关键词: 嗜热链球菌；右旋糖酐酶；异源表达；酶学性质

Abstract: In this study, bioinformatics analysis, heterologous expression and enzymatic characterization of dextranase (StDex) from Streptococcus thermophilus LMG 18311 were conducted to expand the dextranase resource library and lay the foundation for further research on its thermostability mechanism and its application in functional foods. The bioinformatics analysis indicated that StDex contained 166 amino acids with a theoretical molecular mass of 18.60 kDa and an isoelectric point of 6.59. It was a stable hydrophilic protein with a stable low molecular mass. StDex belonged to the glycoside hydrolase (GH) 66 family. Homology modeling showed that its catalytic site characteristics were significantly different from those of other dextranases, indicating that this enzyme might have a unique hydrolysis mechanism. Heterologous soluble expression of StDex was achieved using the pET28a-SUMO vector. After purification by Ni-chelating affinity chromatography, the specific activity of the recombinant StDex reached 90.18 U/mg. The enzymatic characterization indicated that the optimal catalytic conditions for the recombinant StDex were 50 ℃ and pH 7.0. This enzyme retained more than 60% of its original activity after 1 h of incubation at 20–65 ℃, and retained more than 80% of its original activity after 1 h of incubation at pH 4.0–8.0. Dextran T20 was found to be the most suitable substrate for the enzyme, and its relative activities towards dextran T10, T40 and T70 all exceeded 70.36%; its relative activity towards soluble starch was 46.74%. Ca2+, Ba2+, and Fe3+ at concentrations of 1–5 mmol/L had no significant inhibitory effect on the activity of StDex. More than 88% of its activity remained after treatment with 10 mmol/L Ca2+, Fe3+, Ni2+ or Mn2+, and the addition of 0.1% sodium fluoride increased its activity by 14.53%. Compared with dextran T10, T20, T40 and T70, StDex had the smallest Km value of 5.40 μmol/L and the largest kcat/Km value of 90.44 L/(μmol·min) for dextran T500, indicating that it had a higher substrate affinity and catalytic efficiency for dextran with a higher molecular mass. The major products produced from the hydrolysis of dextran T20 and T500 by StDex were both isomaltooligosaccharides ranging from triose to heptaose, and their oligosaccharide products all exhibited high antioxidant activity. The results of this study provide a theoretical basis for the application of StDex in the prevention and treatment of dental caries, the preparation of active oligosaccharides, and the development of functional foods.

Key words: Streptococcus thermophilus; dextranase; heterologous expression; enzymatic properties