Introduction of sulphhydryl groups into the crystalline bacterial cell surface layer protein fromBacillus steatothermophilusPV72 and its application as an immobilization matrix
✍ Scribed by Margit Sára; Uwe B. Sleytr
- Publisher
- Springer
- Year
- 1992
- Tongue
- English
- Weight
- 893 KB
- Volume
- 38
- Category
- Article
- ISSN
- 1432-0614
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✦ Synopsis
The crystalline cell surface layer (S-layer) from Bacillus stearothermophilus PV72 was used as a matrix for reversible immobilization of fl-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°7o 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 gg fl-D-galactosidase could be immobilized per milligram of S-layer protein, which corresponds to one fl-D-galactosidase molecule [relative molecular mass (M0, 116000] per two S-layer subunits (Mr, 130000). At least 90070 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 fl-D-galactosidase was linked to carbodiimide-activated carboxyl groups of the S-layer protein.