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

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

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℃,Co^(2+)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/L IPTG、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。

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 Co^(2+) 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.