采用同源克隆法获得了雪白根霉的脂肪酶基因(rnl),将其连接到pPICZαA载体上,得到重组表达载体pPICZαA-rnl。将表达载体pPICZαA-rnl用限制性内切酶SacI线性化后,电击转入毕赤酵母X-33中。雪白根霉脂肪酶基因在毕赤酵母X-33中成功表达,重组菌株摇瓶培养144 h后,酶活可达48 U/mL。重组酶最适pH值为8.5,最适反应温度为35℃。1 mmol/L的Mn2+,Ca2+和K+以及1%的表面活性剂吐温20,吐温80,曲通100对重组脂肪酶具有激活作用。重组酶的最适底物为三月桂酸甘油酯(C12)。
王建荣
,
刘丹妮
,
李鹏
,
刘金山
,
周平发
,
陈丽芝
,
聂金梅
,
钟开兴
,
李阳源
. 雪白根霉脂肪酶基因在毕赤酵母中的高效表达及其酶学性质研究[J]. 食品与发酵工业, 2014
, 40(02)
: 83
-88
.
DOI: 10.13995/j.cnki.11-1802/ts.2014.02.001
A lipase gene from Rhizopus niveus( rnl) was isolated by homologous cloning method and subcloned into the vector of pPICZαA. The resultant recombinant plasmid pPICZαA-rnl was digested by SacI and transformed into the competent cells of Pichia pastoris strain X-33 through electroporation. The results showed that the gene was expressed successfully in the Pichia pastoris X-33. The optimal pH and temperature of the recombinant lipase were 35℃ and 8. 5,respectively. The activity of the recombinant lipase was stimulated by 1mmol / L Mn2 +,Ca2 +,K+and 1% Tween-20,Tween-80,TritonX-100. The recombinant lipase showed high activity toward trilaurin( C12).
