State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China; China Research Center, DuPont Industrial Bioscience, Shanghai, 200335, PR China.
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China.
Protein Expr Purif. 2020 Nov;175:105721. doi: 10.1016/j.pep.2020.105721. Epub 2020 Aug 5.
Lipomax is a commercialized foldase-dependent Pseudomonas lipase that was previously expressed only in Pseudomonas strains. Here, using Pichia pastoris as the host, we report a new co-expression method that leads to the successful production of Lipomax. The active Lipomax is extracellularly co-expressed with its cognate foldase (LIM); and the purified enzyme mix has the optimum pH at pH 8.0 and an optimal temperature around 40 °C. N-glycosylation was observed for Pichia produced Lipomax, and its reduction was shown to increase the lipolytic activity. With different p-nitrophenyl esters as the substrates, the substrate profiling analyses further indicate that Lipomax prefers esters with middle-long chain fatty acids, showing the highest specific activity to p-nitrophenyl caprylate (C8). The extracellular co-expression of Lipomax and LIM in Pichia will not only increase our ability to investigate additional eukaryotic hosts for lipase expression, but also be of considerable value in analyzing other foldase-dependent lipases.
Lipomax 是一种商业化的折叠酶依赖型假单胞菌脂肪酶，以前仅在假单胞菌菌株中表达。在这里，我们使用巴斯德毕赤酵母作为宿主，报告了一种新的共表达方法，该方法可成功生产 Lipomax。活性 Lipomax 与同源折叠酶（LIM）在细胞外共表达；纯化的酶混合物在 pH8.0 时具有最佳 pH 值，在 40°C 左右时具有最佳温度。观察到巴斯德毕赤酵母产生的 Lipomax 发生了 N-糖基化，并且其还原被证明可以提高脂肪酶活性。用不同的对硝基苯酚酯作为底物进行底物谱分析进一步表明，Lipomax 优先选择具有中长链脂肪酸的酯，对 p-硝基苯辛酸酯（C8）表现出最高的比活性。巴斯德毕赤酵母中 Lipomax 和 LIM 的细胞外共表达不仅将提高我们对其他脂肪酶表达的真核宿主进行研究的能力，而且对分析其他折叠酶依赖型脂肪酶也具有重要价值。
