Lip11 gene from oleaginous yeast Yarrowia lipolytica MSR80 was recombinantly expressed in Pichia pastoris X33. Native secretion signal present in its sequence resulted in 92% expression in comparison to α-secretion factor which resulted to 900 U/L in the extracellular broth. Catalytic triad in Lip11, like most lipases, was formed by serine, histidine, and aspartate residues. While point mutation disrupting putative glycosylation site (N389) present towards the C-terminus ruinously effected its stability and catalytic activity, disruption of the first putative glycosylation site (N17) located towards the N-terminus presented interesting insights. Mutation resulted in a variant N1 exhibiting higher thermal and acid stability; a t_1/2 of 198 min was obtained at 50 °C and it retained almost 80% activity following incubation at pH 3. Catalytic efficiency was improved by 2.7 fold and a 10 °C rise in temperature optima was accompanied by higher relative activity in acidic range. Thermal stability corresponded to convoying structural modifications in the tertiary structure, findings of fluorescence spectroscopy suggested. Thermal fluorescence studies revealed a Tm of 65 °C for both Lip11 and N1 and λmax of Lip11 exhibited a blue shift upon refolding while no shift in the λmax of N1 was observed. A resilient tertiary structure which could fold back to its native confirmation upon thermal denaturation and increase in surface-exposed hydrophobic residues as revealed by ANS binding assay summed up to thermal stability of N1. Furthermore, circular dichroism data disclosed an alternate ratio of alpha-helices and beta-sheets; respective values changed from 36% and 8% to 27% and 19%. Following mutation, substrate specificity remained unaffected and similar to native protein, N1 showed activation in presence of organic solvents and most divalent cations.

产油酵母解脂耶氏酵母a MSR80 的Lip11 基因在毕赤酵母X33中重组表达。与其序列中存在的天然分泌信号导致 92% 的表达相比，α-分泌因子导致细胞外肉汤中的 900 U/L。Lip11 中的催化三联体与大多数脂肪酶一样，由丝氨酸、组氨酸和天冬氨酸残基形成。虽然点突变破坏了存在于 C 端的假定糖基化位点 (N389) 严重影响了其稳定性和催化活性，但破坏位于 N 端的第一个假定糖基化位点 (N17) 提供了有趣的见解。突变导致变体 N1 表现出更高的热稳定性和酸稳定性；在_1/2在 50 °C 下获得了 198 分钟的时间，在 pH 3 下孵育后，它保留了几乎 80% 的活性。催化效率提高了 2.7 倍，最适温度升高 10 °C 伴随着酸性范围内更高的相对活性。荧光光谱的研究结果表明，热稳定性对应于三级结构中的护航结构修改。热荧光研究表明，Lip11 和 N1 的 Tm 均为 65°C，Lip11 的 λmax 在重折叠时呈现蓝移，而 N1 的 λmax 没有观察到变化。如ANS结合测定所揭示的，在热变性和表面暴露的疏水残基增加时可以折叠回其天然确认的弹性三级结构总结为N1的热稳定性。此外，圆二色性数据揭示了α-螺旋和β-折叠的交替比例；相应的值从 36% 和 8% 变为 27% 和 19%。突变后，底物特异性不受影响并且与天然蛋白质相似，N1 在有机溶剂和大多数二价阳离子存在下显示出活化。