Amino acid substitutions that increase the thermal stability of the λ Cro protein

1989 ◽  
Vol 5 (3) ◽  
pp. 202-210 ◽  
Author(s):  
Andrew A. Pakula ◽  
Robert T. Sauer
Keyword(s):  
Thermal Stability ◽  
Amino Acid ◽  
Amino Acid Substitutions ◽  
Cro Protein ◽  
Thermal Stability Of

Effect of single amino acid substitutions on the thermal stability of the .alpha. subunit of tryptophan synthase

Biochemistry ◽  
1980 ◽  
Vol 19 (7) ◽  
pp. 1290-1293 ◽  
Author(s):  
C. R. Matthews ◽  
M. M. Crisanti ◽  
G. L. Gepner ◽  
G. Velicelebi ◽  
J. M. Sturtevant
Keyword(s):  
Thermal Stability ◽  
Amino Acid ◽  
Alpha Subunit ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Tryptophan Synthase ◽  
Thermal Stability Of

scholarly journals Additivity of the Stabilization Effect of Single Amino Acid Substitutions in Triple Mutants of Recombinant Formate Dehydrogenase from the Soybean Glycine max

Acta Naturae ◽  
2015 ◽  
Vol 7 (3) ◽  
pp. 55-64 ◽  
Author(s):  
A. A. Alekseeva ◽  
I. S. Kargov ◽  
S. Yu. Kleimenov ◽  
S. S. Savin ◽  
V I. Tishkov
Keyword(s):  
Thermal Stability ◽  
Glycine Max ◽  
Amino Acid ◽  
Formate Dehydrogenase ◽  
Single Amino Acid ◽  
Inactivation Kinetics ◽  
Amino Acid Substitutions ◽  
Stabilization Effect ◽  
The Stability ◽  
Thermal Stability Of

Recently, we demonstrated that the amino acid substitutions Ala267Met and Ala267Met/Ile272Val (Alekseeva et al., Biochemistry, 2012), Phe290Asp, Phe290Asn and Phe290Ser (Alekseeva et al., Prot. Eng. Des. Select, 2012) in recombinant formate dehydrogenase from soya Glycine max (SoyFDH) lead to a significant (up to 30-100 times) increase in the thermal stability of the enzyme. The substitutions Phe290Asp, Phe290Asn and Phe290Ser were introduced into double mutant SoyFDH Ala267Met/Ile272Val by site-directed mutagenesis. Combinations of three substitutions did not lead to a noticeable change in the catalytic properties of the mutant enzymes. The stability of the resultant triple mutants was studied through thermal inactivation kinetics and differential scanning calorimetry. The thermal stability of the new mutant SoyFDHs was shown to be much higher than that of their precursors. The stability of the best mutant SoyFDH Ala267Met/Ile272Val/Phe290Asp turned out to be comparable to that of the most stable wild-type formate dehydrogenases from other sources. The results obtained with both methods indicate a great synergistic contribution of individual amino acid substitutions to the common stabilization effect.

scholarly journals Engineering Improves Enzymatic Synthesis of L-Tryptophan by Tryptophan Synthase from Escherichia coli

2020 ◽  
Vol 8 (4) ◽  
pp. 519
Author(s):  
Lisheng Xu ◽  
Fangkai Han ◽  
Zeng Dong ◽  
Zhaojun Wei
Keyword(s):  
Thermal Stability ◽  
Amino Acid ◽  
Directed Evolution ◽  
Amino Acid Residue ◽  
Rational Design ◽  
Molecular Engineering ◽  
Mutant Enzyme ◽  
Tryptophan Synthase ◽  
Mutant Library ◽  
Thermal Stability Of

To improve the thermostability of tryptophan synthase, the molecular modification of tryptophan synthase was carried out by rational molecular engineering. First, B-FITTER software was used to analyze the temperature factor (B-factor) of each amino acid residue in the crystal structure of tryptophan synthase. A key amino acid residue, G395, which adversely affected the thermal stability of the enzyme, was identified, and then, a mutant library was constructed by site-specific saturation mutation. A mutant (G395S) enzyme with significantly improved thermal stability was screened from the saturated mutant library. Error-prone PCR was used to conduct a directed evolution of the mutant enzyme (G395S). Compared with the parent, the mutant enzyme (G395S /A191T) had a Km of 0.21 mM and a catalytic efficiency kcat/Km of 5.38 mM−1∙s−1, which was 4.8 times higher than that of the wild-type strain. The conditions for L-tryptophan synthesis by the mutated enzyme were a L-serine concentration of 50 mmol/L, a reaction temperature of 40 °C, pH of 8, a reaction time of 12 h, and an L-tryptophan yield of 81%. The thermal stability of the enzyme can be improved by using an appropriate rational design strategy to modify the correct site. The catalytic activity of tryptophan synthase was increased by directed evolution.

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