D-阿洛酮糖3-差向异构酶在大肠杆菌内的高效可溶性表达及发酵条件研究

将来源于Clostridium bolteae ATCCBAA-613的DAEase基因序列经密码子优化合成，以pCold TF为表达载体，冷休克启动子CspA低温诱导DAEase基因在大肠杆菌（Escherichia coli）BL21 （DE3）中表达，得到高效可溶性的重组Cb-DAEase并利用镍柱亲和层析分离纯化。结果表明，Cb-DAEase最适pH和温度为7.0和55 ℃，Co2+能够显著（P<0.05）增强酶活力。对培养条件进行优化得到，在7 g/L甘油、10 g/L酵母膏、1%接种量、0.25 mmol/LIPTG、诱导前培养5 h的条件下，Cb-DAEase活力达到（10.11±0.02）U/g，比优化前(1.38±0.01) U/g提高了7.33倍；以120 g/L的D-果糖为底物全细胞催化0.5 h后，D-阿洛酮糖产量为（11.47±0.04）g/L，比优化前（1.03±0.02）g/L提高了11.14倍。基于冷休克表达策略构建的重组菌经发酵优化后Cb-DAEase活力显著（P<0.05）提高，为高效制备D-阿洛酮糖提供了理论支持。

Efficient Soluble Expression and Fermentation Conditions of D-Allulose 3-Epimerase in Escherichia coli

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.