Stability of biocatalysts

Polizzi, Karen M.; Bommarius, Andreas S.; Broering, James M.; Chaparro‐Riggers, Javier

doi:10.1016/j.cbpa.2007.01.685

Cited by 382 publications

(281 citation statements)

References 27 publications

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“…Water-miscible organic co-solvents can increase the solubility of nonpolar substrates, suppress hydrolysis reactions, and shift reaction selectivity towards an otherwise unstable product (Castro and Knubovets, 2003;Schmid et al, 2001). One strategy for improving enzyme stability in general is to immobilize the enzyme (Grazu et al, 2005;Miyazaki et al, 2005), which offers the additional advantages of enabling continuous operation of the enzyme reactor and reuse of the enzyme, recycling of substrates, and steady removal of products to alleviate product degradation and inhibition (Bornscheuer, 2003;Polizzi et al, 2007). Immobilization has also been employed to increase enzyme stability in organic co-solvents (Fernandez-Lafuente et al, 2001); however, a common drawback of most immobilization methods is a concomitant reduction in the enzyme's intrinsic activity (Fernandez-Lafuente et al, 2001;Kranz et al, 2007;Wehtje et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

Self‐renaturing enzymes: Design of an enzyme‐chaperone chimera as a new approach to enzyme stabilization

Bergeron

Gomez

Whitehead

et al. 2009

Biotech & Bioengineering

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Molecular chaperones in aqueous-organic mixtures can broaden the utility of biocatalysis by stabilizing enzymes in denaturing conditions. We have designed a self-renaturing enzyme-chaperone chimera consisting of penicillin amidase and a thermophilic chaperonin that functions in aqueous-organic mixtures. The flexible linker separating the enzyme and chaperone domains was optimized and the design was extended to incorporate a chitin binding domain to facilitate immobilization of the chimera to a chitin support. The initial specific activity of penicillin amidase was not compromised by the enzymechaperone fusion or by immobilization. The total turnover number of immobilized chimera for amoxicillin synthesis in aqueous-methanol mixtures was 2.8 times higher after 95 h than the total turnover number of the immobilized penicillin amidase lacking a chaperone domain. Similarly, in 32% methanol the soluble chimera was active for over three times longer than the enzyme alone. This approach could easily be extended to other enzyme systems.

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Section: Introductionmentioning

confidence: 99%

Self‐renaturing enzymes: Design of an enzyme‐chaperone chimera as a new approach to enzyme stabilization

Bergeron

Gomez

Whitehead

et al. 2009

Biotech & Bioengineering

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“…Since biocatalysts are designed to perform their activities in aqueous medium and in organic media, the pH value is important, since it is well established that enzymes "remember" the pH of the last aqueous solution in which they were dissolved and, after water removal, their ionization state remains unchanged (i.e., the optimum to display its maximum activity) [37].…”

Section: Process Of Immobilizationmentioning

confidence: 99%

A Highly Stable Biocatalyst Obtained from Covalent Immobilization of a Non-Commercial Cysteine Phytoprotease

Ceccacci¹

2015

J Bioprocess Biotech

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In this work, araujiain (enzymatic preparation obtained from the latex of Araujia hortorum fruits) was successfully immobilized on glyoxyl-agarose via multipoint covalent attachment. Thus, good efficiency of immobilization and high operational stability of immobilized enzyme were obtained. The activity of araujiain at alkaline pH was significantly improved after immobilization. In addition, immobilized araujiain also showed high activity and good stability, without significant loss in its activity, at temperatures between 37 and 60°C and in the presence of immiscible organic solvents. Immobilized araujiain also showed good performance in a mixture of 50% ethyl acetate in buffer, used for peptide synthesis, with better results than when the free enzyme was used as catalyst. These results indicate that immobilized araujiain via multipoint covalent attachment can be highly stabilized and this method might be used for practical applications of araujiain in hydrolytic and synthetic processes.

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“…The major reason for this is that most mutations, and mutations altering protein function in particular, are destabilizing, increasing the chance that the mutation results in a protein that is not folded (correctly) and thus not functional anymore [10c] . Proteins from thermophilic organisms on the other hand have to encode enzymes that are functional at high temperatures, thus exhibiting a larger free energy difference (ΔGu) between folded (native) and unfolded state than mesophilic enzymes [10d) 11] . This free folding energy can be considered as a 'stability reservoir' that can be consumed by accumulating mutations that encode for new protein functions but are destabilizing in terms of free energy of folding.…”

Section: Introductionmentioning

confidence: 99%

Directed Divergent Evolution of a Thermostable D‐Tagatose Epimerase towards Improved Activity for Two Hexose Substrates

et al. 2015

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Abstract:We exploit the functional promiscuity of an engineered thermostable variant of a promiscuous D-tagatose epimerase (DTE Var8) to morph it into two efficient catalysts for the C3 epimerization of D-fructose to D-psicose and of L-sorbose to L-tagatose. Iterative single-site randomization and screening of 48 residues in the first and second shell around the substrate binding site of Var8 yielded 8-sites mutant IDF8 with an 9-fold improved kcat for the epimerization of D-fructose, and the 6-sites mutant ILS6 with an 14-fold improved epimerization of L-sorbose compared to Var8. Structure analysis of IDF8 revealed a charged patch at the entrance of its active site that supposedly facilitates the entry of the polar substrate, whereas the improvement in variant ILS6 is thought to relate to subtle changes in the hydration of the bound substrate. The structures will inform future engineering efforts of these and other isomerizing enzymes for the production of rare.

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Stability of biocatalysts

Cited by 382 publications

References 27 publications

Self‐renaturing enzymes: Design of an enzyme‐chaperone chimera as a new approach to enzyme stabilization

Self‐renaturing enzymes: Design of an enzyme‐chaperone chimera as a new approach to enzyme stabilization

A Highly Stable Biocatalyst Obtained from Covalent Immobilization of a Non-Commercial Cysteine Phytoprotease

Directed Divergent Evolution of a Thermostable D‐Tagatose Epimerase towards Improved Activity for Two Hexose Substrates

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