Qiao Hanzhen, Zhang Wenfei, Guan Wutai, Chen Fang, Zhang Shihai, Deng Zixiao
South China Agricultural University, College of Animal Science, South China Agricultural University, Wushan Avenue, Tianhe District, Guangzhou, 510642, China.
South China Agricultural University, College of Animal Science, South China Agricultural University, Wushan Avenue, Tianhe District, Guangzhou, 510642, China; SCAU-UniOil Feeding Oil &Fat Research Centre, Guangzhou, 510642, China.
Protein Expr Purif. 2017 Oct;138:34-45. doi: 10.1016/j.pep.2017.05.005. Epub 2017 Jun 3.
Relatively poor heterologous protein yields have limited the commerical applications of Galactomyces geotrichum lipase I (GGl I) efficacy trials. To address this, we have redesigned the GGl I gene to preferentially match codon frequencies of Pichia pastoris (P. pastoris) while retaining the same amino acid sequence. The wild type and codon optimised GGl I (GGl I-wt and GGl I-op) were synthesised and cloned into pPICZαA with an N-terminal 6 × His tag sequence and expressed in P. pastoris X 33. The hydrolytic activity of GGl I-op was 150 U/mL, whereas the activity of the GGl I-wt could not be detected. GGl I-op recombinant proteins were purified by Ni-affinity chromatography and then characterised. The identity and purity of GGl I were confirmed by SDS-PAGE, MALDI-TOF mass spectrometry and Western blot analysis. Enzymatic deglycosylation was used to show that the lipase is a glycosylated protein, containing ∼10% sugar. The molecular weight (MW) of the GGl I secreted by recombinant P. pastoris was approximated at 63 kDa. The optimum pH and temperature of the recombinant lipase were 8.0 and 35 °C, respectively. The enzyme was active over a broad pH range (7.0-9.0) and temperature range (20 °C-45 °C). The lipase showed high activity toward medium- and long-chain fatty acid methyl esters (C8-C16) and retained much of its activity in the presence of Tween-80 and Trition X-100. Lipase activity was stimulated by Mg, Ca, Mn and Cu and inhibited by Fe, Fe, Zn and Co. This lipase may prove useful to the detergent industry and in organic synthesis reactions.
相对较低的异源蛋白产量限制了地丝酵母脂肪酶I（GGl I）功效试验的商业应用。为了解决这个问题，我们重新设计了GGl I基因，使其优先匹配巴斯德毕赤酵母（P. pastoris）的密码子频率，同时保留相同的氨基酸序列。合成了野生型和密码子优化的GGl I（GGl I-wt和GGl I-op），并将其克隆到带有N端6×His标签序列的pPICZαA中，在巴斯德毕赤酵母X33中表达。GGl I-op的水解活性为150 U/mL，而GGl I-wt的活性未检测到。通过镍亲和层析纯化GGl I-op重组蛋白，然后进行表征。通过SDS-PAGE、MALDI-TOF质谱和蛋白质免疫印迹分析确认了GGl I的同一性和纯度。酶促去糖基化表明该脂肪酶是一种糖基化蛋白，含糖量约为10%。重组巴斯德毕赤酵母分泌的GGl I的分子量（MW）约为63 kDa。重组脂肪酶的最适pH和温度分别为8.0和35°C。该酶在较宽的pH范围（7.0-9.0）和温度范围（20°C-45°C）内具有活性。该脂肪酶对中链和长链脂肪酸甲酯（C8-C16）表现出高活性，并且在吐温80和曲拉通X-100存在的情况下保留了大部分活性。脂肪酶活性受到Mg、Ca、Mn和Cu的刺激，而受到Fe、Fe、Zn和Co的抑制。这种脂肪酶可能对洗涤剂工业和有机合成反应有用。
