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

Impact of gene dosage and methanol concentration on Rhizopus chinensis recombinant lipase production in Pichia pastoris

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:拷贝数, 甲醇诱导浓度, 毕赤酵母, 华根霉脂肪酶

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 concentration 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.

Key words:Gene dosage, Methanol induced concentration, Pichia pastoris, Rhizopus chinensis lipase