Use of Physicochemical Tools to Determine the Choice of Optimal Enzyme: Stabilization of -Amino Acid Oxidase (original) (raw)
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Agarose and Its Derivatives as Supports for Enzyme Immobilization
Molecules, 2016
Agarose is a polysaccharide obtained from some seaweeds, with a quite particular structure that allows spontaneous gelation. Agarose-based beads are highly porous, mechanically resistant, chemically and physically inert, and sharply hydrophilic. These features-that could be further improved by means of covalent cross-linking-render them particularly suitable for enzyme immobilization with a wide range of derivatization methods taking advantage of chemical modification of a fraction of the polymer hydroxyls. The main properties of the polymer are described here, followed by a review of cross-linking and derivatization methods. Some recent, innovative procedures to optimize the catalytic activity and operational stability of the obtained preparations are also described, together with multi-enzyme immobilized systems and the main guidelines to exploit their performances.
Journal of Molecular Catalysis B: Enzymatic, 1999
Ž. Three different approaches are proposed to increase the resistance of enzymes against hydrogen peroxide. a Multipoint coÕalent immobilization. Through this technique, enzyme rigidity would be greatly increased and hence, any conformational Ž. change on the enzyme structure involved before or after oxidation with hydrogen peroxide becomes greatly prevented. b Oriented immobilization on supports haÕing large internal surfaces. The immobilization of enzymes, through different areas of their surface on solid supports with internal morphology composed by large surfaces, promotes a certain masking of the enzyme areas that are very close to the support surface. In this way, the accessibility of hydrogen peroxide to such protein Ž. areas becomes greatly restricted. c Additional chemical modification of immobilized enzyme deriÕatiÕes with polymers. By adding thick barriers surrounding the whole enzyme molecule, the effective concentration of hydrogen peroxide in the proximity of the most sensitive residues may be strongly reduced. Multipoint covalently immobilized D-amino acid oxidase Ž. DAAO from Rhodotorula gracilis on glyoxyl-agarose is 11-fold more stable than native enzyme against the deleterious effect of hydrogen peroxide. On the other hand, DAAO from Trigonopsis Õariabilis was not stabilized by rigidification but it could be highly stabilized by an adequate combination of the best orientation on the support plus an additional modification with poly-aldehyde polymers.
Improvement of the stability of alcohol dehydrogenase by covalent immobilization on glyoxyl-agarose
Journal of Biotechnology, 2006
Immobilization of alcohol dehydrogenase (ADH) from Horse Liver inside porous supports promotes a dramatic stabilization of the enzyme against inactivation by air bubbles in stirred tank reactors. Moreover, immobilization of ADH on glyoxyl-agarose promotes additional stabilization against any distorting agent (pH, temperature, organic solvents, etc.). Stabilization is higher when using highly activated supports, they are able to immobilize both subunits of the enzyme. The best glyoxyl derivatives are much more stable than conventional ADH derivatives (e.g., immobilized on BrCN activated agarose). For example, glyoxyl immobilized ADH preserved full activity after incubation at pH 5.0 for 20 h at room temperature and conventional derivatives (as well as the soluble enzyme) preserved less than 50% of activity after incubation under the same conditions. Moreover, glyoxyl derivatives are more than 10 times more stable than BrCN derivatives when incubated in 50% acetone at pH 7.0. Multipoint covalent immobilization, in addition to multisubunit immobilization, seems to play an important stabilizing role against distorting agents. In spite of these interesting stabilization factors, immobilization hardly promotes losses of catalytic activity (keeping values near to 90%). This immobilized preparation is able to keep good activity using dextran-NAD +. In this way, ADH glyoxyl immobilized preparation seems to be suitable to be used as cofactor-recycling enzyme-system in interesting NAD +-mediated oxidation processes, catalyzed by other immobilized dehydrogenases in stirred tank reactors.
Stabilization of different alcohol oxidases via immobilization and post immobilization techniques
Enzyme and Microbial Technology, 2007
The thermal stability of multimeric alcohol oxidases (AOXs) from three different sources (Candida boidinii, Hansenula sp. and Pichia pastoris) was evaluated. AOX from C. boidinii was markedly more unstable than the other two enzymes, while the enzyme from Hansenula sp. was the most stable of the three. The stability of the enzymes was strongly dependent on the enzyme concentration, suggesting that the first inactivation cause could be subunits dissociation.
Journal of Molecular Catalysis B: Enzymatic, 2011
The bioconversion of lignocellulosic biomass to fermentable sugars for production of ethanol requires a multienzyme system named cellulase. This system contains enzymes that act synergistically in the hydrolysis of cellulose: endoglucanase, cellobiohydrolase and -glucosidase. The first two enzymes act directly on cellulose, yielding mainly glucose and cellobiose, which is hydrolyzed into glucose by glucosidase. An industrial process would be more economical by using immobilized systems that allow the reuse of the enzyme and improve the enzyme stability against different inactivation agents. Particularly, the hydrolysis of cellobiose would be performed using immobilized enzyme because cellobiose molecules are soluble in the reaction medium. In this work, -glucosidase was immobilized on agarose matrix derivatized with different reactive groups, e.g. polyethylenimine (PEI), glyoxyl (linear aliphatic aldehydes) and amine-epoxy, trying to optimize the stability and activity of the immobilized enzyme. Using reversible attachment (immobilization by anion exchange), the derivatives were active, but with poor thermal stability, e.g. PEI agarose derivative was approximately 7 times more stable than the soluble -glucosidase. However, these derivatives have important characteristics for an industrial process: reuse of the enzyme and/or the application of continuous systems. Among the activated supports with irreversible attachment (covalent immobilization), glyoxyl agarose did not reach a good thermal stability; it seems that the enzyme surface is very poor in amino groups from lysine residues. Better results were obtained with amine-epoxy agarose supports. -Glucosidase immobilized on that support kept 80% of its activity and was ca. 200 times more stable than the soluble enzyme.
Enzyme and Microbial Technology, 2009
Highly activated glyoxyl-supports rapidly immobilize proteins at pH 10 (where the -amino groups of the Lys groups of the protein surface are very reactive), and stabilize them by multipoint covalent attachment. However, they do not immobilize proteins at pH 8. This paper shows that the enzyme immobilization at this mild pH value is possible by incubation of the enzymes in the presence of different thiolated compounds (dithiothreitol, DTT; was selected as optimal reagent). The thiolated compounds (even the not reducing ones) stabilized the imino bonds formed at pH 8 between the aldehydes in the support and the amino groups of the protein. However, thiolated compounds are unable to reduce the imino bonds or the aldehyde groups and a final reduction step (e.g., using sodium borohydride) was always necessary. After enzyme immobilization through the most reactive amino group of the protein, the further incubation of this immobilized enzyme at pH 10 would improve the reactivity of the -amino groups of the Lys residues of the protein surface. Then, an intense multipoint covalent reaction of the enzyme with the dense layer of glyoxyl groups in the support could be obtained, increasing the stability of the immobilized enzyme. Using three different industrially relevant enzymes (penicillin G acylase from Escherichia coli (PGA), lipase from Bacillus thermocatenulatus (BTL2) and glutaryl acylase from Pseudomonas sp. (GA)), new immobilizedstabilized biocatalysts of the enzymes were produced. After reduction, the preparations incubated at pH 10 were more stable than those that were only immobilized and reduced at pH 8. In the case of the PGA, this preparation was even 4-5-fold more stable than those obtained by direct immobilization at pH 10 (around 40,000-50,000-fold more stable than the soluble enzyme).
Springer eBooks, 2020
Stabilization of enzymes via immobilization techniques is a valuable approach in order to convert a necessary protocol (immobilization) into a very interesting tool to improve key enzyme properties (stabilization). Multipoint covalent attachment of each immobilized enzyme molecule may promote a very interesting stabilizing effect. The relative distances among all enzyme residues involved in immobilization have to remain unaltered during any conformational change induced by any distorting agent. Amino groups are very interesting nucleophiles placed on protein surfaces. The immobilization of enzyme through the region having the highest amount of amino groups (Lys residues) is key for a successful stabilization. Glyoxyl groups are small aliphatic aldehydes that form very unstable Schiff's bases with amino groups, and they do not seem to be useful for enzyme immobilization at neutral pH. However, under alkaline conditions, glyoxyl supports are able to immobilize enzymes via a first multipoint covalent immobilization through the region having the highest amount of lysine groups. Activation of supports with a high surface density of glyoxyl groups and the performance of very intense enzyme-support multipoint covalent attachments are here described.
Biotechnology and Bioengineering, 2002
The cDNA of D-amino acid oxidase (DAO) gene isolated from Trigonopsis variabilis was expressed in Schizosaccharomyces pombe. A clone, ASP327-10, transformed with plasmid vector, pTL2M5DAO, expressed catalytically active DAO in the presence of G418, and converted Cephalosprin C to a-ketoadipyl-7-cephalosporanic acid (KA-7-ACA) and glutaryl-7-aminocephalosporanic acid (GL-7-ACA). Biocatalysts were prepared using ASP327-10 and T. variabilis, and evaluated to demonstrate the feasibility of recombinant S. pombe for industrial application. The cells were immobilized by crosslinking polyethylene imine after glutardialdehyde (GDA) ®xation and permeabilization by alkaline treatment. Although the biocatalyst prepared from ASP327-10 exhibited DAO activity, catalase activity still remained fully even after permeabilization, under which condition, the catalase activity of T. variabilis decreased to 20±30%. Heat treatment was required before cell ®xation by GDA to inactivate the catalase in S. pombe. This improved the ef®ciency of bioconversion to GL-7-ACA, but caused poor mechanical strength in the biocatalyst of S. pombe. To overcome this weakness, a catalase-de®cient host strain was obtained by ethylmethansulfate mutagenesis. Moreover, taking economics into consideration, the integrative vector, pTL2M5DAO-8XL, with multi-copies of expression cassette was constructed to express DAO in S. pombe even in the absence of G418. The newly established integrant, ASP417-7, did not exhibit any catalase activity so that heat treatment was not required. The obtained integrant and its biocatalyst were signi®cantly improved in GL-7ACA conversion ability and mechanical strength. This study demonstrates that the established integrant is a potential candidate as an alternative source of DAO enzyme.
Molecules, 2018
Alcalase was immobilized on glyoxyl 4% CL agarose beads. This permitted to have Alcalase preparations with 50% activity retention versus Boc-l-alanine 4-nitrophenyl ester. However, the recovered activity versus casein was under 20% at 50 °C, as it may be expected from the most likely area of the protein involved in the immobilization. The situation was different at 60 °C, where the activities of immobilized and free enzyme became similar. The chemical amination of the immobilized enzyme or the treatment of the enzyme with glutaraldehyde did not produce any significant stabilization (a factor of 2) with high costs in terms of activity. However, the modification with glutaraldehyde of the previously aminated enzyme permitted to give a jump in Alcalase stability (e.g., with most than 80% of enzyme activity retention for the modified enzyme and less than 30% for the just immobilized enzyme in stress inactivation at pH 7 or 9). This preparation could be used in the hydrolysis of casein a...