Abstract: The DAEase gene sequence derived from Clostridium bolteae ATCCBAA-613 was synthesized by codon optimization. Using pCold TF as the expression vector, the cold-shock promoter CspA induced the expression of the DAEase gene in Escherichia coli BL21(DE3) at low temperature. Then, the highly soluble recombinant Cb-DAEase was obtained and purified by Ni-chelating affinity chromatography. Results showed that, the Cb-DAEase exhibited maximum activity at pH7.0 and 55 ℃. Additionally, the Cb-DAEase showed different sensitivities to the various metal ions when Co²⁺ was able to significantly (P<0.05) enhance its enzyme activity. The optimum fermentation conditions were determined as follows, 7 g/L glycerol, 10 g/L yeast extract, 1% inoculation volume, 0.25 mmol/L IPTG inducer, and incubation 5 h before the induction. Eventually, the secretion level of Cb-DAEase reached (10.11±0.02) U/g, which was 7.33-fold higher than that control (1.38±0.01) U/g. Through optimizing conditions, D-allulose was produced effectively by the whole-cell biotransformation system when 120 g/L D-fructose was used as the substrate for 0.5 h, the yield reached (11.47±0.04) g/L, which was 11.14-fold higher than that control (1.03±0.02) g/L. Overall, the recombinant strain constructed based on cold-shock expression increased significantly (P<0.05) Cb-DAEase activity after fermentation optimization, which would provide a theoretical basis for the efficient preparation of D-allulose.