Isolation and Characterisation of Poly(3-Hydroxybutyrate-Co-3
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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 SHAH1*, Fariha HASAN1, Abdul HAMEED1, Safia AHMED1 1Department 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 oC, 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 CO2 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 4HB)], poly-3-hydroxyoctanoate-co-poly-3-hydroxyhexa- noate [P(3HO-co-3HH)] and poly-3-hydroxybutyrate-co-3- Polyhydroxyalkanoates (PHAs) are natural biodegradable hydroxyvalerate [poly(3HB-co-3HV)] (Williams and Martin, polymers which are synthesised and accumulated intracel- 2002). The copolymer PHBV features good strength prop- lularly by a wide variety of bacteria as a carbon and ener- erties, which vary widely depending on monomer propor- gy reserve material, during unbalanced growth (Calabia tions; hence they have potential use for many applications and Tokiwa, 2006). These polyesters have become the (Madison and Huisman, 1999). focus of widespread attention, as environmentally friendly The ability to degrade extracellular PHA is widely dis- polymers which can be used in a wide range of agricultur- tributed among bacteria, fungi and actinomycetes al, marine and medical applications such as; biodegradable (Mergaert et al., 1995), depending on the secretion or sur- polymer implants and controlled drug release systems face-display of specific PHA depolymerases, which hydrol- (Zinn et al., 2001). Moreover, actual understanding of the yse the polymer by surface erosion to water soluble PHA degradation is very important for the eco-friendly monomers and oligomers (Molitoris et al., 1996), which are management of polymer wastes (Jendrossek and Handrick, metabolised to water and CO2 by these microbes. Aerobic 2002; Kim and Rhee, 2003; Steinbüchel and Lutke- and anaerobic PHA degrading bacteria were isolated from Eversloh, 2003). Two of such polymers, poly(3-hydroxybu- various ecosystems such as soil, compost, aerobic and tyrate) (PHB) as well as copolymers of 3-hydroxybutyrate anaerobic sewage sludge, fresh and marine water, estuar- and 3-hydroxyvalerate (PHBV) have properties similar to ine sediment, and air (Abou-Zeid et al., 2001). Since the petrochemical based thermoplastics (Mergaert et al., 1994) pioneering work of Chowdhury (1963) and Delafield et al. and are biocompatible and biodegradable (Calabia and (1965), biodegradation of PHB and PHBV has been investi- Tokiwa, 2006). PHBV has been receiving commercial inter- gated in different natural environments, such as soils est as a promising candidate for the large-scale production (Mergaert et al., 1993; Kimura et al., 1994), composts of biodegradable and biocompatible thermoplastics (Doi, (Mergaert et al., 1994; Pagga et al., 1995), natural waters 1990). (Doi et al., 1992) and sludge (Briese et al., 1994) as well At the present time, although the known PHAs are quite as under laboratory conditions (Doi et al., 1992; Matavulj diverse, only few of them are being investigated: isomers et al., 1993; Mergaert et al., 1994). and copolymers hydroxybutyrate, poly-3-hydroxybutyrate PHB degrading actinomycetes (Mabrouk and Sabry, [P(3HB)], poly-4-hydroxybutyrate [P(4HB)], poly-3- 2001), Streptomyces, have been isolated from soils and hydroxybutyrate-co-poly-4-hydroxybutyrate [P(3HB-co- compost and they represent nearly a third of the total prokaryotic isolates from those environments (Mergaert et * Corresponding author. Phone: +92-51-90643065; al., 1993). Klingbeil et al. (1996) and Manna et al. (1999) Fax: +92-51-9219888; E-mail: [email protected] have established the versatility of Streptomyces to degrade 584 A.A. Shah et al. P(3HB), P(3HB-co-19%3HV), P(3HB-co-97% 3HV) and 0.001, MnSO4·H2O 0.001, agar 15, pH 7.0, (modified from P(3HB-co-70% 3HD). Nishida and Tokiwa, 1993) was employed to prepare inocu- The microbial degradation rate of poly(3-hydroxybu- lum to be used for the agar plates (optimisation experi- tyrate-co-3-hydroxyvalerate) (PHBV) films in soil appears ments) and liquid medium (enzyme production). to be dependent on the microbial population and distribu- tion, and the degradation ability of the PHBV-degrading Optimisation of culture conditions for PHBV depoly- microorganisms colonizing the surface of incubated PHBV merase production. The influence of time, initial pH (5- films (Sang et al., 2000). Therefore, information about the 9), incubation temperature (30, 37, 40, 45, 50 °C), sub- behavior and characteristics of the microbial consortium on strate concentration (1-4%) and additional carbon sources a film surface will help to clarify the microbial degradation (glucose, fructose, lactose, sucrose) on PHBV degradation of PHAs in soil. was investigated, in terms of formation of clear zones The enzymatic degradation of PHB and its copolymers, around microbial colonies by the production of PHBV PHBV, has been extensively studied using various extracel- depolymerase. lular PHB and PHBV depolymerases purified from several bacteria. PHB and PHBV depolymerases hydrolyse PHB and Production of PHBV depolymerase. Streptoverticillium PHBV to water soluble oligomers, which are metabolised to kashmirense AF1 was cultured in 150 ml of mineral salt liq- water and CO2 by bacteria. A marked characteristic of PHB uid medium containing PHBV (w/v) on a rotary shaker (120 and PHBV depolymerase is concentration dependence of rpm) under optimised conditions. the degradation rate. As well as optimum pH and optimum temperature, some PHB depolymerses have optimum con- PHBV depolymerase assay. Method described by centration (Kasuya et al., 1994). As the concentration of Kobayashi et al. (1999) was used for the determination of enzyme increases, the degradation rate initially increases PHBV depolymerase activity. About 0.3% PHBV was sus- rapidly to maximum and then decreases gradually. On the pended in 50 mM Tris-HCl, pH 8.0. This suspension was basis of the existence of the optimum concentration, sonicated for 20 min in ultrasonic water bath (35 KHz, 285 together with the fact that PHB depolymerases are organ- W) prior to dilution to 0.03% in the same buffer. Culture ised from two domains with substrate binding and catalyt- supernatant (0.1 ml) was added to 0.9 ml of the substrate ic functions and a linker region, the mechanism of enzy- suspension and incubated for 24 h at 30 °C. Activity was matic hydrolysis at the surface of PHB and PHBV film has measured as the decrease in turbidity of PHBV suspension been interpreted in terms of two step model (Mukai et al., at OD650 against substrate buffer blanks. One unit of activ- 1993). The enzymes first adsorb on the film surface by the ity is defined as the activity resulting in a decrease in OD function of the binding domain and then catalyse the at 650 nm per 24 h (Kobayashi et al., 1999). hydrolysis of polymer chains by the function of the catalyt- ic domain. Statistical analysis. The effect of pH, temperature, sub- The extracellular PHB depolymerases of Alcaligenes fae- strate concentration, carbon sources, surfactants and calis, Pseudomonas lemoignei, and Comamonas sp. nitrogen source on PHBV depolymerase activity, was con- (Jendrossek et al., 1993) P(3-hydroxyvalerate) depoly- firmed statistically by applying analysis of variance merases of P. lemoignei (Müller and Jendrossek, 1993) and (ANOVA) to the results. ANOVA test at a family error rate poly(3-hydroxyoctanoate) depolymerase of P. fluorescens of 5% was used to determine the statistical significance of (Schirmer et al., 1993) have been isolated and charac- chemical and biological data. Data were considered to be terised. significantly different between two values if p < 0.05. The The purpose of the present study was to isolate the calculated value of F (Fcal) must also be greater than crit- microorganisms from sewage sludge and evaluate their ical value of F (Fcrit). In such case, the null hypothesis is ability to degrade unique polymer like PHBV under con- rejected. trolled laboratory conditions employing solid-plate assay. Purification of PHBV depolymerase. Following incuba- tion, the cell free culture broth was used as crude enzyme MATERIALS AND METHODS extract. The precipitated (0-100% of ammonium sulphate) enzyme dissolved in 50 mM Tris-HCl