Obtaining high-degree polymerized isomaltose is more difficult while achieving better prebiotic effects. We investigated the mutation specificity and enzymatic properties of SP5-Badex, a dextranase from the GH66 family of Bacillus aquimaris SP5, and determined its mutation sites through molecular docking to obtain five mutants, namely E454K, E454G, Y539F, N369F, and Y153N. Among them, Y539F and Y153N exhibited no enzymatic activity, but their hydrolysates included isomaltotetraose (IMO4). The enzymatic activity of E454G was 1.96 U/ml, which was 3.08 times higher than that before mutation. Moreover, 70% of the enzymatic activity could be retained after holding at 45°C for 180 min, which was 40% higher than that of SP5-Badex. Furthermore, its IMO4 content was 5.62% higher than that of SP5-Badex after hydrolysis at 30°C for 180 min. To investigate the effect of different amino acids on the same mutation site, saturation mutation was induced at site Y153, and the results showed that the enzyme activity of Y153W could be increased by 2 times, and some of the enzyme activity could still be retained at 50°C. Moreover, the enzyme activity increased by 50% compared with that of SP5-Badex after holding at 45°C for 180 min, and the IMO4 content of Y153W was approximately 64.97% after hydrolysis at 30°C for 180 min, which increased by approximately 12.47% compared with that of SP5-Badex. This site is hypothesized to rigidly bind to nonpolar (hydrophobic) amino acids to improve the stability of the protein structure, which in turn improves the thermal stability and simultaneously increases the IMO4 yield.

Intestinal diseases are mainly caused by a decrease in the relative abundance of probiotics and an increase in the number of pathogenic bacteria due to dysbiosis of the intestinal flora. High degree polymerization isomaltooligosaccharide (IMO) can promote probiotic metabolism and proliferation. In this study, the dextranase (PsDex1711) gene of marine bacterial Pseudarthrobacter sp. RN22 was cloned and expressed in Escherichia coli BL21 (DE3). The optimal pH and temperature of the dextranase were 6.0 and 30 °C, respectively, showing the highest stability at 20 °C. The dextran T70 could be hydrolyzed to produce IMO3, IMO4, IMO5, and IMO6 with a high degree of polymerization. The hydrolysate of 1 mg/mL could significantly promote the growth of Lactobacillus and Bifidobacterium after 12 h culture and the formation of biofilms by 58.2%. The hydrolysates could promote the proliferation of probiotics. Furthermore, the IC50 of scavenging rate of DPPH, hydroxyl radical, and superoxide anion was less than 20 mg/mL. This study provides a crucial theoretical basis for the application of dextranase such as pharmaceutical and food industries.

Dextranase is widely used in sugar production, drug synthesis, material preparation, and biotechnology, among other fields. The immobilization of dextranase using nanomaterials in order to make it reusable, is a hot research topic. In this study, the immobilization of purified dextranase was performed using different nanomaterials. The best results were obtained when dextranase was immobilized on titanium dioxide (TiO2), and a particle size of 30 nm was achieved. The optimum immobilization conditions were pH 7.0, temperature 25 °C, time 1 h, and immobilization agent TiO2. The immobilized materials were characterized using Fourier-transform infrared spectroscopy, X-ray diffractometry, and field emission gun scanning electron microscopy. The optimum temperature and pH of the immobilized dextranase were 30 °C and 7.5, respectively. The activity of the immobilized dextranase was >50% even after 7 times of reuse, and 58% of the enzyme was active even after 7 days of storage at 25 °C, indicating the reproducibility of the immobilized enzyme. The adsorption of dextranase by TiO2 nanoparticles exhibited secondary reaction kinetics. Compared with free dextranase, the hydrolysates of the immobilized dextranase were significantly different, and consisted mainly of isomaltotriose and isomaltotetraose. The highly polymerized isomaltotetraose levels could reach >78.69% of the product after 30 min of enzymatic digestion.