Annals of Microbiology, 57 (4) 583-588 (2007) Isolation and characterisation of poly(3-hydroxybutyrate-co-3-hydroxy- valerate) degrading actinomycetes and purification of PHBV depolymerase from newly isolated Streptoverticillium kashmirense AF1 Aamer Ali SHAH 1 *, Fariha HASAN 1 , Abdul HAMEED 1 , Safia AHMED 1 1 Department of Microbiology, Quaid-i-Azam University, Islamabad, Pakistan Received 29 May 2007 / Accepted 18 October 2007 Abstract - Streptoverticillium kashmirense AF1 with the ability to degrade a natural polymer, poly(3-hydroxybutyrate-co-3-hydroxy- valerate) (PHBV) was isolated from municipal sewage sludge by soil burial technique. The PHBV film was degraded by the action of extracellular enzymes secreted by the microorganisms. Degradation of PHBV was evident by the formation of clear zones of hydroly- sis on the polymer containing mineral salt agar plates. The extent of PHBV degradation increased up to 30 days of incubation. Maximum production of PHBV depolymerase was observed both at pH 8 and pH 7, 45 o C, 1% substrate concentration and in the pres- ence of lactose as an additional carbon source. Two types of extracellular PHBV depolymerases were purified from S. kashmirense AF1 by gel permeation chromatography using Sephadex G-75. The molecular weights of the two proteins were found to be 35 and 45 kDa approximately, as determined by SDS-PAGE. The results of the Sturm test also showed more CO 2 production as a result of PHBV degradation, in the test as compared to control. The present findings indicated the degradation capabilities of S. kashmirense AF1. Key words: Streptoverticillium kashmirense AF1, PHBV, biodegradation. INTRODUCTION Polyhydroxyalkanoates (PHAs) are natural biodegradable polymers which are synthesised and accumulated intracel- lularly by a wide variety of bacteria as a carbon and ener- gy reserve material, during unbalanced growth (Calabia and Tokiwa, 2006). These polyesters have become the focus of widespread attention, as environmentally friendly polymers which can be used in a wide range of agricultur- al, marine and medical applications such as; biodegradable polymer implants and controlled drug release systems (Zinn et al., 2001). Moreover, actual understanding of the PHA degradation is very important for the eco-friendly management of polymer wastes (Jendrossek and Handrick, 2002; Kim and Rhee, 2003; Steinbüchel and Lutke- Eversloh, 2003). Two of such polymers, poly(3-hydroxybu- tyrate) (PHB) as well as copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV) have properties similar to petrochemical based thermoplastics (Mergaert et al., 1994) and are biocompatible and biodegradable (Calabia and Tokiwa, 2006). PHBV has been receiving commercial inter- est as a promising candidate for the large-scale production of biodegradable and biocompatible thermoplastics (Doi, 1990). At the present time, although the known PHAs are quite diverse, only few of them are being investigated: isomers and copolymers hydroxybutyrate, poly-3-hydroxybutyrate [P(3HB)], poly-4-hydroxybutyrate [P(4HB)], poly-3- hydroxybutyrate-co-poly-4-hydroxybutyrate [P(3HB-co- 4HB)], poly-3-hydroxyoctanoate-co-poly-3-hydroxyhexa- noate [P(3HO-co-3HH)] and poly-3-hydroxybutyrate-co-3- hydroxyvalerate [poly(3HB-co-3HV)] (Williams and Martin, 2002). The copolymer PHBV features good strength prop- erties, which vary widely depending on monomer propor- tions; hence they have potential use for many applications (Madison and Huisman, 1999). The ability to degrade extracellular PHA is widely dis- tributed among bacteria, fungi and actinomycetes (Mergaert et al., 1995), depending on the secretion or sur- face-display of specific PHA depolymerases, which hydrol- yse the polymer by surface erosion to water soluble monomers and oligomers (Molitoris et al., 1996), which are metabolised to water and CO 2 by these microbes. Aerobic and anaerobic PHA degrading bacteria were isolated from various ecosystems such as soil, compost, aerobic and anaerobic sewage sludge, fresh and marine water, estuar- ine sediment, and air (Abou-Zeid et al., 2001). Since the pioneering work of Chowdhury (1963) and Delafield et al. (1965), biodegradation of PHB and PHBV has been investi- gated in different natural environments, such as soils (Mergaert et al., 1993; Kimura et al., 1994), composts (Mergaert et al., 1994; Pagga et al., 1995), natural waters (Doi et al., 1992) and sludge (Briese et al., 1994) as well as under laboratory conditions (Doi et al., 1992; Matavulj et al., 1993; Mergaert et al., 1994). PHB degrading actinomycetes (Mabrouk and Sabry, 2001), Streptomyces, have been isolated from soils and compost and they represent nearly a third of the total prokaryotic isolates from those environments (Mergaert et al., 1993). Klingbeil et al. (1996) and Manna et al. (1999) have established the versatility of Streptomyces to degrade * Corresponding author. Phone: +92-51-90643065; Fax: +92-51-9219888; E-mail: alishah_75@yahoo.com