2007
DOI: 10.1002/bit.21546
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Further improvement of phosphite dehydrogenase thermostability by saturation mutagenesis

Abstract: Phosphite dehydrogenase represents a new enzymatic system for regenerating reduced nicotinamide cofactors for industrial biocatalysis. We previously engineered a variant of phosphite dehydrogenase with relaxed cofactor specificity and significantly increased activity and stability. Here we performed one round of random mutagenesis followed by comprehensive saturation mutagenesis to further improve the enzyme thermostability while maintaining its activity. Two new thermostabilizing mutations were identified. Th… Show more

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Cited by 62 publications
(51 citation statements)
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“…4d). The half-life (t 1/2 ) of PTDH-K at 45°C was 35 min, which is approximately 25 times higher than that of wild type [8] and three times higher than the E130K mutant of PTDH-WM88 [15]. The most thermostable PTDH known [12] was developed by directed evolution and has a t 1/2 of 8440 min at 45°C.…”
Section: Surface Electrostatic Potential Analysis Of Ptdh-k and Ptdh-mentioning
confidence: 99%
See 1 more Smart Citation
“…4d). The half-life (t 1/2 ) of PTDH-K at 45°C was 35 min, which is approximately 25 times higher than that of wild type [8] and three times higher than the E130K mutant of PTDH-WM88 [15]. The most thermostable PTDH known [12] was developed by directed evolution and has a t 1/2 of 8440 min at 45°C.…”
Section: Surface Electrostatic Potential Analysis Of Ptdh-k and Ptdh-mentioning
confidence: 99%
“…Most studies have concentrated on PTDH-WM88 [8] and its mutants, trying to improve the thermostability and alter the coenzyme preference of PTDH [12][13][14][15]. It is dispensable to comprehensive understand the overall properties of an enzyme before application; however, there are only few studies focused on the physical and chemical factors on PTDH activity and stability, and phosphite dehydrogenase with high stability under both alkaline and acidic conditions has not been reported.…”
Section: Introductionmentioning
confidence: 99%
“…To generate more thermostable mutants of ChKRED20, we subsequently applied combinations of beneficial mutations for possible synergistic or additive effect (Giver et al 1998;Hirokawa et al 2008;McLachlan et al 2008;Pei et al 2011;Zhang et al 2012). After the generation of several double, triple, and quadruple mutants, their residual activities were measured after heat treatment at 70°C for 3 h using the crude enzyme extracts (Fig.…”
Section: Combination Of Beneficial Mutationsmentioning
confidence: 99%
“…Proteins have been previously computationally designed to bind new ligands, 1 proteins, 2 and nucleic acids, 3 to improve protein stability, 4,5 as well as to introduce novel enzymatic activity, 6,7 demonstrating that the fundamental rudiments of molecular recognition can adequately be captured via computational design. The systematic fine-tuning of molecular recognition between proteins and ligands finds many biotechnological applications ranging from improved catalytic activity, 8 improved protein thermostability, [9][10][11] genetic circuits, 12 biosensors, 13,14 chiral separations, 15 the construction of novel enzymes with alternative functionality, 16,17 the creation of gene switches 18 and signal transduction pathways. 19,20 Many of the aforementioned applications require the enzymes to operate under unnatural conditions (e.g., at elevated temperatures or in nonaqueous environments), and/or possess altered cofactor or substrate specificity.…”
Section: Introductionmentioning
confidence: 99%