Calado Cecília R C, Mannesse Maurice, Egmond Maarten, Cabral Joaquim M S, Fonseca Luis P
Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisbon, Portugal.
Biotechnol Bioeng. 2002 Jun 20;78(6):692-8. doi: 10.1002/bit.10252.
This study focused on the growth of Saccha-romyces cerevisiae MM01 recombinant strains and the respective production of three extracellular heterologous cutinases: a wild-type cutinase and two cutinases in which the primary structure was fused with the peptides (WP)(2) and (WP)(4), respectively. Different cultivation and strategies were tested in a 2-L shake flask and a 5-L bioreactor, and the respective cell growth and cutinase production were analyzed and compared for the three yeast strains. The highest cutinase productions and productivities were obtained in the fed-batch culture, where wild-type cutinase was secreted up to a level of cutinase activity per dry cell weight (specific cell activity) of 4.1 Umg(-1) with activity per protein broth (specific activity) of 266 Umg(-1), whereas cutinase-(WP)(2) was secreted with a specific cell activity of 2.1 Umg(-1) with a specific activity of 200 Umg(-1), and cutinase-(WP)(4) with a specific cell activity of 0.7 Umg(-1) with a specific activity of 15 Umg(-1). The results indicate that the fusion of hydrophobic peptides to cutinase that changes the physical properties of the fused protein limits cutinase secretion and subsequently leads to a lower plasmid stability and lower yeast cell growth. These effects were observed under different cultivation conditions (shake flask and bioreactor) and cultivation strategies (batch culture versus fed-batch culture).
本研究聚焦于酿酒酵母MM01重组菌株的生长以及三种细胞外异源角质酶的各自产量：一种野生型角质酶和两种角质酶，其一级结构分别与肽(WP)₂和(WP)₄融合。在2 L摇瓶和5 L生物反应器中测试了不同的培养方法和策略，并对三种酵母菌株的细胞生长和角质酶产量进行了分析和比较。补料分批培养获得了最高的角质酶产量和生产率，其中野生型角质酶的分泌量达到每干细胞重量的角质酶活性（比细胞活性）为4.1 Umg⁻¹，每蛋白肉汤的活性（比活性）为266 Umg⁻¹，而角质酶-(WP)₂的分泌比细胞活性为2.1 Umg⁻¹，比活性为200 Umg⁻¹，角质酶-(WP)₄的分泌比细胞活性为0.7 Umg⁻¹，比活性为15 Umg⁻¹。结果表明，将疏水肽与角质酶融合会改变融合蛋白的物理性质，从而限制角质酶的分泌，进而导致较低的质粒稳定性和较低的酵母细胞生长。在不同的培养条件（摇瓶和生物反应器）和培养策略（分批培养与补料分批培养）下均观察到了这些影响。
