Exploitation of the S-layer self-assembly system for site directed immobilization of enzymes demonstrated for an extremophilic laminarinase from Pyrococcus furiosus.
A fusion protein based on the S-layer protein SbpA from Bacillus sphaericus CCM 2177 and the enzyme laminarinase (LamA) from Pyrococcus furiosus was designed and overexpressed in Escherichia coli. Due to the construction principle, the S-layer fusion protein fully retained the self-assembly capability of the S-layer moiety, while the catalytic domain of LamA remained exposed at the outer surface of the formed protein lattice. The enzyme activity of the S-layer fusion protein monolayer obtained upon recrystallization on silicon wafers, glass slides and different types of polymer membranes was determined colorimetrically and related to the activity of sole LamA that has been immobilized with conventional techniques. LamA aligned within the S-layer fusion protein lattice in a periodic and orientated fashion catalyzed twice the glucose release from the laminarin polysaccharide substrate in comparison to the randomly immobilized enzyme. In combination with the good shelf-life and the high resistance towards temperature and diverse chemicals, these novel composites are regarded a promising approach for site-directed enzyme immobilization.
Tschiggerl
Helga
H
Center for NanoBiotechnology, University of Natural Resources and Applied Life Sciences, Gregor-Mendel-Strasse 33, A-1180 Vienna, Austria.
Breitwieser
Andreas
A
de Roo
Guy
G
Verwoerd
Theo
T
Schäffer
Christina
C
Sleytr
Uwe B
UB
D019843:Archaeal Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D001709:Biotechnology / Q000379:methods*; D002384:Catalysis / Q000187:drug effects; D002480:Cellulase / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003460:Crystallization; D004795:Enzyme Stability / Q000187:drug effects; D004800:Enzymes, Immobilized / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008567:Membranes, Artificial; D020027:Pyrococcus furiosus / Q000187:drug effects / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000648:ultrastructure; D013696:Temperature
D019843:Archaeal Proteins; D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D008562:Membrane Glycoproteins; D008567:Membranes, Artificial; D011993:Recombinant Fusion Proteins; C055764:S-layer proteins; D002480:Cellulase