编码米曲霉果糖基转移酶基因在大肠杆菌中的重组表达

Recombinant Expression of Fructosyl Transferase Gene of Aspergillus oryzae in Escherichia coli

摘要:

为了大量获得果糖基转移酶，本文通过RT-PCR扩增获得米曲霉果糖基转移酶基因，连接到大肠杆菌pEASY-E1质粒上，并转化到大肠杆菌BL21-DE3菌株中成功表达，通过对重组菌诱导表达条件的优化，最终确定在诱导温度为25 ℃，诱导剂异丙基硫代半乳糖苷（IPTG）浓度为1.0 μmol/mL的条件下，该重组酶可以顺利表达。利用重组质粒PEASY-S1上的6×组氨酸标签，用亲和层析的方法纯化能够得到较高纯度的果糖基转移酶。以蔗糖为底物，用该酶进行果糖基转化生产低聚果糖的实验结果表明：重组果糖基转移酶具有一定催化能力，其酶活力达到59.0 U/g，且在低温诱导时稳定表达，具备一定的潜在开发利用价值。本实验成功在大肠杆菌中重组表达出果糖基转移酶，并且能够短时间内、大量表达具有催化活性的果糖基转移酶，可望为果糖基转移酶的工业化提供理论基础。

Abstract:

To obtain a higher yield of fructosyl transferase during industrial production, the fructosyl transferase gene from Aspergillus oryzae SBB201 strain was amplified using reverse transcriptase-polymerase chain reaction (RT-PCR) technique and cloned into plasmid pEASY-E1, which was then transformed into E. coli BL21-DE3 strain to achieve successful expression. Optimal conditions for inducing the expression of recombinant genes were ultimately determined for successful expression of fructosyl transferase, namely, induction temperature of 25 ℃ and a dose of 1.0 μmol/mL isopropylthiogalactoside (IPTG,) as an inducer. Taking advantage of the 6 × histidine-tag at the amino terminus of recombinant plasmid PEASY-S1, the expressed fructosyl transferase was purified by chelating affinity chromatography. Sucrose was used as substrate to test the catalyzing ability of fructosyl transferase. The results indicated that the recombinant fructosyl transferase had catalyzing ability and its enzyme activity reached 59.0 U/g. In addition, this recombinant fructosyl transferase could be stably expressed at low temperature. Thus, it has great potential for development and application. In this study, recombinant expression of fructosyl transferase in E. coli was successful, achieving rapid expression of a large amount of fructosyl transferase with catalyzing ability. This study could provide a theoretical basis for industrial production of fructosyl transferase.