1.20.1.1 A146S increases the half-life of thermal inactivation at 45°C from around 1 min to 8 min 691424 1.20.1.1 A176R strongly decreased Km for NADP+ 657956 1.20.1.1 A319E increases the half-life of thermal inactivation at 45°C from around 1 min to 2 min 691424 1.20.1.1 A319E/T101A increases the half-life of thermal inactivation at 45°C from around 1 min to 5 min 691424 1.20.1.1 A325V thermostability almost identical to that of the wild-type enzyme 691424 1.20.1.1 C336D increases solubility and activity, thermostability almost identical to that of the wild-type enzyme 691424 1.20.1.1 D13E increases solubility and activity, thermostability almost identical to that of the wild-type enzyme 691424 1.20.1.1 D13E/M26I/E175A/E332N/C336D mutant obtained by directed evolution, round 4 673144 1.20.1.1 D13E/M26I/E175A/T181S/A308T/E332N/C336D mutant obtained by directed evolution, round 6, strong decrease in KM value for NADP compared to wild-type 673144 1.20.1.1 D13E/M26I/E175A/T181S/E332N/C336D mutant obtained by directed evolution, round 5 673144 1.20.1.1 D13E/M26I/E332N/C336D mutant obtained by directed evolution, round 3 673144 1.20.1.1 D13E/M26I/V71I/E130K/Q132R/Q137R/I150F/E175A/Q215L/R275Q/L276Q/I313L/V315A/A319E/A325V/E332N/C336D thermostable mutant TS-PTDH which contains 12 mutations that result in considerably increased thermostability with minimal change in activity, and four additional mutations that increase its activity. Mutation E175A leads to relaxation of cofactor specificity 726982 1.20.1.1 D79A significant differences in its kinetic constants compared to the wild-type enzyme. 2600fold decrease in catalytic efficiency. Pre-steady-state rates are approximately the same as the steady-state rates -, 690884 1.20.1.1 D79N has kinetic parameters more similar to those of wild-type 690884 1.20.1.1 E130K increases the half-life of thermal inactivation at 45°C from around 1 min to 12.5 min 691424 1.20.1.1 E130Q increases the half-life of thermal inactivation at 45°C from around 1 min to 7 min 691424 1.20.1.1 E130R increases the half-life of thermal inactivation at 45°C from around 1 min to 9 min 691424 1.20.1.1 E175A increases solubility and activity, thermostability almost identical to that of the wild-type enzyme 691424 1.20.1.1 E175A mutant with significantly increased kcat value for NADP+ 673144 1.20.1.1 E175A strongly decreased Km for NADP+ 657956 1.20.1.1 E175A the mutation allows the enzyme to use both NAD+ and NADP+ -, 744691 1.20.1.1 E175A/A176R double mutant, 3.6-fold higher efficiency with NAD+, 1000-fold higher efficiency with NADP+, 3-fold favor for NADP+ over NAD+ as cofactor -, 657956, 658259 1.20.1.1 E175A/A176R mutant utilizes both NAD+ and NADP+, kinetic isotope effects study, hydride transfer step is partially rate determining 673526 1.20.1.1 E266Q higher activity, steady-state and pre-steady-state rates are comparable -, 690884 1.20.1.1 E266Q significant increase in Km for both substrates, increase in turnover -, 658120 1.20.1.1 E332N increases solubility and activity, thermostability almost identical to that of the wild-type enzyme 691424 1.20.1.1 F198I leads to low activity 691424 1.20.1.1 F198M increases the half-life of thermal inactivation at 45°C from around 1 min to 2 min 691424 1.20.1.1 H292F almost complete loss of activity 658120 1.20.1.1 H292F no activity, NAD+ binding is abolished -, 658259 1.20.1.1 H292K almost complete loss of activity 658120 1.20.1.1 H292K no activity, NAD+ binding is abolished -, 658259 1.20.1.1 H292N almost complete loss of activity -, 658120 1.20.1.1 H292N no activity, NAD+ binding is abolished -, 658259 1.20.1.1 H292Q no activity, NAD+ binding is abolished 658259 1.20.1.1 I150F increases the half-life of thermal inactivation at 45°C from around 1 min to 7 min 691424 1.20.1.1 I150F thermostable mutant, half-life at 45°C 7.0 min compared to 1.4 min of wild-type 671425 1.20.1.1 I313L thermostability almost identical to that of the wild-type enzyme 691424 1.20.1.1 K330* mutant obtained by directed evolution, round 1 673144 1.20.1.1 K76A pre-steady-state rates are approximately the same as the steady-state rates -, 690884 1.20.1.1 K76A significant increase in Km for both substrates -, 658120 1.20.1.1 K76C significant increase in Km for both substrates 658120 1.20.1.1 K76M significant increase in Km for both substrates -, 658120 1.20.1.1 K76R significant increase in Km for both substrates -, 658120 1.20.1.1 L276C increases the half-life of thermal inactivation at 45°C from around 1 min to 12 min 691424 1.20.1.1 L276H increases the half-life of thermal inactivation at 45°C from around 1 min to 2 min 691424 1.20.1.1 L276Q increases the half-life of thermal inactivation at 45°C from around 1 min to 3.5 min 691424 1.20.1.1 L276R increases the half-life of thermal inactivation at 45°C from around 1 min to 8 min 691424 1.20.1.1 L276S increases the half-life of thermal inactivation at 45°C from around 1 min to 3 min 691424 1.20.1.1 M26I increases solubility and activity, thermostability almost identical to that of the wild-type enzyme 691424 1.20.1.1 M26I/E332N/C336D mutant obtained by directed evolution, round 2 673144 1.20.1.1 M53A the KM values for phosphonate and NAD+ are little different from the parent enzyme, while kcat is reduced by a factor of 50 -, 744691 1.20.1.1 M53N the KM values for phosphonate and NAD+ are little different from the parent enzyme, while kcat is reduced by a factor of 200 -, 744691 1.20.1.1 additional information 12 x PTDH mutant, saturation mutagenesis performed separately on each of the following residues in the parent PTDH template: V71, E130, Q132, Q137, I150, Q215, R275, L276, I313, V315, A319, and A325. The most thermostabilizing mutation discovered for each particular site is incorporated into the 12 x PTDH mutant, performed for K132, H137, L275, and C276, forming an optimized thermally stable phosphite dehydrogenase termed Opt12. Addition of A146S to Opt12 leads to the Opt13 variant, and the further addition of F198M leads to Opt14 691424 1.20.1.1 additional information mutant 17x-PTDH is based on a thermostable mutant TS-PTDH which contains 12 mutations that result in considerably increased thermostability with minimal change in activity, and four additional mutations that increase its activity, plus mutation E175A that allows this mutant to use both NAD+ and NADP+ -, 726981 1.20.1.1 additional information thermostable mutant 12X-PTDH with higher solubility than the wild-type. Thermostable mutant with dual cofactor specificity NADP-12X NAD shows pre-steady-state behavior very similar to that observed with 12X-PTDH and the wild-type enzyme. Pre-steady-state traces of thermostable mutant with dual cofactor specificity NADP-12X NADP shows curvature with NADP, particularly with protiated phosphite -, 690884 1.20.1.1 Q132K increases the half-life of thermal inactivation at 45°C from around 1 min to 3 min 691424 1.20.1.1 Q132R increases the half-life of thermal inactivation at 45°C from around 1 min to 2 min 691424 1.20.1.1 Q132R thermostable mutant, half-life at 45°C 2.3 min compared to 1.4 min of wild-type 671425 1.20.1.1 Q132R/Q137R/I150F/Q215L/R275Q thermostable mutant, half-life at 45°C 161 min compared to 1.4 min of wild-type 671425 1.20.1.1 Q137H increases the half-life of thermal inactivation at 45°C from around 1 min to 4.5 min 691424 1.20.1.1 Q137R increases the half-life of thermal inactivation at 45°C from around 1 min to 4 min 691424 1.20.1.1 Q137R thermostable mutant, half-life at 45°C 3.8 min compared to 1.4 min of wild-type 671425 1.20.1.1 Q137R/I150F/Q215L/R275Q thermostable mutant, half-life at 45°C 200 min compared to 1.4 min of wild-type 671425 1.20.1.1 Q137R/I150F/Q215L/R275Q/A319E thermostable mutant, half-life at 45°C 567 min compared to 1.4 min of wild-type 671425 1.20.1.1 Q137R/I150F/Q215L/R275Q/D162N/V315A thermostable mutant, half-life at 45°C 614 min compared to 1.4 min of wild-type 671425 1.20.1.1 Q137R/I150F/Q215L/R275Q/L276Q thermostable mutant, half-life at 45°C 437 min compared to 1.4 min of wild-type 671425 1.20.1.1 Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A thermostable mutant, half-life at 45°C 1421 min compared to 1.4 min of wild-type 671425 1.20.1.1 Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/A325V thermostable mutant, half-life at 45°C 2315 min compared to 1.4 min of wild-type 671425 1.20.1.1 Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/E130K thermostable mutant, half-life at 45°C 1515 min compared to 1.4 min of wild-type 671425 1.20.1.1 Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/I313L thermostable mutant, half-life at 45°C 1765 min compared to 1.4 min of wild-type 671425 1.20.1.1 Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/Q132R thermostable mutant, half-life at 45°C 2350 min compared to 1.4 min of wild-type 671425 1.20.1.1 Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/Q132R/V71I/E130K/I313L/A325V thermostable mutant, half-life at 45°C 8440 min compared to 1.4 min of wild-type 671425 1.20.1.1 Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/Q132R/V71I/E130K/I313L/A325V/A176R highly stable and active mutant engineered for regeneration of NADPH and enzyme membrane reactors 672712 1.20.1.1 Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/V71I thermostable mutant, half-life at 45°C 2000 min compared to 1.4 min of wild-type 671425 1.20.1.1 Q215L increases the half-life of thermal inactivation at 45°C from around 1 min to 9 min 691424 1.20.1.1 Q215L thermostable mutant, half-life at 45°C 8.7 min compared to 1.4 min of wild-type 671425 1.20.1.1 Q215M increases the half-life of thermal inactivation at 45°C from around 1 min to 2.5 min 691424 1.20.1.1 R237H almost complete loss of activity 658120 1.20.1.1 R237K low activity, absence of a significant burst in the pre-steady-state -, 690884 1.20.1.1 R237K significant increase in Km for both substrates 658120 1.20.1.1 R237K strong increase in Km for both substrates, reduced Vmax -, 658259 1.20.1.1 R237L almost complete loss of activity 658120 1.20.1.1 R237Q almost complete loss of activity 658120 1.20.1.1 R275L increases the half-life of thermal inactivation at 45°C from around 1 min to 9 min 691424 1.20.1.1 R275Q increases the half-life of thermal inactivation at 45°C from around 1 min to 4.5 min 691424 1.20.1.1 R275Q thermostable mutant, half-life at 45°C 4.6 min compared to 1.4 min of wild-type 671425 1.20.1.1 R301A mutant based on a thermostable mutant TS-PTDH which contains 12 mutations that result in considerably increased thermostability with minimal change in activity, and four additional mutations that increase its activity, plus mutation E175A that allows this mutant to use both NAD+ and NADP+. Mutation R301A leads to sharp decrease in activity, while kcat value is similar to wild-type -, 726981 1.20.1.1 R301A the mutant shows about 100fold loss in kcat and a 500fold increased Km value for phosphonate. The Ki value of sulfite with the mutant is 400fold increased compared to the wild type enzyme -, 746256 1.20.1.1 R301K mutant based on a thermostable mutant TS-PTDH which contains 12 mutations that result in considerably increased thermostability with minimal change in activity, and four additional mutations that increase its activity, plus mutation E175A that allows this mutant to use both NAD+ and NADP+. Mutation R301K leads to sharp decrease in activity, while kcat value is similar to wild-type -, 726981 1.20.1.1 R301K the mutant exhibits a slightly higher kcat than the wild type enzyme and a 20fold increased Km value for phosphonate -, 746256 1.20.1.1 S295A mutant based on a thermostable mutant TS-PTDH which contains 12 mutations that result in considerably increased thermostability with minimal change in activity, and four additional mutations that increase its activity, plus mutation E175A that allows this mutant to use both NAD+ and NADP+. Mutation S295A leads to sharp decrease in activity, while kcat value is similar to wild-type 726981 1.20.1.1 T101A increases the half-life of thermal inactivation at 45°C from around 1 min to 4.5 min 691424 1.20.1.1 V315A thermostability almost identical to that of the wild-type enzyme 691424 1.20.1.1 V71Ia thermostability almost identical to that of the wild-type enzyme 691424 1.20.1.1 W134A mutant based on a thermostable mutant TS-PTDH which contains 12 mutations that result in considerably increased thermostability with minimal change in activity, and four additional mutations that increase its activity, plus mutation E175A that allows this mutant to use both NAD+ and NADP+. Mutation W134A leads to sharp decrease in activity, while kcat value is similar to wild-type -, 726981 1.20.1.1 W134F mutant based on a thermostable mutant TS-PTDH which contains 12 mutations that result in considerably increased thermostability with minimal change in activity, and four additional mutations that increase its activity, plus mutation E175A that allows this mutant to use both NAD+ and NADP+. Mutation W134F leads to sharp decrease in activity, while kcat value is similar to wild-type 726981 1.20.1.1 Y139F mutant based on a thermostable mutant TS-PTDH which contains 12 mutations that result in considerably increased thermostability with minimal change in activity, and four additional mutations that increase its activity, plus mutation E175A that allows this mutant to use both NAD+ and NADP+. Mutation Y139F leads to sharp decrease in activity, while kcat value is similar to wild-type -, 726981