One-step affinity purification of amidase from mutant strains of Pseudomonas aeruginosa.
Amidases (acylamide amidohydrolase EC 3.5.1.4) from mutant strains (i.e., B6, AI3, AIU1N, OUCH 4 and L10) of Pseudomonas aeruginosa were purified in one-step by ligand affinity chromatography using Epoxy-activated Sepharose 4B-acetamide. The yields of the purified enzymes were about 90% for all mutant strains with purification factors of about 10 and were apparently homogeneous when analysed by SDS-PAGE and native PAGE. The protein bands on native PAGE coincided with the stained band of enzyme activity for all amidase preparations. Affinity columns had a maximum binding capacity of 0.5 mg amidase protein/ml of sedimented gel and could be regenerated and reused several times without any loss of binding capacity and resolution. Affinity gels containing either semicarbazide or urea were also found useful for the isolation of amidase. The differences in substrate specificity of these amidases reported previously were also observed in the elution behaviour of these enzymes from the affinity columns.
Domingos
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A
LNETI/DTIQ-Bioquimica, Estrada das Palmeiras, Queluz, Portugal.
Karmali
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A
Brown
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PR
D000081:Acetamides; D000581:Amidohydrolases / Q000235:genetics / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004853:Epoxy Resins; D008970:Molecular Weight; D009154:Mutation; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D012685:Sepharose; D014508:Urea
D000081:Acetamides; D004853:Epoxy Resins; D014508:Urea; D012685:Sepharose; D000581:Amidohydrolases