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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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| 本文描述了以畢赤酵母為宿主的枯草桿菌植酸酶基因克隆和表達(dá)。同時(shí)本文還描述了N-糖基化對(duì)枯草桿菌植酸酶生化性質(zhì)的影響,pH值對(duì)天然枯草桿菌植酸酶和重組酶的影響,以及兩種酶對(duì)抗消化酶和豬胰蛋白酶的活性。在搖瓶中浸泡甲醇48 h后,挑選出的重組菌種產(chǎn)生并分泌出了0.82 U/ml(71mg/升)的重組酶。這種植酸酶是N-糖基化的,N-去糖基化后的分子量為39 kDa,在pH2.5 到 9和25 到70°C下表現(xiàn)出了活性,在80°C下10分鐘、pH 5.5和5 mM CaCl2存在下的活性為85% 2%,且對(duì)消化酶和豬胰蛋白酶是有對(duì)抗作用的。盡管重組枯草桿菌植酸酶在相同pH值和溫度下的未糖基化的天然植酸酶相似,但是熱穩(wěn)定性并不相同,盡管都為鈣濃度和pH依賴性。 |
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