Directed evolution of formate dehydrogenase from <i>Candida boidinii</i> for improved stability during entrapment in polyacrylamide

Ansorge‐Schumacher, Marion B.; Slusarczyk, Heike; Schümers, Julia; Hirtz, Dennis

doi:10.1111/j.1742-4658.2006.05395.x

Cited by 38 publications

(18 citation statements)

References 27 publications

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“…[54] The best mutant had a 4.4-fold higher activity compared to the free enzyme immobilized in the gel. The stabilization resulted from a substitution of lysine, glutamic acid and cysteine residues remote from the active site.…”

Section: Hydrogelsmentioning

confidence: 98%

Enzyme Immobilization: The Quest for Optimum Performance

2007

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Immobilization is often the key to optimizing the operational performance of an enzyme in industrial processes, particularly for use in non-aqueous media. Different methods for the immobilization of enzymes are critically reviewed. The methods are divided into three main categories, viz. (i) binding to a prefabricated support (carrier), (ii) entrapment in organic or inorganic polymer matrices, and (iii) cross-linking of enzyme molecules. Emphasis is placed on relatively recent developments, such as the use of novel supports, e.g., mesoporous silicas, hydrogels, and smart polymers, novel entrapment methods and cross-linked enzyme aggregates (CLEAs).

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Section: Hydrogelsmentioning

confidence: 98%

Enzyme Immobilization: The Quest for Optimum Performance

2007

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“…Due to advances in molecular biology it is now also possible to modify enzymes (through directed evolution or site directed mutagenesis) to enhance immobilisation, such as generation of additional binding residues (Mateo et al 2007c) or adaptation to a particular support (Ansorge-Schumacher et al 2006). …”

Section: Enhanced Performancementioning

confidence: 99%

Advances in enzyme immobilisation

Brady

Jordaan²

2009

Biotechnol Lett

750

482

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Improvements in current strategies for carrier-based immobilisation have been developed using hetero-functionalised supports that enhance the binding efficacy and stability through multipoint attachment. New commercial resins (Sepabeads) exhibit improved protein binding capacity. Novel methods of enzyme self-immobilisation have been developed (CLEC, CLEA, Spherezyme), as well as carrier materials (Dendrispheres), encapsulation (PEI Microspheres), and entrapment. Apart from retention, recovery and stabilisation, other advantages to enzyme immobilisation have emerged, such as enhanced enzyme activity, modification of substrate selectivity and enantioselectivity, and multi-enzyme reactions. These advances promise to enhance the roles of immobilisation enzymes in industry, while opening the door for novel applications.

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“…strain 101 also decreases significantly after the replacement of C255 (5). Cysteine residues are frequently found within functional (regulatory, catalytic, or binding) sites in proteins and play an important role in impartation of specialized properties for key sites (6, 7). Thus, the replacement of the free cysteine residues of FDHs is not the best strategy to improve enzyme chemical stability.…”

Section: Introductionmentioning

confidence: 99%

Elimination of a Free Cysteine by Creation of a Disulfide Bond Increases the Activity and Stability of Candida boidinii Formate Dehydrogenase

Zheng

Yang

Zhou

et al. 2017

Appl Environ Microbiol

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NAD+-dependent formate dehydrogenase (FDH; EC 1.2.1.2) is an industrial enzyme widely used for NADH regeneration. However, enzyme inactivation caused by the oxidation of cysteine residues is a flaw of native FDH. In this study, we relieved the oxidation of the free cysteine of FDH from Candida boidinii (CboFDH) through the construction of disulfide bonds between A10 and C23 as well as I239 and C262. Variants A10C, I239C, and A10C/I239C were obtained by the site-directed mutagenesis and their properties were studied. Results showed that there were no significant changes in the optimum temperature and pH between variants and wild-type CboFDH. However, the stabilities of all variant enzymes were improved. Specifically, the CboFDH variant A10C (A10Cfdh) showed a significant increase in copper ion resistance and acid resistance, a 6.7-fold increase in half-life at 60°C, and a 1.4-fold increase in catalytic efficiency compared with the wild type. Asymmetric synthesis of l-tert-leucine indicated that the process time was reduced by 40% with variant A10Cfdh, which benefited from the increase in catalytic efficiency. Circular dichroism analysis and molecular dynamics simulation indicated that variants that contained disulfide bonds lowered the overall root mean square deviation (RMSD) and consequently increased the protein rigidity without affecting the secondary structure of enzyme. This work is expected to provide a viable strategy to avoid the microbial enzyme inactivation caused by the oxidation of the free cysteine residues and improving their performances.IMPORTANCE FDH is widely used for NADH regeneration in dehydrogenase-based synthesis of optically active compounds to decrease the cost of production. This study highlighted a viable strategy that was used to eliminate the oxidation of free cysteine residues of FDH from Candida boidinii by the introduction of disulfide bonds. Using this strategy, we obtained a variant FDH with improved activity and stability. The improvement of activity and stability of FDH is expected to reduce its price and then further to decrease the cost of its application.

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Directed evolution of formate dehydrogenase from Candida boidinii for improved stability during entrapment in polyacrylamide

Cited by 38 publications

References 27 publications

Enzyme Immobilization: The Quest for Optimum Performance

Enzyme Immobilization: The Quest for Optimum Performance

Advances in enzyme immobilisation

Elimination of a Free Cysteine by Creation of a Disulfide Bond Increases the Activity and Stability of Candida boidinii Formate Dehydrogenase

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