Efficient Production Of L-sorbose By Whole Cell Immobilization Of Gluconobacter Oxydans

In this sutdy, Gluconobacter oxydans WSH-003, a producer of L-sorbose, was chosen tostudy the effects of the immobilized cells and D-sorbitol oxidation pathway on production ofL-sorbose. And fed-batch fermentation was established with the combination of the researchand fermentation engineering. A recombinant strain was obtained by enhancing the oxidationpathway of D-sorbitol by metabolic engineering. The strain was utilized to improve thecontinuous and effective accumulation of L-sorbose by fed-batch fermentation. Main resultswere described as follows:(1) The effect of immobilizing G. oxydans WSH-003on a novel combination carrier toproduce L-sorbose was investigated. A combination carrier of sodium alginate (25g L-1) anddiatomite (5g L-1) was shown to be biocompatible and durable. Optimal conditions ofpreparation for immobilized cells were determined to be concentrations of15g L-1CaCl2,2.0-mm bead diameter and the beads were placed in the CaCl2solution and stored at4°C for3h. Under optimal conditions, the final D-sorbitol conversion rate was93.5%after36h.Additionally, the immobilized cells showed excellent stability and were maintained for morethan15days in a bioreactor. The D-sorbitol conversion rate was more than80%in theprevious nine runs of repeated-batch fermentation.(2) Enhanced the productivity of L-sorbose with immobilized cells by the fed-batchfermentation. On the basis of extensive analyses of the L-sorbose fermentation withimmobilized cells, the effect of feeding methods on the yield of L-sorbose was investigated.Our results showed that the fed-batch fermentation is more conducive than constant-fedfermentation. Besides, kinetic models of L-sorbose formation and D-sorbitol consumption inbatch fermentation with immobilized cells are proposed. According to the kinetic models, a21-day fed-batch fermentation was completed. The D-sorbitol consumption rates was up to90%and maintained for20days in the21-day fed-batch fermentation in a bioreactor.(3) Enhanced the productivity of L-sorbose in G. oxydans WSH-003by regulation of theD-sorbitol oxidation pathway. To increase the D-sorbitol dehydrogenase activity, the sldhABgene, which encodes the sorbitol dehydrogenase in G. oxydans, was overexpressed with astrong promoter, PtufB. Besides, the mRNA stability of the sldhAB gene was enhanced bylinking a series of poly(A/T) tails. The sldhAB expression levels of G. oxydans SLDH6wereabout6.2-fold and1.7-fold of those of the control and G. oxydans SLDH. The L-sorbose yeildof G. oxydans SLDH6was about1.4-fold of the control. In addition, fed-batch fermentationwith immobilized G. oxydans SLDH6cells was examined. The long-term performance ofimmobilized cells in a1-L fermenter over a period of21-day showed that the long-termculture process could further amplify the advantage gained by the presence of the poly(A/T)tails. The L-sorbose yield was improved by33.7%using the immobilization of G. oxydansSLDH6cells.