The combined cross-linked enzyme aggregates (combi-CLEAs) containing galactitol dehydrogenase (Gdh) and NADH oxidase (Nox) were prepared for l-tagatose synthesis. To prevent the excess consumption of cofactor, Nox in the combi-CLEAs was used to in situ regenerate NAD⁺. In the immobilization process, ammonia sulfate and glutaraldehyde were used as the precipitant and cross-linking reagent, respectively. The preparation conditions were optimized as follows: 60% ammonium sulfate, 1:1 (molar ratio) of Gdh to Nox, 20:1 (molar ratio) of protein to glutaraldehyde, and 6 h of cross-linking time at 35 °C. Under these conditions, the activity of the combi-CLEAs was 210 U g⁻¹. The combi-CLEAs exhibited higher thermostability and preserved 51.5% of the original activity after eight cycles of reuses at 45 °C. The combi-CLEAs were utilized for the preparation of l-tagatose without by-products. Therefore, the combi-CLEAs have the industrial potential for the bioconversion of galactitol to l-tagatose.

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