Efficient expression of γ-glutamyl transpeptidase in Bacillus subtilis via CRISPR/Cas9n and its immobilization.

In this study, we successfully applied the strategy of combining tandem promoters and tandem signal peptides with overexpressing signal peptidase to efficiently express and produce γ-glutamyl peptidase (GGT) enzymes (BsGGT, BaGGT, and BlGGT) from Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus licheniformis in Bacillus subtilis ATCC6051Δ5. In order to avoid the problem of instability caused by duplicated strong promoters, we assembled tandem promoters of different homologous genes from different species. To achieve resistance marker-free enzyme in the food industry, we first removed the replication origin and corresponding resistance marker of Escherichia coli from the expression vector. The plasmid was then transformed into the B. subtilis host, and the Kan resistance gene in the expression plasmid was directly edited and silenced using the CRISPR/Cas9n-AID base editing system. As a result, a recombinant protein expression carrier without resistance markers was constructed, and the enzyme activity of the BlGGT strain during shake flask fermentation can reach 53.65 U/mL. The recombinant BlGGT was immobilized with epoxy resin and maintained 82.8% enzyme activity after repeated use for 10 times and 87.36% enzyme activity after storage at 4 °C for 2 months. The immobilized BlGGT enzyme was used for the continuous synthesis of theanine with a conversion rate of 65.38%. These results indicated that our approach was a promising solution for improving enzyme production efficiency and achieving safe production of enzyme preparations in the food industry. KEY POINTS: • Efficient expression of recombinant proteins by a combination of dual promoter and dual signal peptide. • Construction of small vectors without resistance markers in B. subtilis using CRISPR/Cas9n-AID editing system. • The process of immobilizing BlGGT with epoxy resin was optimized.

In-situ formed Cyclodextrin-functionalized graphene oxide / poly (N-isopropylacrylamide) nanocomposite hydrogel as an recovery adsorbent for phenol and microfluidic valve.

Phenolic compounds are important industrial raw materials for various industrial applications, but phenol-containing wastewater creates significant environmental and biological hazards. To address these issues, a three-dimensional network graphene oxide-cyanoethyltriethoxysilane-ß-cyclodextrin/poly (N-isopropylacrylamide) (GO-CTES-ß-CD/PNIPAM) nanocomposite hydrogel as a phenol recovery adsorbent is prepared herein by in-situ polymerization. Double graft modification on the graphene oxide (GO) via the silane coupling agent 2-cyanoethyltriethoxysilane (CTES) and single (6-tetraethylenepentamine-6-deoxy)-ß-cyclodextrin (NH-ß-CD) compensated the loss of the active sites on both GO and N-isopropylacrylamide (NIPAM), and the hydrogel shows excellent mechanical properties as the chemical crosslinking and physical entanglement of the two components. Consequently, the composite hydrogel achieved an adsorption capacity of 131.64 mg·g-1 for the common environmental toxin 4-NP. After five repeated adsorption-desorption cycles, the hydrogel retained 74% of the initial 4-NP removal ratio. The adsorption results followed pseudo-first-order kinetics, corresponding to heterogeneous multilayer adsorption, which was regulated by a combination of surface adsorption and intra-particle diffusion mechanisms. In general, the nanocomposite hydrogel shows promising application in the field of recycling phenols from wastewater. Also, high photothermal conversion and temperature-sensitive properties are also demonstrated, which makes the hydrogel possessing great potential to be applied in smart microvalves.