low EBC performance and poor durability. 9 The disadvantages are attributed to the low immobilization ratio of GOx molecules, slow GOx-related reaction rate, low GOx activity and easy GOx denaturation. Of them, low GOx immobilization has been considered the main problem.

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Preparation, optimization, and structures of cross‐linked enzyme aggregates (CLEAs)

Cited by 449 publications

References 20 publications

Imobilização De Beta-Galactosidase Utilizando a Técnica Clea (Cross-Linked Enzyme Aggregates)

Imobilização De Beta-Galactosidase Utilizando a Técnica Clea (Cross-Linked Enzyme Aggregates)

Use of high activity enzyme preparations in neat organic solvents for organic synthesis

A new biocatalyst employing pyrenecarboxaldehyde as an anodic catalyst for enhancing the performance and stability of an enzymatic biofuel cell

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