Probing the Role of Two Critical Residues in Inulin Fructotransferase (DFA III-Producing) Thermostability from <i>Arthrobacter</i> sp. 161MFSha2.1

Maltooligosyltrehalose synthase (MTSase) is a key enzyme in trehalose production. MTSase from Arthrobacter ramosus has poor thermostability, limiting its industrial use. In this study, mutant G415P was obtained by directed evolution and S361R/S444E was subsequently generated based on a structure analysis of the region around G415. The t 1/2 of G415P and S361R/S444E at 60 °C increased by 3.0-and 3.2-fold, respectively, compared with the wild-type enzyme. A triple mutant (G415P/S361R/S444E) was obtained through a combination of the above mutants, and its t 1/2 significantly increased by 19.7fold. Kinetic and thermodynamic stability results showed that the T 50 and T m values of the triple mutant increased by 7.1 and 7.3 °C, respectively, compared with those of the wild-type enzyme. When the triple mutant was used in trehalose production, the yield reached 71.6%, higher than the 70.3% achieved with the wild-type. Thus, the mutant has a potential application for industrial trehalose production.

Section: Discussionmentioning

confidence: 99%

Improved Thermostability of Maltooligosyltrehalose Synthase from Arthrobacter ramosus by Directed Evolution and Site-Directed Mutagenesis

Chen

et al. 2019

“…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 ).…”

Section: Resultsmentioning

confidence: 99%

“…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. extruding loop, which might be of conformational importance for substrate binding (Figure 3).…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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

Cai

Jiang

Zhang

et al. 2018

Self Cite

l-Citrulline is a nonessential amino acid with a variety of physiological functions and can be enzymatically produced by arginine deiminase (ADI, EC 3.5.3.6). The enzymatic-production approach is of immense interest because of its mild conditions, high yield, low cost, and environmental benignity. However, the major hindrances of l-citrulline industrialization are the poor thermostability and enzyme activity of ADI. Hence, in this work, directed evolution and site-directed mutagenesis aided with in silico screening, including the use of b-factor values and HoTMuSiC, were applied to a previously identified ADI from Enterococcus faecalis SK23.001 ( EfADI), and a triple-site variant R15K-F269Y-G292P was obtained. The triple-site variant displays a 2.5-fold higher specific enzyme activity (333 U mg), a lower K value of 6.4 mM, and a 6.1-fold longer half-life ( t = 86.7 min) than wild-type EfADI. This work provides a protein-engineering strategy to improve enzyme activity and thermostability, which might be transferrable to other ADIs and enzymes.

“…The molecular modeling procedure was similar to that of our previous work. 28 Briefly, the models were constructed by SWISS-MODEL server 29−31 using the structure of DFA III-forming IFTase (BsIFTase, PDB ID: 2INV) 32 as a template (sequence identity = 48%) and verified by SAVES server (https:// services.mbi.ucla.edu/SAVES/). Moreover, the structural energy minimization was implemented by Discovery Studio package (Accelrys, San Diego, CA, U.S.A.).…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…The site-directed mutagenesis was implemented according to the TaKaRa MutantBEST Kit protocol, which can be found in our previous work. 28 The mutated residues can be divided into three groups. The residues in group I including G236, A257, G281, and A314 are located in the central tunnel formed by three identical subunits.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

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

Zhang

Zhu

et al. 2017

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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.