Martinić Cezar Tea, Paić Antonia, Prekpalaj Stefani, Teparić Renata, Žunar Bojan, Stuparević Igor
Laboratory for Biochemistry, Department of Chemistry and Biochemistry, Faculty of Food Technology and Biotechnology, University of Zagreb, Croatia.
FEBS Open Bio. 2025 Apr;15(4):573-579. doi: 10.1002/2211-5463.13886. Epub 2024 Aug 28.
Yeast surface display is a promising biotechnological tool that uses genetically modified yeast cell wall proteins as anchors for enzymes of interest, thereby transforming yeast cell wall into a living catalytic material. Here, we present a comprehensive protocol for quantifying surface-displayed β-lactamase on the cell wall of model yeast Saccharomyces cerevisiae. We use β-lactamase as a reporter enzyme, which we tagged to be anchored to the cell wall closer to its N or C terminus, through the portion of the Pir2 or Ccw12 cell wall proteins, respectively. The catalytic activity of surface-displayed β-lactamase is assessed by its ability to hydrolyze nitrocefin, which produces a colorimetric change that is quantitatively measured by spectrophotometric analysis at 482 nm. This system enables precise quantification of the potential of S. cerevisiae strains for surface display, continuous real-time monitoring of enzyme activity, and facilitates the study of enzyme kinetics and interactions with inhibitors within the cell's native environment. In addition, the system provides a platform for high-throughput screening of potential β-lactamase inhibitors and can be adapted for the visualization of other enzymes, making it a versatile tool for drug discovery and bioprocess development.
酵母表面展示是一种很有前景的生物技术工具，它利用基因改造的酵母细胞壁蛋白作为目标酶的锚定物，从而将酵母细胞壁转化为一种活性催化材料。在此，我们展示了一种全面的方案，用于定量模型酵母酿酒酵母细胞壁上表面展示的β-内酰胺酶。我们使用β-内酰胺酶作为报告酶，分别通过Pir2或Ccw12细胞壁蛋白的部分区域将其标记，使其分别在靠近其N端或C端锚定到细胞壁上。表面展示的β-内酰胺酶的催化活性通过其水解头孢硝噻吩的能力来评估，头孢硝噻吩水解会产生比色变化，通过在482nm处的分光光度分析进行定量测量。该系统能够精确量化酿酒酵母菌株表面展示的潜力，对酶活性进行连续实时监测，并有助于在细胞的天然环境中研究酶动力学以及与抑制剂的相互作用。此外，该系统为潜在的β-内酰胺酶抑制剂提供了一个高通量筛选平台，并且可以适用于其他酶的可视化，使其成为药物发现和生物工艺开发的通用工具。
