Multiple approaches of loop region modification for thermostability improvement of 4,6-α-glucanotransferase from Limosilactobacillus fermentum NCC 3057.

4,6-α-glucanotransferase (4,6-α-GT), as a member of the glycoside hydrolase 70 (GH70) family, converts starch/maltooligosaccharides into α,1-6 bond-containing α-glucan and possesses potential applications in food, medical and related industries but does not satisfy the high-temperature requirement due to its poor thermostability. In this study, a 4,6-α-GT (ΔGtfB) from Limosilactobacillus fermentum NCC 3057 was used as a model enzyme to improve its thermostability. The loops of ΔGtfB as the target region were optimized using directed evolution, sequence alignment, and computer-aided design. A total of 11 positive mutants were obtained and iteratively combined to obtain a combined mutant CM9, with high resistance to temperature (50 °C). The activity of mutant CM9 was 2.08-fold the activity of the wild type, accompanied by a 5 °C higher optimal temperature, a 5.76 °C higher melting point (Tm, 59.46 °C), and an 11.95-fold longer half-life time (t1/2). The results showed that most of the polar residues in the loop region of ΔGtfB are mutated into rigid proline residues. Molecular dynamics simulation demonstrated that the root mean square fluctuation of CM9 significantly decreased by "Breathing" movement reduction of the loop region. This study provides a new strategy for improving the thermostability of 4,6-α-GT through rational loop region modification.

Trehalose promotes high-level heterologous expression of 4,6-α-glucanotransferase GtfR2 in Escherichia coli and mechanistic analysis.

4,6-α-Glucanotransferases (4,6-α-GTs) hold great potential for applications in the food and medical industries because of their efficient transglycosylation ability. However, it is relatively difficult to achieve high soluble expression because of their high molecular weight and multidomain nature. In this study, 4,6-α-GT of Burkholderia sp. (GtfR2) was successfully expressed in E. coli, and the activity attained 1.55 × 104 U/mL by traditional fermentation optimization. However, a large number of inactive inclusion bodies of GtfR2 were still present due to aggregation and precipitation. The trehalose-mediated strategy was first proposed and applied in the fermentation process of GtfR2. Trehalose addition significantly reduced inclusion bodies, resulting in an increase in GtfR2 activity (6.48 × 104 U/mL), which was 4.20 times higher than that of the control group. Our molecular dynamics simulations revealed that trehalose could spontaneously stabilize the conformational dynamics of GtfR2 by binding to the groove, loop, α-helix and N-terminal unstable regions on the surface. This strategy was also available to enhance the soluble expression of other 4,6/4,3-α-GTs, which were increased by 3.03-77.19 times. This study is the first to observe that trehalose can inhibit the aggregation and precipitation of GtfR2, which provides a new perspective for the recombinant expression of 4,6/4,3-α-GTs.