Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany.
Microb Cell Fact. 2024 Oct 5;23(1):263. doi: 10.1186/s12934-024-02544-5.
The β-galactosidase from Paenibacillus wynnii (β-gal-Pw) is a promising candidate for lactose hydrolysis in milk and dairy products, as it has a higher affinity for the substrate lactose (low K value) compared to industrially used β-galactosidases and is not inhibited by the hydrolysis-generated product D-galactose. However, β-gal-Pw must firstly be produced cost-effectively for any potential industrial application. Accordingly, the yeast Komagataella phaffii was chosen to investigate its feasibility to recombinantly produce β-gal-Pw since it is approved for the regulated production of food enzymes. The aim of this study was to find the most suitable way to produce the β-gal-Pw in K. phaffii either extracellularly or intracellularly.
Firstly, 11 different signal peptides were tested for extracellular production of β-gal-Pw by K. phaffii under the control of the constitutive GAP promoter. None of the signal peptides resulted in a secretion of β-gal-Pw, indicating problems within the secretory pathway of this enzyme. Therefore, intracellular β-gal-Pw production was investigated using the GAP or methanol-inducible AOX1 promoter. A four-fold higher volumetric β-galactosidase activity of 7537 ± 66 µkat/L was achieved by the K. phaffii clone 27 using the AOX1 promoter in fed-batch bioreactor cultivations, compared to the clone 5 using the GAP promoter. However, a two-fold higher specific productivity of 3.14 ± 0.05 µkat/g/h was achieved when using the GAP promoter for β-gal-Pw production compared to the AOX1 promoter. After partial purification, a β-gal-Pw enzyme preparation with a total β-galactosidase activity of 3082 ± 98 µkat was obtained from 1 L of recombinant K. phaffii culture (using AOX1 promoter).
This study showed that the β-gal-Pw was produced intracellularly by K. phaffii, but the secretion was not achieved with the signal peptides chosen. Nevertheless, a straightforward approach to improve the intracellular β-gal-Pw production with K. phaffii by using either the GAP or AOX1 promoter in bioreactor cultivations was demonstrated, offering insights into alternative production methods for this enzyme.
来自巨大芽孢杆菌（Paenibacillus wynnii）的β-半乳糖苷酶（β-gal-Pw）是一种很有前途的用于水解牛奶和乳制品中乳糖的候选酶，因为与工业上使用的β-半乳糖苷酶相比，它对乳糖（低 K 值）具有更高的亲和力，并且不受水解生成的产物 D-半乳糖的抑制。然而，β-gal-Pw 必须首先以具有成本效益的方式生产，才能用于任何潜在的工业应用。因此，选择酿酒酵母（Komagataella phaffii）来研究其用于重组生产β-gal-Pw 的可行性，因为它被批准用于受监管的食品酶生产。本研究的目的是找到最适合的方法在酿酒酵母中胞外或胞内生产β-gal-Pw。
首先，通过酿酒酵母在组成型 GAP 启动子的控制下，测试了 11 种不同的信号肽用于β-gal-Pw 的胞外生产。没有一种信号肽能导致β-gal-Pw 的分泌，这表明该酶的分泌途径存在问题。因此，使用 GAP 或甲醇诱导的 AOX1 启动子研究了胞内β-gal-Pw 的生产。在补料分批生物反应器培养中，使用 AOX1 启动子的 K. phaffii 克隆 27 的比容β-半乳糖苷酶活性达到 7537±66 μkat/L，是使用 GAP 启动子的克隆 5 的四倍。然而，当使用 GAP 启动子生产β-gal-Pw 时，比活达到 3.14±0.05 μkat/g/h，是使用 AOX1 启动子的两倍。经过部分纯化，从 1 L 重组酿酒酵母培养液（使用 AOX1 启动子）中获得了一种总β-半乳糖苷酶活性为 3082±98 μkat 的β-gal-Pw 酶制剂。
本研究表明，β-gal-Pw 是由酿酒酵母在细胞内产生的，但选择的信号肽未能实现其分泌。然而，通过在生物反应器培养中使用 GAP 或 AOX1 启动子，展示了一种简单的方法来提高酿酒酵母细胞内β-gal-Pw 的产量，为该酶的替代生产方法提供了思路。
