## 4 Discussion  
In order to develop CDH expression system suitable for whole-cell biocatalysis and directed evolution by high-throughput screening systems, cdh gene from _Phanerochaete chrysosporium_ was cloned in pCTCON2 vector for expression as a chimera with Aga2 protein on the surface of _Saccharomyces cerevisiae_ EBY100 yeast cells. After expression on the cell surface activity of the wild type CDH could not be detected. Significant decrease in specific activity of enzymes expressed on the surface of yeast cells compared with their soluble counterparts was described previously for other enzymes like glucose oxidase [30]. Since CDH has much lower specific activity compared to glucose oxidase [31] further decrease in specific activity of CDH due to the expression in the immobilized form on the surface of yeast cells could be the reason why we did not detect wild type CDH activity. During agar plate screening of yeast colonies expressing epPCR gene library of wtCDH, a triple mutant (D20N, A64T, and V592M) of CDH that was showing activity was found. All three substitutions were located on the surface of CDH protein, with two of them in heme domain (D20N and A64T) near the active site containing heme, Figure 7.  
Third substitution V592M was within FAD domain and also very close to the entrance of the active site containing FAD in it, Figure 8.  
Both A64T and V592M substitutions are in the region where there two domains interact with each other and these interactions are important for the activity of CDH [3]. Any change in this region can significantly influence activity of CDH and these two substitutions could be the reason tmCDH showed measurable activity on the surface of yeast cells, while at the same time wtCDH activity could not be detected. Figure 7: Presentation of the structure of the heme domain of CDH from _P. chrysosporium_ (PDB accession code 1D7C) with labelled substitutions. The picture was made with UCSF Chimera 1.13.1. (University of California, San Francisco, CA, US). Expression of tmCDH in liquid culture was further optimized and the highest activity was obtained at 25 \({}^{\circ}\)C after 24 h of galactose induction (Supplementary material; Figure S2). Optimal temperature for enzyme expression was lower than usually reported 30 \({}^{\circ}\)C, while expression time of 24 h was longer than optimal 16 h previously determined for glucose oxidase expression in the same host [32]. This could be result of higher molecular weight of CDH protein compared to GOx that very often requires lower expression rate of protein in order to properly fold. Some other proteins like eGFP when expressed in yeast also require lower expression temperature of 20 \({}^{\circ}\)C [33]. Due to the decrease in specific activity of surface displayed enzymes, we wanted to have more sensitive enzymatic assay than usually used spectrophotometric assays based on 2,6-dichlorophenol-indophenol and methylene green [34] and that could also be used with flow cytometry. Therefore, we tested fluorogenic resazurin dye as a substrate for detection of CDH activity on the surface of yeast cells. Resazurin reduction to resorufin that can be measured by red fluorescence was used previously for measuring viability of living cells [24] and blood glucose concentration [35]. The CDH assay using resazurin was optimized with respect to resazurin, cellobiose/lactose concentration, and pH of the reaction medium. Since resorufin has the highest fluorescence at pH above 7.5 when it is in anionic form [36] and CDH has an acidic pH optimum around pH 4-5 with only 20% of residual activity at pH 7.5 [37], optimal pH for resazurin assay was determined to be at pH 5.5 (Figure 1). Above this pH resorufin has higher fluorescence but enzyme is less active, while below this pH the enzyme is more active but due to protonation of resorufin fluorescence of that product is decreased. Similar results were obtained also during development of resazurin based assay for dihydrorotate dehydrogenase that also has an acidic pH optimum [38].