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Gene coding for sucrose phosphorylase (SP) from Leuconostoc mesenteroides (Lm) was inserted into pET21b with N- (N, His6-LmSP) or C-terminal (C) His₆-tag (LmSP-His₆) and rigid linker (L) consisted of repetitive sequences of (EAAAK)n with n = 6 (~5 nm) and 14 (~10 nm) or n = 6 (~5 nm), 14 (~10 nm) and 19 (~15 nm), respectively. Fused genes and the plasmid were used for electroporation of E. coli (BL21)DE3 and constructs were expressed, isolated, and purified by affinity chromatography. Since inclusion bodies were observed with N-terminally fused linkers, only Cterminally His6-tagged constructs with higher specific activity were considered for immobilization experiments. The immobilization of the control and LmSP-CL₅nm on SEPABEADSTM EC-EP was successful without any unbound protein in the supernatant. Due to its size, the rigid linker of LmSPCL₅nm completely prevented Cu²⁺(SEPABEADSTM EC-EP) and His₆ (the construct) coupling. The control and three LmSP-CL constructs were readily bound to Ni-NTA Superflow and had a higher immobilization yield than the control, with the highest immobilization efficiency of LmSP-CL₁₅nm. Gen koji kodira saharozu fosforilazu (SF) iz Leuconostoc mesenteroides (Lm) je insertiran u pET21b sa N- (N, His₆-LmSF) ili C-terminalnim (C) His₆-tag-om (LmSF-His6) i rigidnim linkerom (L), koji je načinjen od ponavljajućih sekvenci (EAAAK)n, gdje je, redom, n = 6 (~5 nm) i 14 (~10 nm) ili n = 6 (~5 nm), 14 (~10 nm) i 19 (~15 nm). Geni fuzirani u plazmid korišteni su za elektroporaciju E. coli (BL21)DE3 i ovi konstrukti su eksprimirani, izolirani i pročišćeni afinitetnom kromatografijom. Inkluzijska tijela su formirana kod linkera na N-kraju proteina, pa su samo konstrukti sa His₆-tag-om na C-kraju proteina sa višom specifičnom aktivnosti korišteni u eksperimentima imobilizacije. Imobilizacija kontrole i LmSP-CL₅nm na nosaču SEPABEADSTM EC-EP bila je uspješna, te nevezani proteini nisu detektirani u supernatantu. Zbog svoje veličine, rigidni linker u LmSP-CL₁₅nm potpuno je spriječio povezivanje Cu²⁺(SEPABEADSTM EC-EP) i His₆ (s konstrukta). Kontrola i tri konstrukta LmSP-CL direktno su se vezali na Ni-NTA Superflow i, u usporedbi s kontrolom, uspješnija je bila imobilizacija ovih konstrukata, a najveća imobilizacijska učinkovitost je procijenjena kod LmSP-CL₁₅nm. |
