## 4 Discussion
Optimal concentration of resazurin dye was determined to be 0.1 mM since at higher concentrations it behaves as an inhibitor of CDH activity (Figure 2). Drop in fluorescence signal produced by enzymatic action at higher concentrations of resazurin (above 0.025 mM) was also previously reported for diaphorase coupled detection of glucose [35]. Drop in the enzyme activity at higher resazurin concentrations were results of substrate inhibition phenomenon that is often caused by nonproductive binding of the substrate for the enzyme active site. Lactose and cellobiose that were used as CDH substrates in the assay also showed inhibition of fluorescence signal at higher concentrations. Substrate inhibition of CDH at higher concentrations due to non-productive binding of two disaccharide units is probably the result of two binding sites for hexoses that are present in the active site of CDH [39]. Substrate inhibition was observed only  
Figure 8: Presentation of the structure of the FAD domain of CDH from _P.chrysosporium_ (PDB accession code 1 NAA) with labelled substitutions. The picture was made with UCSF Chimera 1.13.1. (University of California, San Francisco, CA, US). with cellobiose and not for lactose, probably because of much higher affinity of binding subsites for glucose than for galactose, leading to higher probability of non-productive binding of two molecules of cellobiose for one molecule of the enzyme. This difference in affinity was confirmed by much lower CDH K\({}_{\text{m}}\) value for cellobiose than for lactose. Therefore, optimal substrate concentration was determined to be 10 mM for lactose and 0.08 mM for cellobiose. The resazurin assay showed linear correlation between amount of added enzyme and fluorescence intensity, showing that it could be used for detection of enzymatic variants with different activities usually created in directed evolution experiments. The assay was also tested and optimized for measuring CDH activity on the surface of yeast cells that is particularly important for developing high-throughput screening systems based on flow cytometry. Similar for soluble enzyme, substrate inhibition was detected, but due to the diffusional limitations that immobilized enzymes usually show, optimal substrate concentrations were higher, and for lactose it was 20 mM, while for cellobiose was 0.1 mM. Also, with CDH expressed on the cell surface the assay showed a linear response to the number of added cells. In order to test the resazurin assay as a high-throughput screening system in MTP, a tmCDH gene library was generated by epPCR using 0.05 mM MnCl\({}_{2}\) and around 2000 colonies were screened in MTPs by resazurin assay for higher activity. Two mutants with activity higher than the parent gene were found and characterized by sequencing and measuring kinetic parameters in immobilized form on the surface of yeast cells. The expression rate of Aga1 protein that acts as an anchor for CDH did not change in the CDH gene library and was reported to be 10\({}^{5}\) copies per single cell [13]. Therefore, the number of CDH mutants expressed on the surface of yeast cells should remain constant (10\({}^{5}\) per cell) and all kinetic data normalized to cell density of washed cells (OD600 nm) should not be influenced by mutant expression rate. Mutant H5 showed additional mutation V22A on the surface of CDH heme domain (Figure 7) that was very close to the already mutated D20N position in tmCDH. This could mean that mutations in this region can increase activity of CDH. Mutant H9 showed additional T84A substitution in heme domain that is located inside the protein molecule and A261P, E674G, N715S substitutions on the surface of FAD domain of CDH compared to the parent tmCDH (D20N, A64T, and V592M). Since substitutions in internal part of protein molecule can have profound influence on activity and stability, change of T84 residue to alanine, as one of the most acceptable substitutions after serine based on BLOSUM62 matrix table, can have positive influence on activity of CDH.