Abstract: The lipase from Thermoanaerobacter tengcongensis (LipA) is a kind of ester-bond hydrolysis enzyme catalyst of triacylglycerol. The LipA genes (lipA) were cloned into pET28a (T7 promoter) and pTrc99A (Trc promoter)，yielding pET28a-lipA and pTrc99A-lipA，and transformed into the Escherichia coli BL21 (DE3) and Top10，respectively. The strain harboring pTrc99A-lipA produced more recombinant LipA than the strain harboring pET28a-lipA. The recombinant LipA was purified to homogeneity from E. coli by a simple two-step procedure involving heat treatment and DEAE-Sephacel. The purified recombinant LipA reached 1.9U/mg. The recombinant LipA had a molecular mass of 42kDa on SDS-PAGE. The maximum activity was at pH 4.5 and 80℃. The purified enzyme was stable from pH 4.0—6.0，and retained approx. 60% of its activity after 2h at 85℃. The LipA activity were decreased 38.9% and 69.2% by Cu2+ and Zn2+ (5mmol/L)，respectively. Mn2+，Co2+ and Tween-20 had positive effect on the activity of LipA. The recombinant LipA had a Km of 1.5 mmol/L and kcat of 34.5s?1 for p-nitrophenyl-laurate.

Key words: Thermoanaerobacter tengcongensis, lipase, enzymatic properties, p-nitrophenyl-laurate, Thermoanaerobacter tengcongensis, lipase, enzymatic properties, p-nitrophenyl-laurate

摘要： 将来自腾冲菌的脂肪酶（LipA）基因（lipA）克隆到大肠杆菌表达载体pET28a（含T7启动子）和pTrc99A（含Trc启动子）中，转入大肠杆菌表达，发现Trc启动子更适合LipA的表达。通过热处理和DEAE-Sepharose阴离子柱纯化过程，重组LipA得到纯化，比酶活达到1.9U/mg，重组LipA分子量为42kDa。重组LipA在80℃、pH 4.5时酶活最高，经85℃保温2h，酶活保持60%以上；在pH值4.0～6.0之间，LipA具有较好的稳定性；Cu2+和Zn2+对酶活力分别有38.9%和69.2%的抑制作用，Mn2+、Co2+和Tween-20对该酶有较大的激活作用；以p-nitrophenyl-laurate (p-NP-C12)为底物时，该酶的Km值为1.5mmol/L，kcat为34.5s?1。

关键词: 腾冲菌, 脂肪酶, 酶学性质, 对硝基苯十二酸酯, 腾冲菌, 脂肪酶, 酶学性质, 对硝基苯十二酸酯