In this study, based on the codon preference of Escherichia coli BL21 (DE3), base optimization was carried out on the tannase encoding gene CsTanA of Camellia sinensis. In the optimized gene, the GC content was 51.9%, the codon adaption index (CAI) value was 0.8, and the consistency of the gene sequence before and after optimization was 77.8%. Recombinant expression and enzymatic characterization of the optimized gene using expression vector pET30a were investigated. The recombinant strain, with an A_{600 nm} value of approximately 0.6, was cultured at 30 ℃ in the presence of the inducer isopropyl-β-D-thiogalactoside (IPTG). In the supernatant from the broken cells, the recombinant tannase rCsTanA had a specific activity of 0.35 U/mg, and it was purified 4.4 folds by Ni-NTA affinity chromatography with a specific activity of 1.53 U/mg. The apparent molecular mass of the purified rCsTanA was about 39 kDa, and its optimal reaction temperature and pH were 40 ℃ and 7.0, respectively. The effects of different metal ion concentrations on the enzyme activity were different. At low concentration (1 mmol/L), K⁺ enhanced the enzyme activity of rCsTanA by 37.28%, while Ag⁺, Cu²⁺ and Fe³⁺ reduced the enzyme activity by more than 80%. At high concentration (5 mmol/L), Cu²⁺ completely inhibited the activity of rCsTanA. The surfactants sodium dodecyl sulfate (SDS), Tween-80 and cetyltrimethyl ammonium bromide (CTAB), and the polar solvents methanol, ethanol, glycerin, isopropanol and acetone had strong inhibitory effects on rCsTanA activity. The results of this study can provide theoretical support for the further application of plant tannase.

The aim of this study was to achieve efficient expression of mannanase CtMan26 from Chaetomium thermophilum in Pichia pastoris and to investigate the enzymatic properties of CtMan26 and its potential in the preparation of mannooligosaccharides (MOS). The mannanase CtMan26 contained a total of 448 amino acids and consisted of the family 35 carbohydrate-binding module (CBM35), Linker and the catalytic domain module of the GH26 family. Asp-395 and Leu-396 in the catalytic domain and Leu-100 in the auxiliary module of CBM35 were subjected to multiple hydrophobic forces and could form multiple hydrogen bonds with amino acids in the Linker. Recombinant enzyme CtMan26 was expressed with N-glycosylation modification. CtMan26 showed an apparent molecular mass of about 55 kDa. The optimum temperature of the recombinant enzyme was 55 ℃, and the optimum pH was 5.0. The enzymatic activity was maintained at more than 90% of its original value after being incubated at 50 and 60 ℃ for 1 hour, and it showed good stability at pH ranging from 4.0 to 12.0. The specific activity of CtMan26 was 23.15 U/mg, and its K_m and V_max values were 4.75 mg/mL and 27.17 μmol/(min·mg), respectively, when konjac glucomannan was used as the substrate. MOS prepared with the recombinant enzyme mainly consisted of mannose (14.2 mg/L), mannose disaccharides (20.1 mg/L), mannose trisaccharides (6.1 mg/L) and mannose tetrasaccharides (2.5 mg/L). The MOS exhibited good antioxidant capacity, which scavenged (90.80 ± 0.65)% of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, (80.7 ± 1.07)% of 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) cation radical, (69.10 ± 1.10)% of hydroxyl radical and (76.7 ± 3.14)% of superoxide anion radical at 2.4 mg/mL concentration. Meanwhile, the MOS had obvious prebiotic properties, which could inhibit the growth of Escherichia coli and promote the growth of the beneficial bacteria Pediococcus pentosaceus and Lactobacillu plantarum the effect being more pronounced with increasing concentration and incubation time. In conclusion, the recombinant mannanase CtMan26 has good enzyme properties and therefore has potential application in the preparation of MOS.

Polygalacturonases (PGA) have important application prospects in the field of food processing. This study aims to heterologously express and characterize the thermophilic polygalacturonase AfPGA from Aspergillus fumigatus HBFH5 and explore its application for the preparation of pectin oligosaccharides (POS) form pineapple peel. The results showed that the polygalacturonase AfPGA belongs to the GH28 family, with a molecular mass of 39.2 kDa and an optimal temperature of 70 ℃. After treatment at 55 ℃ for 1 h, the enzyme’s activity remained stable. The optimal pH value was 5.0, and over 80% of the initial activity remained after treatment for 1 hour within the pH range of 2.0–12.0. It had strong resistance to various metal ions and chemical reagents. When using polygalacturonic acid as the substrate, the K_m and V_max of the recombinant enzyme were 1.05 mg/mL and 52.6 μmol/(min·mg), respectively. Molecular dynamics (MD) simulation analysis found that the root mean square deviation (RMSD) and root mean square fluctuation (RMSF) of the mesophilic polygalacturonase ADPG2 were higher than those of AfPGA. In addition, POS prepared with AfPGA had strong antibacterial activity, which could significantly inhibit the growth of Salmonella, Staphylococcus aureus, Escherichia coli, and Shigella. POS also had good antioxidant properties. In summary, this study enriches the resources of thermophilic PGA, providing theoretical support for the application of this enzyme in POS preparation.