Development of an improved in vitro activity assay for medium chain length PHA polymerases based on CoenzymeA release measurements.
An improved activity assay for polyhydroxyalkanoate (PHA) polymerases from Pseudomonas oleovorans GPo1 was developed. The activity assay is based on the detection of released Coenzyme A (CoA) using 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), a compound which specifically reacts with thiol groups. The formed adduct was measured spectrophotometrically with high sensitivity and accuracy. The assay was used to study the effect of several additives on the activity of granule-associated PHA polymerase. Mild non-ionic detergents such as Tween-20, Triton X-100, CHAPS and Hecameg all appeared to be strongly inhibitory. In contrast, bovine serum albumin (BSA) had a strong stimulatory effect on the activity and stability of the PHA polymerases. Using optimized conditions, activities up to 5.8 U/mg granule-bound polymerase have been measured.
de Roo
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G
Institute of Biotechnology, Swiss Federal Institute of Technology, Zürich, Switzerland.
Ren
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Q
Witholt
B
B
Kessler
B
B
D000217:Acyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins; D003065:Coenzyme A / Q000378:metabolism*; D003902:Detergents; D004228:Dithionitrobenzoic Acid; D006801:Humans; D011549:Pseudomonas / Q000201:enzymology*; D012680:Sensitivity and Specificity; D013439:Sulfhydryl Reagents
D001426:Bacterial Proteins; D003902:Detergents; D013439:Sulfhydryl Reagents; D004228:Dithionitrobenzoic Acid; D000217:Acyltransferases; C067032:poly(3-hydroxyalkenoate)polymerase; D003065:Coenzyme A