Measuring the capacity of yeast for surface display of cell wall-anchored protein isoforms by using ß-lactamase as a reporter enzyme.

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 ß-lactamase on the cell wall of model yeast Saccharomyces cerevisiae. We use ß-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 ß-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 ß-lactamase inhibitors and can be adapted for the visualization of other enzymes, making it a versatile tool for drug discovery and bioprocess development.

First Molecular Evidence of Seewis Virus in Croatia.

Orthohantaviruses are mainly carried and transmitted by wild rodents, although during the last decade, they have also been identified in multiple species of shrews and moles. Orthohantavirus, Orthohantavirus seewisense (Seewis virus, SWSV), first detected in Switzerland in a single Sorex araneus (Eurasian common shrew) specimen, has been further described in several European countries, including Croatia's neighboring Slovenia and Hungary. Croatia is a well-known endemic region for several zoonotic agents including three different orthohantaviruses: Orthohantavirus puumalaense (PUUV), Orthohantavirus dobravaense (DOBV), and Orthohantavirus tulaense (TULV). In this study, nine shrews were tested and SWSV RNA was detected in liver, lung, and kidney belonging to two shrews (22.22%), one collected on Medvednica mountain in Zagreb County, and the other in the Stara Gradiska area in lowland Croatia. The phylogenetic analysis of the complete S segment's open reading frame (ORF) and partial L-segment revealed that the Croatian sequences, when compared to sequences from the adjacent geographic regions, form a specific genetic lineage. Two SWSV-positive shrew species-Sorex araneus and Neomys milleri (Mediterranean water shrew)-were identified using barcode-based sequence analysis. Therefore, the SWSV detection in N. milleri throughout the course of this study is seen as a rare find in this shrew species. To our knowledge, this is the first molecular and phylogenetic analysis of SWSV in Croatia.