Significantly Improving the Thermostability and Catalytic Efficiency of Streptomyces mobaraenesis Transglutaminase through Combined Rational Design

Abstract

Streptomyces mobaraenesis transglutaminase has been widely used in food processing. We here significantly improved the catalytic properties of S2P-S23V-Y24N-S199A-K294L (TGm1), a highly stabilized variant of the transglutaminase. First, a virtual proline scan was performed based on folding free energy changes to obtain TGm1 variants with enhanced thermostability. Second, the residues within 15 Å of Cys64 in the enzyme–substrate complex of TGm1 were subjected to virtual saturation mutagenesis to generate the variants with reduced binding free energy and increased activity. After combining the favorable mutations, we obtained the variant FRAPD-TGm1-E28T-A265P-A287P (FRAPD-TGm2), exhibiting 66.9 min of half-life at 60 °C (t 1/2(60 °C)), 67.8 °C of melting temperature (T m), and 71.8 U/mg of specific activity, which are 2-fold, 2.6 °C, and 43.8% higher than those of FRAPD-TGm1, respectively. At last, to increase the surface negative net charge of FRAPD-TGm2, we introduced the mutations N96E-S144E-N163D-R183E-R208E-K325E, yielding FRAPD-TGm3. The latter’s t 1/2(60 °C), T m, and specific activity were 122.9 min, 68.6 °C, and 83.7 U/mg, which are 83.8%, 0.8 °C, and 16.6% higher than the former, respectively. FRAPD-TGm3 is thus a robust candidate for transglutaminase application.

Significant Improvement of Both Catalytic Efficiency and Stability of Fructosyltransferase from Aspergillus niger by Structure-Guided Engineering of Key Residues in the Conserved Sequence of the Catalytic Domain

“…In the middle and late stages of MD simulations, the values tend to be stable and the average rmsd of mutant C43N can be found to be lower than that of WT, which indicates that mutant C43N has a more stable protein backbone. Many studies reported that enzymes with lower rmsd values had enhanced thermostability. , …”

“…Many studies reported that enzymes with lower rmsd values had enhanced thermostability. 39,40 The RMSF reflects the possibility of movements of each amino acid of an enzyme, and a higher RMSF value of an amino acid indicates that the residue has a higher degree of freedom. 38 The analysis of RMSF plots at 325 K for WT and mutant C43N shows that most parts of the variant are similar (Figure 5B).…”

“…Moreover, the relative activities (percentage) of FruSG or its mutants were obtained by making a comparison with the highest activity of the same enzyme at other temperatures. The thermal stabilities of FruSG and its mutants were determined by incubating enzymes at different temperatures (30,40,50,60, and 70 °C) in Mcllvaine buffer (100 mmol/L, pH 5.5) for 2 h. After incubation, FruSG activities were measured at 45 °C in Mcllvaine buffer (100 mmol/L, pH 5.5). For one enzyme, the residual activities of FruSG or its mutants after 2 h of incubation at 30 °C were taken as 100%.…”

Significantly Enhanced Thermostability of Aspergillus niger Xylanase by Modifying Its Highly Flexible Regions

“…The increase in structural rigidity is one of the main factors for enzyme stability. 39 Compared to the wild-type enzyme, xynAm1 showed a remarkable decrease in RMSD (Figure 5A), and the total number of hydrogen bonds and hydrophobic and electrostatic interactions were increased accordingly (Table 2). These results suggested that the enhanced structural rigidity plays an important role in the stabilization of xynAm1.…”

DUnet: A deep learning guided protein-ligand binding pocket prediction

“…To measure the specific activity of smTG, 60 μL of pre-warmed (at 37□ for 5 min) sample protein was added to 150 μL substrate solution (200 mM Tris-HCl, 100 mM hydroxylamine, 10 mM GSH, 30 mM CBZ-Gln-Gly, pH 6.0), and the reaction was terminated by 60 μL of termination solution (termination solution achieved by mixing same volume of 3 M HCl, 12% trichloroacetic acid and 5% FeCl 3 ·H 2 O) after 10 min incubation under 37□ [29]. One unit of smTG activity was defined as 1 μmol L-glutamic acid γ-monohydroxamate produced per min.…”