Journal of Methods Microbiological Journal of Microbiological Methods 41 (2000) 1–8 www.elsevier.com / locate / jmicmeth Development of an improved in vitro activity assay for medium chain length PHA polymerases based on CoenzymeA release measurements 1 * Guy de Roo, Qun Ren , Bernard Witholt , Birgit Kessler ¨ ¨ ¨ Institute of Biotechnology, Swiss Federal Institute of Technology, ETH Zurich, Honggerberg HPT, CH-8093 Zurich, Switzerland Received 15 September 1999; received in revised form 19 November 1999; accepted 20 January 2000 Abstract 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 5,59-dithiobis (2-nitrobenzoic acid) (DTNB), a compound which specifically reacts with thiol groups. The formed adduct was measured spectro- photometrically 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.  2000 Elsevier Science B.V. All rights reserved. Keywords: PHA polymerase; In vitro activity assay; Medium chain length polyhydroxyalkanoates; Pseudomonas oleovorans 1. Introduction considerable attention for many years due to their biodegradable and thermoplastic properties. Poly-3-hydroxyalkanoate (PHA) is a linear, opti- Pseudomonads synthesize mainly mcl-PHAs, cally active polyoxoester which is a widespread formed of monomers of 6 to 14 carbons (Huisman et intracellular storage compound among prokaryotic al., 1989). To date, more than 100 different mono- organisms (for reviews, see Anderson and Dawes, mers have been found in PHA in which the side ¨ 1990; Steinbuchel, 1991a,b). PHAs have attracted chains contain a wide variety of functional groups ¨ (Steinbuchel and Valentin, 1995). The PHA com- position is influenced by the substrate added to the growth media, and determines the physical properties of the plastic (de Koning et al., 1996). Inclusion of different functional groups into PHA can extend their *Corresponding author. Tel.: 141-1-633-3286; fax: 141-1-633- physical properties and potential applications. 1051. PHA polymerases are the key enzymes of the E-mail address: bw@biotech.biol.ethz.ch (B. Witholt). 1 polymerisation reaction which use CoA thioesters as Present address: Institute of Microbiology, Swiss Federal Institute ¨ ¨ of Technology, ETH Zurich, CH 8092 Zurich, Switzerland. substrates. They can be distinguished in at least two 0167-7012 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0167-7012(00)00129-9