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Acetobacter diazotrophicus levansucrase (LsdA) is a potential new candidate enzyme for kestose production from sucrose. Culture conditions for maximal LsdA yield were investigated. Variations in the medium pH had the most significant influence on LsdA production. The highest yield (32 mg l−1) was achieved at an initial pH of 8.0, although optimal growth occurred under acidic conditions. The introduction of extrachromosomal copies of the levansucrase gene increased the enzyme yield to 72 mg l−1. In the genetically modified A. diazotrophicus strain, levansucrase represented more than 95% of total secreted proteins showing an overall activity of 189 units ml−1.