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wd519wd706

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Expression of a Bacillus Phytase C Gene in Pichia pastoris and
Properties of the Recombinant Enzyme

The cloning and expression of a native gene encoding a Bacillus subtilis phytase using Pichia pastoris as the host is described. In addition, the inﬂuence of N-glycosylation on the biochemical properties of the B. subtilis phytase, the inﬂuence of pH on the thermostability of the recombinant and native B. subtilis phytases, and the resistance of both phytases to shrimp digestive enzymes and porcine trypsin are also described. After 48 h of methanol induction in shake ﬂasks, a selected recombinant strain produced and secreted 0.82 U/ml (71mg/liter) recombinant phytase. This phytase was N-glycosylated, had a molecular mass of 39 kDa after N-deglycosylation, exhibited activity within a pH range of 2.5 to 9 and at temperatures of 25 to 70°C, had high residual activity (85% 2%) after 10 min of heat treatment at 80°C and pH 5.5 in the presence of 5 mM CaCl2,
and was resistant to shrimp digestive enzymes and porcine trypsin. Although the recombinant Bacillus phytase had pH and temperature activity proﬁles that were similar to those of the corresponding nonglycosylated native phytase, the thermal stabilities of the recombinant and native phytases were different, although both were calcium concentration and pH dependent

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1楼 2010-11-12 00:42:54

已阅回复此楼关注TA 给TA发消息送TA红花 TA的回帖

wangyan820

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wd519wd706(金币+8, 翻译EPI+1):感谢你 2010-11-14 00:24:45

本文描述了以毕赤酵母为宿主的枯草杆菌植酸酶基因克隆和表达。同时本文还描述了N-糖基化对枯草杆菌植酸酶生化性质的影响，pH值对天然枯草杆菌植酸酶和重组酶的影响，以及两种酶对抗消化酶和猪胰蛋白酶的活性。在摇瓶中浸泡甲醇48 h后，挑选出的重组菌种产生并分泌出了0.82 U/ml(71mg/升)的重组酶。这种植酸酶是N-糖基化的，N-去糖基化后的分子量为39 kDa，在pH2.5 到 9和25 到70°C下表现出了活性，在80°C下10分钟、pH 5.5和5 mM CaCl2存在下的活性为85% 2%，且对消化酶和猪胰蛋白酶是有对抗作用的。尽管重组枯草杆菌植酸酶在相同pH值和温度下的未糖基化的天然植酸酶相似，但是热稳定性并不相同，尽管都为钙浓度和pH依赖性。