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.

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

Directed Evolution of Immune-Escaping Adeno-Associated Virus Vectors

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3532-3532
Author(s):  
Stephan Maersch ◽  
Anke Huber ◽  
Michael Hallek ◽  
Hildegard Buening ◽  
Luca Perabo
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gene Transfer ◽  
Directed Evolution ◽  
Neutralizing Antibodies ◽  
Rational Design ◽  
Multiple Point ◽  
Repeated Treatment ◽  
Therapeutic Gene ◽  
Adeno Associated Virus

Abstract Efficiency of therapeutic gene transfer by adeno-associated virus of serotype 2 (AAV-2) vectors is hampered in patients with pre-existing immunity against the natural virus. Genetic engineering by rational design or directed evolution has been employed in the last 3 years to generate capsids that escape antibody neutralization and has led to identify several amino acid residues of the capsid proteins that can be mutated in order to decrease antibody recognition (Perabo et al., 2006; Maheshri et al, 2006; Lochrie et al., 2006). In this novel study, we aimed to exploit the comprehensive knowledge gathered so far by generating novel capsid variants that carried multiple point mutations at these previously identified sites. Capsid libraries were generated by codon randomization of several immunogenic residues and screened to isolate mutants that most efficiently infected human cells despite the presence of anti-AAV2 neutralizing antibodies. Besides testing novel combinations of concomitant mutations at these sites, this approach allowed for the first time an exhaustive scanning of combinations of all 20 natural amino acids at each position. We identified several novel capsid mutants that remain highly infectious even when incubated with serum concentrations that completely neutralize wild type AAV2. Our results demonstrate that combining mutations at several sites it is possible to improve the immune-escaping ability of the capsid. In addition, we show that escaping ability and other biological characteristics of these mutants are strongly dependent on the type of amino acid substituted, demonstrating that an exact choice of substituted amino acids is essential to maximize stealth properties and minimize loss of packaging ability, particle stability and transduction efficacy. These vectors can be used for therapeutic gene transfer to patients with pre-existing immunity, or for repeated treatment after antibodies are generated upon first application.

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 Improved Insights into Protein Thermal Stability: From the Molecular to the Structurome Scale

2016 ◽  
Author(s):  
Fabrizio Pucci ◽  
Marianne Rooman
Keyword(s):  
Thermal Stability ◽  
Amino Acid ◽  
Protein Stability ◽  
Large Scale ◽  
Molecular Level ◽  
Temperature Dependent ◽  
Natural Evolution ◽  
Multi Scale ◽  
Large Scale Analysis ◽  
Thermal Stability Of

AbstractDespite the intense efforts of the last decades to understand the thermal stability of proteins, the mechanisms responsible for its modulation still remain debated. In this investigation, we tackle this issue by showing how a multi-scale perspective can yield new insights. With the help of temperature-dependent statistical potentials, we analyzed some amino acid interactions at the molecular level, which are suggested to be relevant for the enhancement of thermal resistance. We then investigated the thermal stability at the protein level by quantifying its modification upon amino acid substitutions. Finally, a large scale analysis of protein stability - at the structurome level - contributed to the clarification of the relation between stability and natural evolution, thereby showing that the mutational profile of thermostable and mesostable proteins differ. Some final considerations on how the multi-scale approach could help unraveling the protein stability mechanisms are briefly discussed.

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