Detection of polyhydroxyalkanoate synthase activity on a polyacrylamide gel.
This study presents a method to detect active polyhydroxyalkanoate (PHA) synthase on a polyacrylamide gel that combines the polyhydroxybutyrate (PHB) polymerization reaction with Sudan Black B staining. After separation of the protein samples on a modified sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the slab gel was submerged in a buffer containing beta-hydroxybutyryl-coenzyme A (3-HBCoA) as substrate and incubated at room temperature for in vitro PHB polymerization. The active PHA synthase catalyzed 3-HBCoA into the PHB polymer and was stained with Sudan Black B. The active PHA synthase appeared as a dark blue band. The activity staining was of high sensitivity, capable of detecting 3.9 ng (0.273 mU) of Cupriavidus necator H16 PHA synthase purified from recombinant Escherichia coli. The detection sensitivity of activity staining was comparable to that of Western blotting analysis. Furthermore, the high sensitivity of activity staining enabled specific detection of the active PHA synthase in the crude extract of wild-type strain C. necator H16. This study provides a rapid, sensitive, and highly specific method for detecting active PHA synthase in gel. The method could be applied to detecting PHA synthase from wild-type bacteria and to the process of enzyme purification.
Sheu
Der-Shyan
DS
Department of Marine Biotechnology, National Kaohsiung Marine University, Kaohsiung 81143, Taiwan. sds@mail.nkmu.edu.tw
Lai
Yung-Wei
YW
Chang
Rey-Chang
RC
Chen
Wen-Ming
WM
D000180:Acrylic Resins; D000217:Acyltransferases / Q000032:analysis* / Q000378:metabolism; D049921:Cupriavidus / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D012680:Sensitivity and Specificity
D000180:Acrylic Resins; C016680:polyacrylamide gels; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase