Introduction of sulphhydryl groups into the crystalline bacterial cell surface layer protein from Bacillus stearothermophilus PV72 and its application as an immobilization matrix.
The crystalline cell surface layer (S-layer) from Bacillus stearothermophilus PV72 was used as a matrix for reversible immobilization of beta-D-galactosidase via disulphide bonds. In order to obtain an immobilization matrix stable towards acid, alkali and reducing agents such as dithiothreitol (DTT), the S-layer subunits were first cross-linked with glutaraldehyde. This was done in a way whereby 75% of the free amino groups remained unmodified, and then could be completely converted into sulphhydryl groups upon reaction with the monofunctional imidoester iminothiolane. After activation of the sulphhydryl groups with 2,2'-dipyridyldisulphide, 550 micrograms beta-D-galactosidase could be immobilized per milligram of S-layer protein, which corresponds to one beta-D-galactosidase molecule [relative molecular mass (M(r)), 116,000] per two S-layer subunits (M(r), 130,000). At least 90% of the sulphhydryl groups from the S-layer protein could be regenerated for further activation by cleaving the disulphide bonds with DTT. In comparative studies beta-D-galactosidase was linked to carbodiimide-activated carboxyl groups of the S-layer protein.
Zentrum für Ultrastrukturforschung, Universität für Bodenkultur, Vienna, Austria.
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Sleytr
D002473:Cell Wall / Q000737:chemistry; D003432:Cross-Linking Reagents; D004220:Disulfides / Q000737:chemistry; D001411:Geobacillus stearothermophilus / Q000737:chemistry*; D013439:Sulfhydryl Reagents / Q000737:chemistry*
D003432:Cross-Linking Reagents; D004220:Disulfides; D013439:Sulfhydryl Reagents