Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, Guangdong Province, PR China; Wuliangye Yibin Co. Ltd., Yibin, 644007, Sichuan Province, PR China.
Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, Guangdong Province, PR China.
Protein Expr Purif. 2021 Apr;180:105804. doi: 10.1016/j.pep.2020.105804. Epub 2020 Dec 2.
Lipase from Rhizomucor miehei (RML) is a promising biocatalyst used in food industry, fine chemicals, and biodiesel production. Yeast surface display allows direct application of lipase in form of whole-cell biocatalyst, avoiding purification and immobilization process, but the protease of the host cell may affect the activity of displayed lipase. Herein, we used the protease-deficient Pichia pastoris, PichiaPink™ as host to display RML efficiently. RML gene, GCW21 gene and α-factor gene were co-cloned into plasmid pPink LC/HC and transformed into protease-deficient P. pastoris. After inducution expression for 96 h, the lipase activity of displayed RML reached 121.72 U/g in proteinase-A-deficient P. pastoris harboring high-copy plasmid, which exhibited 46.7% higher than recombinant P. pastoris without protease defect. Displayed RML occurred the maximum activity at pH 8.0 and 45 °C and the optimal substrate was p-nitrophenyl octanoate. Metal ions Li, Na, K, and Mg of 1-10 mM had activation towards displayed RML. Displayed RML was effectively improved in PichiaPink™ protease-deficient system, which may promote the further research and development for the industrial application of RML.
里氏木霉脂肪酶（RML）是一种有应用前景的生物催化剂，用于食品工业、精细化工和生物柴油生产。酵母表面展示允许脂肪酶直接作为全细胞生物催化剂应用，避免了纯化和固定化过程，但宿主细胞的蛋白酶可能会影响展示脂肪酶的活性。本文使用蛋白酶缺陷型毕赤酵母 PichiaPink™作为宿主，有效地展示 RML。RML 基因、GCW21 基因和α因子基因被共克隆到质粒 pPink LC/HC 中，并转化到蛋白酶缺陷型 P. pastoris 中。诱导表达 96 小时后，在携带高拷贝质粒的蛋白酶缺陷型 P. pastoris 中，展示的 RML 的酶活达到 121.72 U/g 蛋白，比没有蛋白酶缺陷的重组 P. pastoris 高 46.7%。展示的 RML 在 pH8.0 和 45°C 时活性最高，最佳底物为对硝基苯辛酸酯。1-10mM 的金属离子 Li、Na、K 和 Mg 对展示的 RML 有激活作用。在 PichiaPink™蛋白酶缺陷型系统中，展示的 RML 得到了有效改善，这可能促进了 RML 的进一步研究和开发，以实现其工业应用。
