This study focused on the cloning, overexpression, and characterization of the gene encoding L-asparaginase (ansZ) from a nonpathogenic strain of Bacillus subtilis B11-06. The recombinant enzyme showed high thermostability and low affinity to L-glutamine. The ansZ gene, encoding a putative L-asparaginase II, was amplified by PCR and expressed in B. subtilis 168 using the shuttle vector pMA5. The activity of the recombinant enzyme was 9.98 U/mL, which was significantly higher than that of B. subtilis B11-06. The recombinant enzyme was purified by a two-step procedure including ammonium sulfate fractionation and hydrophobic interaction chromatography. The optimum pH and temperature of the recombinant enzyme were 7.5 and 40 °C, respectively. The enzyme was quite stable at a pH range of 6.0-9.0 and exhibited about 14.7 and 9.0% retention of activity following 2 h incubation at 50 or 60 °C, respectively. The Km for L-asparagine was 0.43 mM, and the Vmax was 77.51 μM/min. Results of this study also revealed the potential industrial application of this enzyme in reducing acrylamide formation during the potato frying process.

In the current work, the in vitro synthetic system for rare sugars was successfully transformed into an engineered Escherichia coli with a plasmid containing both the aldolase RhaD and phosphatase YqaB. By taking advantage of the inherent biosynthetic pathways in E. coli, this approach permits the use of simple and cheap glycerol for the synthesis of DHAP in vivo. Moreover, the introduction of the phosphatase into the E. coli system allows for the removal of the phosphate group on the synthetic intermediate to yield the neutral rare sugars, which can be readily secreted to the medium without accumulation in the cell.