Semi-rational evolution of ω-transaminase from Aspergillus terreus for enhancing the thermostability / 生物工程学报

ω-transaminase (ω-TA) is a natural biocatalyst that has good application potential in the synthesis of chiral amines. However, the poor stability and low activity of ω-TA in the process of catalyzing unnatural substrates greatly hampers its application. To overcome these shortcomings, the thermostability of (R)-ω-TA (AtTA) from Aspergillus terreus was engineered by combining molecular dynamics simulation assisted computer-aided design with random and combinatorial mutation. An optimal mutant AtTA-E104D/A246V/R266Q (M3) with synchronously enhanced thermostability and activity was obtained. Compared with the wild- type (WT) enzyme, the half-life t1/2 (35 ℃) of M3 was prolonged by 4.8-time (from 17.8 min to 102.7 min), and the half deactivation temperature (T1050) was increased from 38.1 ℃ to 40.3 ℃. The catalytic efficiencies toward pyruvate and 1-(R)-phenylethylamine of M3 were 1.59- and 1.56-fold that of WT. Molecular dynamics simulation and molecular docking showed that the reinforced stability of α-helix caused by the increase of hydrogen bond and hydrophobic interaction in molecules was the main reason for the improvement of enzyme thermostability. The enhanced hydrogen bond of substrate with surrounding amino acid residues and the enlarged substrate binding pocket contributed to the increased catalytic efficiency of M3. Substrate spectrum analysis revealed that the catalytic performance of M3 on 11 aromatic ketones were higher than that of WT, which further showed the application potential of M3 in the synthesis of chiral amines.

CSPGs loaded in a gelatin sleeve prevent regenerated axons escaping from the sciatic nerve suture after nerve transection in rats / 中华显微外科杂志

Objective:To observe the effect that covering the nerve suture with CSPGs-gelatin sleeve after the sciatic nerve transection in SD rats, and evaluate its effect to improve nerve regeneration in peripheral nerve transection model.Methods:Covered and protected the sciatic nerve by end-to-end suture in the SD rats with CSPGs-gelatin sleeve. From July, 2019 to September, 2019, 24 SD rats were randomly and evenly divided into 3 groups, which were direct suture group, gelatin sleeve group without CSPGs (blank group), and CSPGs-gelatin sleeve group (CSPGs group). In each group, 3 rats were used to mark fluorescent gold at 5 weeks after operation. At 6 weeks after operation, histological and electrophysiological tests were performed to evaluate the tissue regeneration in the end to end suture and the effect of peripheral nerve regeneration after transection in the other 5 rats. One-way ANOVA was used for data analysis. If the difference between groups was statistically significant, LSD method would be further used for pairwise comparison. P<0.05 was considered statistically significant. Results:The escape distances in direct suture groups, blank group and CSPGs group groups were (787.19±213.77) μm, (547.17±167.71) μm and (350.60±68.58) μm, respectively; The numbers of the axons that grow into the distal basement membrane tube in 3 groups were (6 360±736.89) /mm 2, (8 040±673.05) /mm 2 and (9 000±644.20) /mm 2, respectively; The numbers of sensory nerve cells that were marked by fluorescent gold in the dorsal root ganglion were (124.35±25.88) /mm 2, (165.36±30.74) /mm 2 and (208.98±20.51) /mm 2 in 3 groups, respectively. The differences were significant compared with the CSPGs-gelatin sleeve group ( P<0.05). Thus, it led to a better nerve regeneration after transection in CSPGs-gelatin sleeve group according to the electrophysiological test and histological section observation of gastrocnemius muscles( P<0.05). Conclusion:CSPGs loaded in the gelatin sleeve can inhibit axons regeneration, and prevent the regenerated axons escaping from the end to end suture that may cause disorder axons regeneration or traumatic neuroma, and therefore improve the effective nerve regeneration after sciatic nerve injury in SD rat.