Improving the Catalytic Behavior of DFA I-Forming Inulin Fructotransferase from Streptomyces davawensis with Site-Directed Mutagenesis

Abstract

Previously, a α-d-fructofuranose-β-d-fructofuranose 1,2':2,1'-dianhydride (DFA I)-forming inulin fructotransferase (IFTase), namely, SdIFTase, was identified. The enzyme does not show high performances. In this work, to improve catalytic behavior including activity and thermostability, the enzyme was modified using site-directed mutagenesis on the basis of structure. The mutated residues were divided into three groups. Those in group I are located at central tunnel including G236, A257, G281, T313, and A314S. The group II contains residues at the inner edge of substrate binding pocket including I80, while group III at the outer edge includes G121 and T122. The thermostability was reflected by the melting temperature (T) determined by Nano DSC. Finally, the T values of G236S/G281S/A257S/T313S/A314S in group I and G121A/T122L in group III were enhanced by 3.2 and 4.5 °C, and the relative activities were enhanced to 140.5% and 148.7%, respectively. The method in this work may be applicable to other DFA I-forming IFTases.

Thermostability and Specific-Activity Enhancement of an Arginine Deiminase fromEnterococcus faecalisSK23.001 via Semirational Design forl-Citrulline Production

“…Figure d represents the space-filling model of M1’s structure, where the locations of these 10 residues are clearly shown on the surface of M1, and thus, they may have a direct relation to the rise in the optimum temperature. Previous studies have shown that the rigidity of surface residues is of great importance in the maintenance of protein thermostability. ,, Therefore, it is a common strategy to modify the surface amino acids for enhanced enzyme thermostability. , In addition, much protein-engineering research has revealed that besides the catalytic triads, the amino acids around the active site are crucial to the enzyme activity. , To achieve a thermostability-improved variant with minimum time and cost, three residues, Gly292, Phe269, and Gly264, were chosen as sites for point mutagenesis because (i) Gly292 possesses the highest b -factor value, indicating its flexibility in the structure, and (ii) Phe269 and Gly264 are close to the active site and located on the extruding loop, which might be of conformational importance for substrate binding (Figure ).…”

“…Recent studies have shown that double-site and even triple-site variants exhibit superior properties compared with single-site variants. , Therefore, on the basis of the results of enzyme activity, pH stability, thermostability, and kinetics of the double-site variants (R15K–G292P, R15K–F269Y, and R15K–G264P), a triple-site variant, R15K–F269Y–G292P, of Ef ADI was constructed to achieve an enzyme with improved enzyme activity and thermostability.…”

“…28,30,31 Therefore, it is a common strategy to modify the surface amino acids for enhanced enzyme thermostability. 19,20 In addition, much proteinengineering research has revealed that besides the catalytic triads, the amino acids around the active site are crucial to the enzyme activity. 33,34 To achieve a thermostability-improved variant with minimum time and cost, three residues, Gly292, Phe269, and Gly264, were chosen as sites for point mutagenesis because (i) Gly292 possesses the highest b-factor value, indicating its flexibility in the structure, and (ii) Phe269 and Gly264 are close to the active site and located on the The three amino acid residues chosen for subsequent site-directed mutagenesis are highlighted in bold.…”

“…Protein engineering can be divided into three main categories: irrational design, semirational design, and rational design . The combination of irrational and rational design constitutes the semirational design, which is an effective approach that intends to generate small but functionally rich mutant libraries. − Therefore, we employed a combinatorial approach (directed evolution and site-directed mutagenesis) on our previously constructed recombinant ADI from E. faecalis SK23.001 ( Ef ADI) expressed in Escherichia coli BL21­(DE3), which displayed a specific enzyme activity of 131.2 U mg –1 on l -arginine at pH 5.5 and retained 50% of its initial activity after 14.3 min at 45 °C, with the intention of achieving thermostability-improved variants. In this study, directed evolution via error-prone polymerase chain reaction (epPCR), in silico screening ( b -factor values and the HoTMuSiC algorithm), and site-directed mutagenesis were successively applied to wild-type Ef ADI, resulting in variants with enhanced enzyme activities and thermostabilities.…”