基因拷贝数和甲醇浓度对重组毕赤酵母产华根霉脂肪酶的影响
李飞 喻晓蔚 沙冲 徐岩*
江南大学生物工程学院 教育部工业生物技术重点实验室 江苏 无锡 214122
摘 要: 将华根霉脂肪酶基因克隆到甲基营养型毕赤酵母中表达, 以甲醇利用快型菌株为宿主, 在7 L发酵罐水平对脂肪酶基因拷贝数分别为3、5、6的3株基因重组菌--XY RCL-3、XY RCL-5、XY RCL-6进行高密度发酵调控, 同时研究了甲醇浓度对表达华根霉脂肪酶的影响。结果表明, XY RCL-5在相同条件下发酵产酶能力高于XY RCL-6和XY RCL-3, 最适甲醇诱导浓度控制在0.1%±0.02%时, 酶活可达到12 500 U/mL, 菌体干重达到204 g/L, 蛋白浓度也能达到8.02 g/L。
关键词: 基因拷贝数, 甲醇诱导浓度, 毕赤酵母, 华根霉脂肪酶
生物谷推荐英文摘要：
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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