Advances in the design of new epoxy supports for enzyme immobilization–stabilization

2007 ◽  
Vol 35 (6) ◽  
pp. 1593-1601 ◽  
Author(s):  
C. Mateo ◽  
V. Grazú ◽  
B.C.C. Pessela ◽  
T. Montes ◽  
J.M. Palomo ◽  
...  
Keyword(s):  
Physical Adsorption ◽  
Immobilized Enzymes ◽  
Covalent Immobilization ◽  
Covalent Attachment ◽  
Industrial Enzymes ◽  
Protein Surface ◽  
Strong Stabilization ◽  
Support Surfaces ◽  
Multipoint Covalent Attachment ◽  
Epoxy Groups

Multipoint covalent immobilization of enzymes (through very short spacer arms) on support surfaces promotes a very interesting ‘rigidification’ of protein molecules. In this case, the relative positions of each residue of the enzyme involved in the immobilization process have to be preserved unchanged during any conformational change induced on the immobilized enzyme by any distorting agent (heat, organic solvents etc.). In this way, multipoint covalent immobilization should induce a very strong stabilization of immobilized enzymes. Epoxy-activated supports are able to chemically react with all nucleophile groups placed on the protein surface: lysine, histidine, cysteine, tyrosine etc. Besides, epoxy groups are very stable. This allows the performance of very long enzyme–support reactions, enabling us to get very intense multipoint covalent attachment. In this way, these epoxy supports seem to be very suitable to stabilize industrial enzymes by multipoint covalent attachment. However, epoxy groups exhibit a low intermolecular reactivity towards nucleophiles and hence the enzymes are not able to directly react with the epoxy supports. Thus a rapid physical adsorption of enzymes on the supports becomes a first step, followed by an additional rapid ‘intramolecular’ reaction between the already adsorbed enzyme and the activated support. In this situation, a suitable first orientation of the enzyme on the support (e.g. through regions that are very rich in nucleophiles) is obviously necessary to get a very intense additional multipoint covalent immobilization. The preparation of different ‘generations’ of epoxy supports and the design of different protocols to fully control the first interaction between enzymes and epoxy supports will be reviewed in this paper. Finally, the possibilities of a directed immobilization of mutated enzymes (change of an amino acid by cysteine on specific points of the protein surface) on tailor-made disulfide-epoxy supports will be discussed as an almost-ideal procedure to achieve very intense and very efficient rigidification of a desired region of industrial enzymes.

Epoxy Sepabeads: A Novel Epoxy Support for Stabilization of Industrial Enzymes via Very Intense Multipoint Covalent Attachment

2002 ◽  
Vol 18 (3) ◽  
pp. 629-634 ◽  
Author(s):  
C. Mateo ◽  
O. Abian ◽  
G. Fernandez-Lorente ◽  
J. Pedroche ◽  
R. Fernandez-Lafuente ◽  
...  
Keyword(s):  
Covalent Attachment ◽  
Industrial Enzymes ◽  
Multipoint Covalent Attachment

scholarly journals Improving the Thermostability and Optimal Temperature of a Lipase from the Hyperthermophilic ArchaeonPyrococcus furiosusby Covalent Immobilization

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Roberta V. Branco ◽  
Melissa L. E. Gutarra ◽  
Jose M. Guisan ◽  
Denise M. G. Freire ◽  
Rodrigo V. Almeida ◽  
...  
Keyword(s):  
Pyrococcus Furiosus ◽  
Enzyme Stability ◽  
Enantiomeric Excess ◽  
Residual Activity ◽  
Covalent Bond ◽  
Covalent Immobilization ◽  
Covalent Attachment ◽  
Optimal Temperature ◽  
Thermostable Lipase ◽  
Multipoint Covalent Attachment

A recombinant thermostable lipase (Pf2001Δ60) from the hyperthermophilic ArchaeonPyrococcus furiosus(PFUL) was immobilized by hydrophobic interaction on octyl-agarose (octyl PFUL) and by covalent bond on aldehyde activated-agarose in the presence of DTT at pH = 7.0 (one-point covalent attachment) (glyoxyl-DTT PFUL) and on glyoxyl-agarose at pH 10.2 (multipoint covalent attachment) (glyoxyl PFUL). The enzyme’s properties, such as optimal temperature and pH, thermostability, and selectivity, were improved by covalent immobilization. The highest enzyme stability at 70°C for 48 h incubation was achieved for glyoxyl PFUL (around 82% of residual activity), whereas glyoxyl-DTT PFUL maintained around 69% activity, followed by octyl PFUL (27% remaining activity). Immobilization on glyoxyl-agarose improved the optimal temperature to 90°C, while the optimal temperature of octyl PFUL was 70°C. Also, very significant changes in activity with different substrates were found. In general, the covalent bond derivatives were more active than octyl PFUL. TheEvalue also depended substantially on the derivative and the conditions used. It was observed that the reaction of glyoxyl-DTT PFUL using methyl mandelate as a substrate at pH 7 presented the best results for enantioselectivityE=22and enantiomeric excess (ee (%) = 91).

scholarly journals Stabilization of Penicillin G Acylase from Escherichia coli: Site-Directed Mutagenesis of the Protein Surface To Increase Multipoint Covalent Attachment

2004 ◽  
Vol 70 (2) ◽  
pp. 1249-1251 ◽  
Author(s):  
Olga Abian ◽  
Valeria Grazú ◽  
Juan Hermoso ◽  
Ramón González ◽  
José Luis García ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Penicillin Acylase ◽  
Site Directed Mutagenesis ◽  
Covalent Attachment ◽  
Penicillin G ◽  
Directed Mutagenesis ◽  
Kinetic Properties ◽  
Protein Surface ◽  
Improved Stability ◽  
Multipoint Covalent Attachment

ABSTRACT Three mutations on the penicillin acylase surface (increasing the number of Lys in a defined area) were performed. They did not alter the enzyme's stability and kinetic properties; however, after immobilization on glyoxyl-agarose, the mutant enzyme showed improved stability under all tested conditions (e.g., pH 2.5 at 4°C, pH 5 at 60°C, pH 7 at 55°C, or 60% dimethylformamide), with stabilization factors ranging from 4 to 11 compared with the native enzyme immobilized on glyoxyl-agarose.

scholarly journals Stabilization of Enzymes by Multipoint Covalent Attachment on Aldehyde-Supports: 2-Picoline Borane as an Alternative Reducing Agent

Catalysts ◽  
2018 ◽  
Vol 8 (8) ◽  
pp. 333 ◽  
Author(s):  
Alejandro H. Orrego ◽  
Maria Romero-Fernández ◽  
María Millán-Linares ◽  
María Yust ◽  
José Guisán ◽  
...  
Keyword(s):  
Immobilized Enzymes ◽  
Aliphatic Aldehyde ◽  
Reducing Agent ◽  
Covalent Attachment ◽  
Significant Activity ◽  
Borohydride Reduction ◽  
Secondary Amino ◽  
Alternative Methodology ◽  
Multipoint Covalent Attachment ◽  
Aldehyde Groups

Enzyme immobilization by multipoint covalent attachment on supports activated with aliphatic aldehyde groups (e.g., glyoxyl agarose) has proven to be an excellent immobilization technique for enzyme stabilization. Borohydride reduction of immobilized enzymes is necessary to convert enzyme–support linkages into stable secondary amino groups and to convert the remaining aldehyde groups on the support into hydroxy groups. However, the use of borohydride can adversely affect the structure–activity of some immobilized enzymes. For this reason, 2-picoline borane is proposed here as an alternative milder reducing agent, especially, for those enzymes sensitive to borohydride reduction. The immobilization-stabilization parameters of five enzymes from different sources and nature (from monomeric to multimeric enzymes) were compared with those obtained by conventional methodology. The most interesting results were obtained for bacterial (R)-mandelate dehydrogenase (ManDH). Immobilized ManDH reduced with borohydride almost completely lost its catalytic activity (1.5% of expressed activity). In contrast, using 2-picoline borane and blocking the remaining aldehyde groups on the support with glycine allowed for a conjugate with a significant activity of 19.5%. This improved biocatalyst was 357-fold more stable than the soluble enzyme at 50 °C and pH 7. The results show that this alternative methodology can lead to more stable and active biocatalysts.

Immobilization-Stabilization of Enzymes by Multipoint Covalent Attachment on Supports Activated With Epoxy Groups

2006 ◽  
pp. 47-55 ◽  
Author(s):  
Cesar Mateo ◽  
Olga Abian ◽  
Gloria Fernández-Lorente ◽  
Benevides C. C. Pessela ◽  
Valeria Grazu ◽  
...  
Keyword(s):  
Covalent Attachment ◽  
Multipoint Covalent Attachment ◽  
Epoxy Groups

Preservation of epoxy groups on surfaces in the covalent attachment of butadiene monoxide on Si(111)-(7×7): the effect of a vinyl substituent

2015 ◽  
Vol 51 (75) ◽  
pp. 14195-14198
Author(s):  
Wei Mao ◽  
Jing Hui He ◽  
Jia Qiang Gu ◽  
Wei Chen ◽  
Kai Wu ◽  
...  
Keyword(s):  
Epoxy Group ◽  
Covalent Attachment ◽  
Epoxy Groups

A vinyl substituent ensures that butadiene monoxide selectively binds to Si(111)-(7×7) through a [2+2]-like cycloaddition, maintaining an epoxy group in the cycloadduct.

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