The use of phenyl-Sepharose for the affinity purification of proteinases.
Phenyl-Sepharose is most often used as an adsorbent for hydrophobic interaction chromatography (HIC). We report on its effective use for the affinity purification of some extracellular thermostable proteinases from bacterial sources. Proteinases belonging to the serine, aspartate and metallo mechanistic classes were effectively retained by the media. Purification factors in the range of 2.9-60 and enzyme activity yields in excess of 88% were obtained. In some cases homogeneous enzyme was obtained from culture supernatants in a single step. A number of other proteinases from mammalian sources were also retained. The specificity of the enzyme/support interaction was studied. Proteinases complexed with peptide inhibitors (pepstatin and chymostatin) showed reduced binding to phenyl Sepharose indicating interaction with the active site cleft whereas modification with low molecular weight active site directed inactivators such as PMSF and DAN did not, indicating that binding may not be dependent on the catalytic site. Pepsinogen and the pro-enzyme form of the serine proteinase from the thermophilic Bacillus sp. strain Ak.1 were not retained by the media and could be resolved in an efficient manner from their active counterparts.
Prescott
M
M
Thermophile Research Unit, University of Waikato, Hamilton, New Zealand.
Peek
K
K
Veitch
D P
DP
Daniel
R M
RM
D001419:Bacteria / Q000201:enzymology; D002846:Chromatography, Affinity; D003470:Culture Media; D004792:Enzyme Precursors / Q000302:isolation & purification; D004795:Enzyme Stability; D006358:Hot Temperature; D010447:Peptide Hydrolases / Q000737:chemistry / Q000302:isolation & purification*; D012685:Sepharose / Q000031:analogs & derivatives* / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry
D003470:Culture Media; D004792:Enzyme Precursors; C020927:phenyl-sepharose; D012685:Sepharose; D010447:Peptide Hydrolases; D012697:Serine Endopeptidases