3.4 Recyclable synthesis of isomaltulose under optimized conditions in 5 L fermenter
Based on the above results, one-batch whole-cell biocatalysis process was applied toC. glutamicum /pXMJ19/pdsi V280L/S499F cells to transform sucrose to isomaltulose in a 5 L fermenter. The other details were as follows: reaction volume 2 L, rotational speed 200 r.min−1, 1‰ (w/v) Triton X-100, and 500 g/L sucrose as substrate. The reaction time-course curves, consisting of isomaltulose production, as well as by-product glucose and fructose accumulation, are illustrated in Figure 3D. The concentration of isomaltulose increased rapidly for the first 6 h and gradually reached a plateau after 10 h. Specifically, the maximum yield of isomaltulose reached 451 g.L−1 in 10 h with a conversion rate of 90.2% (w/w) and a productivity of 45.1 g/L/h. At the same time, 7.2 ± 1.5 g/L of glucose and 10.6 ± 0.8 g/L of glucose fructose were concurrently produced as a byproduct during the reactions.
To increase isomaltulose productivity and save the cost of culturing bacteria, continuous catalytic reaction of recombinant cells was evaluated by measuring the conversion rate of 500 g/L sucrose in a 5 L fermentor biotransformation system. As depicted in Figure 3E, the whole-cell C. glutamicum /pXMJ19/pdsi V280L/S499F exhibited robust and great operational stability by reaching a total reaction batches of 180 h (every batch took 12 h, including 2 h for substrate preparation and cell collection) and maintaining more than 83.2 ± 2.1% of the initial isomaltulose productivity even after 15 batches of repeated utilization at 30 °C, but the conversion rate of WT decreased 63.3 ± 1.6% after 15 batches. Wu and Li also reported the use ofB. subtilis and Yarrowia lipolytica as whole-cell biocatalyst to synthesize isomaltulose respectively, while the conversion rate dropped to about 80% after 12 batches of biotransformation.[26, 28] Therefore, we successfully obtained a recombinant GRAS strain, which had the highest operational stability, and the productivity was also the highest reported in food-grade strains to date (details listed in the Table S3).