Isomaltulose

The gene encoding sucrose isomerase (NCBI accession number YP_004505648.1) from S. plymuthica AS9 was optimized based on the preferred codon usage of E. coli and synthesized by Shanghai Generay Biotech Co. Ltd. (Shanghai, China). This synthetic gene was incorporated into the modified secretion-expression vector pET-24a-ompA, which was constructed using pET-24a(+) (Novagen; Madison, WI, USA) and ompA as backbone and secretion signal peptide coding sequence respectively, to generate the expression vector pET-24a-ompA/palI (S1 and S2 Files). E. coli JM109 (TakaRa, Dalian, China) was used as host for gene cloning, while E. coli BL21(DE3) (Novagen) was used for sucrose isomerase expression. The restriction enzymes, T₄ DNA ligase, Dpn I, agarose gel DNA purification kit, pMD18-T simple vector and E. coli JM109 were purchased from TaKaRa (Dalian, China). Isomaltulose and trehalulose were purchased from Sigma (Shanghai, China). All other chemicals were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China).

An HPLC system Dionex Ultimate 3000 (ThermoFisher Scientific, Waltham, MA, USA), with a Varian 380-LC (Varian, Palo Alto, CA, USA) evaporative light-scattering detector was employed. The column was HALO Penta-HILIC (AMT, Wilmington, DE, USA), 150 × 4.1 mm, with particles diameter 2.7 μm. No precolumn was used.
Chemicals were purchased from various sources: acetonitrile of a gradient-HPLC grade, ammonium formate, ammonium bicarbonate, ammonium acetate, formic acid, acetic acid, standards of glucose, isomaltulose, saccharose, cellobiose, trehalose were from Sigma-Aldrich (Merck, Germany). Maltodextrin DE 12-19 was purchased from 4fitness.cz (Brno, Czech Republic).
Samples containing isomaltulose available in the market of the Czech Republic were bought from various producers: Rossmann—Burgwedel, Germany; Heaven Labs—Prague, Czech Republic; Penco—Prague, Czech Republic; Nutrend—Olomouc, Czech Republic; Extrifit—Dolní Újezd, Czech Republic; Amix—Mnichovo Hradiště, Czech Republic; Energikakan—Skeppsbron, Sweden; Vitamin center—Bologna, Italy; for additional information, see Table 1.