节杆菌10137β-呋喃果糖苷酶基因克隆及在大肠杆菌表达

Cloning and Expression of β-fructofuranosidase Gene from Arthrobacter 10137 in E.coli

以一株高产β-呋喃果糖苷酶的节杆菌10137基因组为模板,成功地扩增出β-呋喃果糖苷酶基因,基因片段长度为1488bp,编码496个氨基酸。将扩增出的目的片段克隆到pFL-B13cl载体上,通过双酶切鉴定,确定该基因已成功克隆至表达载体。研究不同表达条件对β-呋喃果糖苷酶活性的影响,IPTG浓度0.5mmol/L,诱导温度22℃,诱导12h,比酶活可达1412.30U/g,SDS-PAGE电泳显示融合蛋白分子质量大小约为67u,亲和层析纯化后比酶活为2207.48U/g。将重组大肠杆菌在2L发酵罐中培养,诱导10h后β-呋喃果糖甘酶的比酶活为1208.23U/g。

A β-fructofuranosidase gene from Arthrobacter 10137 was cloned successfully and heterologously expressed in Escherichia coli BL21. The cloned fragment contained 1488bp and the protein consisted of 496 amino acids. We also studied the different culture conditions of recombinant E. coll. The results showed that the enzyme activity attain to maximum 1 412.30U/g when induced with 0.5 mmol/L IPTG for 12h at 22℃. The molecular weight was 67 ku by SDS-PAGE identified, and the purified enzyme activity was about 2 207.48 U/g . Bioreactor culture test in 2L bioreactor showed that the activity of β-fructofuranosidase was 1 208.23U/g after inducing 10h.