2000
DOI: 10.1046/j.1432-1327.2000.01123.x
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Stabilization of NAD‐dependent formate dehydrogenase from Candida boidinii by site‐directed mutagenesis of cysteine residues

Abstract: The gene of the NAD-dependent formate dehydrogenase (FDH) from the yeast Candida boidinii was cloned by PCR using genomic DNA as a template. Expression of the gene in Escherichia coli yielded functional FDH with about 20% of the soluble cell protein. To confirm the hypothesis of a thiol-coupled inactivation process, both cysteine residues in the primary structure of the enzyme have been exchanged by site-directed mutagenesis using a homology model based on the 3D structure of FDH from Pseudomonas sp. 101 and f… Show more

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Cited by 173 publications
(133 citation statements)
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“…Here we report the directed evolution of a highly thermostable PTDH mutant for NAD(P)H regeneration. Three rounds of errorprone PCR and high-throughput screening coupled with sitedirected mutagenesis yielded a PTDH mutant with higher thermostability than the most thermostable Candida boidinii FDH reported previously (17).…”
mentioning
confidence: 84%
See 1 more Smart Citation
“…Here we report the directed evolution of a highly thermostable PTDH mutant for NAD(P)H regeneration. Three rounds of errorprone PCR and high-throughput screening coupled with sitedirected mutagenesis yielded a PTDH mutant with higher thermostability than the most thermostable Candida boidinii FDH reported previously (17).…”
mentioning
confidence: 84%
“…The t 1/2 of FDH at 50°C is in good agreement with values reported elsewhere for the wild-type and recombinant FDH (t 1/2 Ն300 min at 50°C in 100 mM potassium phosphate buffer, pH 7.5) (17). Slusarczyk and coworkers improved the oxidative stability of the wild-type FDH by replacing two cysteine resi- dues using site-directed mutagenesis, but this resulted in several mutants with significantly lower thermostability (17). Thus, the 12x mutant is substantially more thermostable than these FDH variants.…”
Section: Reagentsmentioning
confidence: 99%
“…The binding experiments were performed on an SPRbased Biacore T200 instrument (GE, Uppsala, Sweden) at 25°C with a flow rate of 30 …”
Section: Protein-protein Interaction Studies With Sprmentioning
confidence: 99%
“…Sequence Space-To demonstrate the applicability of our QAW strategy, an evolutional design approach, for simultaneous improvement of particular properties, PHBH was used as a model protein in this study to improve the characteristics in terms of main activity, sub-activity, reaction specificity, and thermal stability, using a diminished amino acid component such as Cys-and Met-reduced (41)(42)(43). The merit of the QAW strategy is the great reduction in the number (no more than 10 4 ) of mutants to be tested in sequence space, and therefore the method can be, in principal, applied to improve any particular characteristics (1).…”
Section: Protein Design By Means Of An Adaptive Walk Search Inmentioning
confidence: 99%