Fig. 4

Production optimization of the industrially relevant proteins tyrosine ammonia lyase (TAL) and sialidase (SIA). a A His-tagged sialidase-encoding sequence was translationally coupled to the chloramphenicol resistance gene. A TIR library was constructed, transformed into B. subtilis and grown with different chloramphenicol concentrations on agar plates. Colony forming units (CFUs) were counted for all concentrations for the library (TIR^lib) and the original, non-randomized clone (TIR^Orig) as control. b A Strep-tagged tal-encoding sequence was translationally coupled to the chloramphenicol resistance gene. A TIR library was constructed, transformed into L. lactis and grown with different chloramphenicol concentrations on agar plates. CFUs were counted for all concentrations for the library (TIR^lib) and the original, non-randomized clone (TIR^orig) as control. c The sialidase production level in the culture supernatant of the best performing clone (TIR^opt) was analyzed by Western blot, using an antibody against a His-tag (left panel). The increase in production was analyzed by densitometry and plotted as the relative protein production compared to the original clone (right panel). d TAL production level of the best performing clone (TIR^opt) was analyzed by Western blotting using an antibody against Strep-tag (left panel). The increase in production was analyzed by densitometry and plotted as the relative protein production compared to the original clone (right panel). e Sialidase (TIR^opt) was purified via a Ni²⁺-NTA column and purified product concentration was estimated using a BCA assay (left panel). Product per cell mass and per culture volume was calculated (right panel). The original construct (TIR^orig) was used for comparison