乳糖诱导D塔格糖3差向异构酶基因在大肠杆菌中的表达
Expression of recombinant D-tagatose 3-epimerase in E.coli BL21/ (DE3) induced by lactose
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摘要: 以D-塔格糖3-差向异构酶高效表达菌株BL21（DE3）/pET22b-cbdte为研究对象,考察乳糖作为诱导剂诱导D-塔格糖3-差向异构酶在大肠杆菌BL21（DE3）中表达的可行性。在摇瓶发酵条件下,从诱导时机、诱导剂浓度、诱导时间及诱导温度等四个方面,研究诱导条件对目的蛋白表达的影响。结果表明,工程菌培养至对数生长中期OD值为1.0左右后,加入终浓度为5g/L的乳糖,于20℃的条件下诱导7h,可获得最大酶活。与异丙基-β-D-硫代吡喃半乳糖苷（IPTG）最适诱导条件相比,优化后的乳糖诱导酶活提高82.7%,可溶性目的蛋白的表达量提高99.5%,菌体生物量提高30.5%。研究结果为乳糖作为诱导剂应用于D-塔格糖3-差向异构酶的工业化生产提供有益的参考和借鉴。Abstract: The feasibility of using lactose as an inducer to substitute the common inducer IPTG ( isopropyl-β-D-thiogalactopyranoside) to induce the expression of D-tagatose 3-epimerase in E.coli BL21 ( DE3 ) was investigated.The effects of the main factors of induction such as the optimal time of induction, the concentration of the inducer, the duration and temperature of induction were analyzed.The result showed that the optimal condition was to add 5g/L ( final concentration) lactose at the mid-log-phase of cell growth ( OD600 = 1.0) and incubate at 20℃ for 7h.Under this condition, the enzyme activity, the soluble recombinant protein expression and final biomass induced by lactose was 82.7% , 99.5% , and 30.5% higher than induced by IPTG, respectively.The results indicated that lactose could be used as a promising inducer in the production of recombined D-tagatose 3-epimerase.
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