Abstract
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.
Highlights
Tryptophan is a precursor of serotonin, an important neurotransmitter, and an essential amino acid in the human body
The tryptophan synthase from Pyrococcus furiosus was modified via directed evolution, and the ability of the enzyme to synthesize 5-bromo-L-tryptophan and 6-hydroxy-L-tryptophan was promoted by modification of the tryptophan synthase β subunit [5]
The temperature factor values of the amino acids at the entrance of the substrate channel were sorted, and the top 20 amino acid residue sites were identified (Table 2). Among these 20 amino acid residues, we selected glycine 395 (G395), as it was the site with the largest temperature factor value for molecular modification
Summary
Tryptophan is a precursor of serotonin, an important neurotransmitter, and an essential amino acid in the human body. The enzyme can synthesize L-tryptophan with indole and L-serine as substrates. The substrate spectra of tryptophan synthase derived from Salmonella, including fluorine, chlorine, bromine, and iodine-substituted indole derivatives, have been reported in the literature. This enzyme can synthesize L-tryptophan derivatives [4]. The active sites of tryptophan synthase in Pyrococcus furiosus mainly include Asp300, Glu104, Lys, and Ala106, among which hydrogen bond formation between substrate complex E(Aex1) and Asp300 plays an important role in the catalytic process. The tryptophan synthase from Pyrococcus furiosus was modified via directed evolution, and the ability of the enzyme to synthesize 5-bromo-L-tryptophan and 6-hydroxy-L-tryptophan was promoted by modification of the tryptophan synthase β subunit [5].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
