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

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。

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.