Purification, Characterization, and Hydrolysate Analysis of Dextranase From Arthrobacter oxydans G6-4B

Abstract

Dextran has aroused increasingly more attention as the primary pollutant in sucrose production and storage. Although enzymatic hydrolysis is more efficient and environmentally friendly than physical methods, the utilization of dextranase in the sugar industry is restricted by the mismatch of reaction conditions and heterogeneity of hydrolysis products. In this research, a dextranase from Arthrobacter oxydans G6-4B was purified and characterized. Through anion exchange chromatography, dextranase was successfully purified up to 32.25-fold with a specific activity of 288.62 U/mg protein and a Mw of 71.12 kDa. The optimum reaction conditions were 55°C and pH 7.5, and it remained relatively stable in the range of pH 7.0-9.0 and below 60°C, while significantly inhibited by metal ions, such as Ni⁺, Cu²⁺, Zn²⁺, Fe³⁺, and Co²⁺. Noteworthily, a distinction of previous studies was that the hydrolysates of dextran were basically isomalto-triose (more than 73%) without glucose, and the type of hydrolysates tended to be relatively stable in 30 min; dextranase activity showed a great influence on hydrolysate. In conclusion, given the superior thermal stability and simplicity of hydrolysates, the dextranase in this study presented great potential in the sugar industry to remove dextran and obtain isomalto-triose.

Keywords: Arthrobacter oxydans; dextran; hydrolysis; isomalto-oligosaccharides; purification.

FIGURE 1

Transparent zone formed by G6-4B on the plate containing blue dextran (A) and scanning electron micrograph (B).

FIGURE 2

Phylogenetic tree based on 16S rDNA gene sequences.

FIGURE 3

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of dextranase from G6-4B. M, protein marker; Lane 1, purified dextranase.

FIGURE 4

Effects of temperature (A) and pH (B) on dextranase activity and stability (C) and (D).

FIGURE 5

Hydrolysates of different hydrolysis systems by HPLC. (A) standards from G1 to G7 were glucose, maltose, isomalto-triose, isomalto-tetraose, isomalto-pentaose, isomalto-hexaose, and isomalto-heptaose, respectively. (B) Hydrolysates of 3% T20, 3%T40, and 3%T70 after 1 h. (C) Hydrolysates of 3% T20 after 30 min, 1 h, and 3 h. (D) Hydrolysates of 0.5%T20 by different dextranase activity (0.5, 1, and 5 U/ml).