This part is the CDS of a dextranase from a psychrophile organism (Gelidibacter algens) codon optimized for expression in Escherichia coli. Following reports that the Streptococcus mutans homologous dextranase has a higher activity when its N terminal (residues 1-99) and C-terminal (residues 733-850) are deleted (Kim et al., 2011), we removed them from the CDS submitted to the registry. Accordingly, our final CDS contains the region of the G. algens dextranase sequence that aligns to residues 104 - 734 of the PDB structure of the S. mutans dextranase (Suzuki et al., 2012). The dextranase of G. algens was successfully expressed in E. coli using the pET28a expression vector.


We determined that the optimal conditions for the expression of this protein with this expression system is induction with 1mM IPTG at 18°C for 18 hours. Afterward, we were able to purify the protein with IMACS chromatography.

Figure 1: SDS-PAGE 12% gels with full extract aliquots from the expression optimization experiment of the G. algens dextranase. The expected MW was 63.5kDa for the G.algens dextranase. We see the increase in expression through induction time and the effect of temperature and IPTG concentration on expression. The induction conditions in which the expression was the best were 0.
Figure 2: SDS-PAGE 12% gels showing verification of solubility and purification of G. algens dextranase. A. We compare the soluble fraction induced with 0.4mM and 1mM IPTG to verify which allows a higher concentration of the soluble enzyme. We observed that the 1mM condition confears a larger amount of dextranase in the soluble fraction. B. Gel showing the steps of IMACS purification. The purified protein seems to be quite pure. However, throughout the purification we lose a lot of dextranase.

We performed the Somogyi-Nelson enzymatic assays (Borkowska et al., 2019) to assess the reducing sugars resulting from the degradation of dextran by the dextranase. Those tests allowed us to conclude that the optimal conditions for the activity of this enzyme is pH 6.5 at 40°C and that its stability is not significantly affected in the pH range we tested, but that it is optimal at 15°C.

Figure 3: Activity and stability of the G. algens’ dextranase in function of the pH at 18 °C in function of the temperature at pH 6.5 using micro-SNT enzymatic assays. The enzyme appears to be more active at pH 6.5 and more stable at pH 7.0 while being more active at 40 °C and more stable at 15 °C and below.

Then, our team used the dextranase of G. algens to degrade dextran in ropy maple syrup. Rheological assays were performed to assess the diminution in viscosity of the ropy syrup caused by the dextranase. Indeed, we showed that our product is active in ropy maple syrup and allows to decrease its viscosity.

Figure 4: Viscosity of ropy maple syrup with and without added buffer and G. algens’ dextranase using rheological assays. The assays were performed at 20 °C and 10 °C with prior incubation of the ropy syrup with the dextranase. The control of the viscosimetric measurement was done with the same amount of buffer added to the ropy syrup. At 20 °C, the ropy syrup mixed buffer had a viscosity of 0.25 Pa•s while this value was 0.22 Pa•s when mixed with dextranase. The ropy syrup alone increases in viscosity with increasing fluctuations during the sampling time. At 10 °C, the ropy syrup mixed buffer had a viscosity of 0.47 Pa•s while this value was 0.42 Pa•s when mixed with dextranase.

References Borkowska, M., Białas, W., Kubiak, M., & Celińska, E. (2019). Rapid micro-assays for amylolytic activities determination: customization and validation of the tests. Applied Microbiology and Biotechnology, 103 (5), 2367–2379.
ddd Kim, Y.-M., Shimizu, R., Nakai, H., Mori, H., Okuyama, M., Kang, M.-S., Fujimoto, Z., Funane, K., Kim, D., & Kimura, A. (2011). Truncation of N- and C-terminal regions of Streptococcus mutans dextranase enhances catalytic activity. Applied Microbiology and Biotechnology, 91 (2), 329–339.
ddd Suzuki, N., Kim, Y.-M., Fujimoto, Z., Momma, M., Okuyama, M., Mori, H., Funane, K., & Kimura, A. (2012). Structural Elucidation of Dextran Degradation Mechanism by Streptococcus mutans Dextranase Belonging to Glycoside Hydrolase Family 66*. The Journal of Biological Chemistry, 287 (24), 19916–19926.