基因拷貝數(shù)和甲醇濃度對重組畢赤酵母產(chǎn)華根霉脂肪酶的影響
李飛 喻曉蔚 沙沖 徐巖*
江南大學生物工程學院 教育部工業(yè)生物技術重點實驗室 江蘇 無錫 214122
摘 要: 將華根霉脂肪酶基因克隆到甲基營養(yǎng)型畢赤酵母中表達, 以甲醇利用快型菌株為宿主, 在7 L發(fā)酵罐水平對脂肪酶基因拷貝數(shù)分別為3,、5、6的3株基因重組菌--XY RCL-3,、XY RCL-5,、XY RCL-6進行高密度發(fā)酵調(diào)控, 同時研究了甲醇濃度對表達華根霉脂肪酶的影響。結(jié)果表明, XY RCL-5在相同條件下發(fā)酵產(chǎn)酶能力高于XY RCL-6和XY RCL-3, 最適甲醇誘導濃度控制在0.1%±0.02%時, 酶活可達到12 500 U/mL, 菌體干重達到204 g/L, 蛋白濃度也能達到8.02 g/L,。
關鍵詞: 基因拷貝數(shù), 甲醇誘導濃度, 畢赤酵母, 華根霉脂肪酶
生物谷推薦英文摘要：
Impact of gene dosage and methanol concentration on Rhizopus chinensis recombinant lipase production in
Pichia pastoris
LI Fei YU Xiao-Wei SHA Chong XU Yan*
(Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, China)
Abstract: The Rhizopus chinensis lipase genes was cloned and expressed in Mut+ methylotrophic Pichia pastoris with different gene dosage. The high-density fermentation of the recombinant Pichia pastoris with gene dosage of three (XY RCL-3), five (XY RCL-5) and six (XY RCL-6) was investigated on 7-liter fermenter. The impact of methanol concentration on Rhizopus chinensis lipase expression was also explored. Our results indicated that more Rhizopus chinensis lipase was produced in XY RCL-5 produced than that of XY RCL-6 and XY RCL-3 under the same conditions. When methanol concentra-tion was controlled at 0.10%±0.02%, the enzyme activity of Rhizopus chinensis lipase produced by XY RCL-5 could achieve 12 500 U/mL, and the dry weight of XY RCL-5 and protein concentration could reach 204 g/L and 8.02 g/L, respectively.
Keywords: Gene dosage, Methanol induced concentration, Pichia pastoris, Rhizopus chinensis lipase
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