Existing industrial fermentations for Gluconobacter oxydans require a rich nitrogen source, which results in a relatively high production cost. In present study, amino acids utilization from yeast extract (YE) (20 g.L-1) was investigated, revealing that glutamate is disproportionately utilized, and consequently becomes a rate-limiting factor for the activity of membrane-bound sorbitol dehydrogenase (mSLDH). Additional supplementation of glutamate (0.1%) improved the abundance of coenzyme pyrroloquinoline quinone (PQQ) by 1.51-fold. Meanwhile, enzyme activity of mSLDH was improved by 1.21-fold, which was mainly dependent on the induction of PQQ. Partial substitution of YE (15 g.L-1) with glutamate (2 g.L-1) as nitrogen source was tested in G. oxydans fermentations and the thus-obtained resting-cells exhibited an high mSLDH activity towards the miglitol precursor N-2-hydroxyethyl-glucamine with an increased productivity (59.2 vs. 42.1 mg.(g DCW)(-1) h(-1)) and an equally high yield (81.56% vs. 80.35%) compared with cells fermented with 25 g.L-1 YE. Taken together, the results provide a feasible strategy for economical fermentation of G. oxydans in achieving high mSLDH activity for biotransformation applications in large-scale.