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Isolation and characterisation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) degrading actinomycetes and purification of PHBV depolymerase from newly isolatedStreptoverticillium kashmirense AF1

Isolation and characterisation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) degrading... 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 1 1 1 1 Aamer Ali SHAH *, Fariha HASAN , Abdul HAMEED , Safia AHMED 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 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 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 CO 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 al., 1993). Klingbeil et al. (1996) and Manna et al. (1999) * Corresponding author. Phone: +92-51-90643065; have established the versatility of Streptomyces to degrade Fax: +92-51-9219888; E-mail: alishah_75@yahoo.com 584 A.A. Shah et al. P(3HB), P(3HB-co-19%3HV), P(3HB-co-97% 3HV) and 0.001, MnSO ·H O 0.001, agar 15, pH 7.0, (modified from 4 2 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 CO 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 OD 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 buffer, pH 8.0, was Polymer studied. PHBV containing 5% 3-hydroxyvalerate purified on Sephadex G-75 column. Each fraction (3 ml) (3HV) was obtained in the form of powder from Sigma- eluted from the column was analysed for total protein Aldrich Chemie, Germany. (absorbance at 280 nm), protein estimation (Lowry et al., 1951) and PHBV depolymerase activity (Kobayashi et al., Microorganism. The sewage sludge was collected from the 1999). The specific activity (enzyme activity/total protein, Sewage Treatment Plant, Rawalpindi, Pakistan, and was U/mg) of each fraction was also calculated. The fractions used as a source for isolating microorganisms having the with high specific activity were pooled, concentrated and ability to degrade plastics. An actinomycetes strain was iso- used for relative molecular mass determination by SDS- lated by enrichment technique from sewage sludge which PAGE on a 12% gel according to the method of Laemmli was able to degrade PHBV and was identified according to (1970). Gel was stained with Coomassie brilliant blue R- Bergey’s Manual of Determinative Bacteriology (Holt, 1993). 250 and calibrated with molecular mass markers for 14.4- 97.4 kDa. Inoculum preparation. Arginine glycerol salt medium containing (AGS) (g/l): arginine 1, glycerol 12.5, K HPO 1, Analysis of PHBV Biodegradation by Sturm Test. CO 2 4 2 KH PO 0.2, NaCl 1, CaCl 0.01, MgSO ·7H O 0.5, evolution as a result of PHBV biodegradation was deter- 2 4 2 4 2 FeSO ·6H O 0.01, CuSO ·5H O 0.001, ZnSO ·7H O, mined by Sturm Test. CO evolved as a result of degrada- 4 2 4 2 4 2 2 Ann. Microbiol., 57 (4), 583-588 (2007) 585 tion of polymeric chain was trapped in the absorption bot- due to inhibition of diffusion of extracellular enzymes. tles containing KOH. Barium chloride solution was added to Manna and Paul (2000) have reported that maximum zone the CO containing KOH bottles and as a result precipitates of inhibition was obtained after 12 days of incubation. With of barium carbonate (using CO released by breakdown of further incubation, there was no increase in the size of zone polymer) were formed. CO produced can be calculated diameter. gravimetrically by measuring amount (g/ml) of CO precip- itates evolved by addition of BaCl . Difference in the Optimisation of culture conditions for PHBV depoly- weights of precipitates both in test and control was merase production observed (Müller et al., 1992). Most of the PHBV degrading microorganisms grow better at either ambient or mesophilic temperatures, whereas, only a few species like Bacillus strain TT96 (Tansengco and RESULTS AND DISCUSSION Tokiwa, 1998) and Streptomyces strain MG (Tokiwa and Calabia, 2004) were reported to degrade it at higher tem- PHBV degrading microorganism peratures. There is little information on microbial degrada- The PHBV degrading actinomycetes strain was isolated, tion of PHBV at high temperatures (Takeda et al., 1998). In attached to PHBV film, buried in soil. The isolate was iden- the present study it was observed that the tified as Streptoverticillium kashmirense AF1 on the basis Streptoverticillium kashmirense AF1 was much proficient in of morphological and biochemical characteristics (Holt, degrading PHBV at 45 °C with zone diameter of about 2.2 1993). cm (p > 0.05) after 3 weeks of incubation (Fig. 1A). PHBV degrading microorganisms have earlier been iso- Increased microbial degradation of PHAs at 40 °C has also lated from various habitats by different researchers. Imam been reported by Mergaert et al. (1994). et al. (1999) have isolated a Pseudoalteromonas sp. strain Streptoverticillium kashmirense AF1 was able to NRRL B-30083 isolated from water. Few species of marine degrade the polymer with maximum zone of hydrolysis bacteria, including Acinetobacter johnsoniae, Comamonas (2.6 cm), at pH 8 (p > 0.05) followed by pH 7 (Fig. 1B). testosteroni, Flavobacterium johnsoniae, Vibrio ordalii and After purification and characterisation, it was confirmed Zoogloea ramigera (Mergaert et al., 1992; Mukai et al., that there were two types of PHBV depolymerases with dif- 1993), Pseudoalteromonas haloplanktis (Mergaert et al., ferent molecular sizes, produced under both alkaline and 1996) and Alcaligenes sp. (Nojiri and Saito, 1997) have acidic conditions. PHB depolymerase from municipal been identified as PHA-degraders. Mergaert et al. (1993) sewage sludge at different pH ranges has been isolated, at isolated various PHB and PHBV degrading bacterial strains pH 7 from Alcaligenes faecalis (Tanio et al., 1982), at pH 8 from soil, including; Bacillus strains, Streptomyces, from Pseudomonas lemoignei (Schirmer et al., 1993). Aspergillus fumigatus, Penicillium strains, Gram negative The optimum substrate concentration required for the bacteria including Acidovorax facilis and Variovorax para- production of PHBV depolymerase production was reported doxus. Khan et al. (2002) isolated PHBV degrading b- as 1% (w/v) as shown by clear zone of hydrolysis (2.4 cm) Proteobacteria closely related to Acidovorax, Comamonas, (p < 0.05) (Fig. 1C). The enzyme activity was decreased Aeromonas, Pseudomonas, g-Proteobacteria from sewage with further increase in polymer concentration due to sat- treatment plants. Abou-Zeid et al. (2001) also isolated few uration of extracellular depolymerases by the substrate in strains resembling Clostridium sp. from sewage sludge. the immediate vicinity of the microbial growth. Substrate Ghanem et al. (2005) isolated PHBV and PHB degrading concentration above the optimum level as a rule suppress- Nocardiopsis aegyptia from marine seashore environment. es the activity of the enzyme (Manna and Paul, 2000). The microbial degradation rate of poly(3-hydroxybutyrate- Jendrossek et al. (1993) have mentioned that most co-3-hydroxyvalerate) (PHBV) films in soil appears to be PHAs-degrading bacteria repress PHA depolymerase gene dependent on the microbial population and distribution, expression in the presence of a soluble carbon source that and the degradative ability of the PHBV-degrading microor- permits high growth rates. After exhaustion of the readily ganisms colonising the surface of incubated PHBV films available nutrients, the synthesis of PHA depolymerases is (Sang et al., 2000). Bacteria and actinomycetes required derepressed in many strains and halo formation begins much time to occupy the entire surface, due to their slow (Manna and Paul, 2000). In our study, maximum degrada- surface growth rate (Sang et al., 2002). Coleman et al. tion of PHBV was observed in the media supplemented with (1989) reported that in some soils fungi are the dominant easily consumable carbon source, such as lactose (2.7 cm) decomposers of organic matter, and increase their popula- (Fig. 1D). It is important to mention, that carbon catabolite tion abruptly for the utilisation of organic matter. repression (CCR) by lactose was observed for the organ- isms degrading polyhydroxyalkanoates, i.e. cells sense the Degradation of PHBV by Streptoverticillium kash- presence of a favourable carbon source, in this case lac- mirense AF1 tose, and transmit the information to the relevant control The degradation of PHBV by S. kashmirense AF1 on poly- units. Consequently, PHBV as a C-source is not depoly- mer containing agar plates was recorded on weekly basis; merised to act as a catabolite. The degradation efficiency there was increase in degradation with the increase in incu- obtained with similar organisms but different nutritional bation period. Maximum degradation, as evident by zone of environments may alter. Results of supplementation stud- hydrolysis, was observed within 30 days of incubation, ies have indicated that the synthesis of PHBV depoly- beyond which no further degradation was recorded. Such merases by the isolates is regulated by soluble carbon an increase in clear zone formation could be explained by sources. In addition, the degradation potential of the dif- the release of extracellular PHBV depolymerases by S. ferent isolated organisms was greatly influenced through kashmirense AF1, up to a period of 3-4 weeks. variation/optimisation of the growth medium, since differ- Furthermore, no increase was observed beyond 4 weeks, ent organisms exhibit different nutritional requirements 586 A.A. Shah et al. (Abou-Zeid et al., 2001). According to Manna and Paul Purification of PHBV depolymerase (2000) degradation of PHB by bacterial strains isolated Streptoverticillium kashmirense AF1 was used to produce from soil and sewage sludge was affected significantly enzyme under optimised conditions and was purified. when the PHB containing medium was supplemented with Protein was precipitated at 80% of ammonium sulphate. easily consumable carbon sources. The enzyme precipitates were dissolved in minimal quan- tity of buffer and were purified by gel permeation (Sephadex G-75). As a result two peaks having the depolymerase activity were obtained (Figs. 2, 3) and two 2.5 bands of 35 and 45 kDa, isolated through SDS-PAGE con- firmed the two peaks obtained by gel permeation (Fig. 4). The results suggested the presence of two different types 1.5 of depolymerases having different molecular weights. It can be stated that two depolymerases are encoded by two different genes, which can be further sequenced. Most PHA-degrading bacteria apparently contain only one 0.5 depolymerase, P. lemoignei has at least five, which differ slightly in their biochemical properties. PHA depolymeras- 30 37 40 45 50 es purified from recombinant Escherichia coli appeared as Temperature (°C) four bands on SDS-PAGE with molecular masses calculat- ed as 49, 46, 44 and 65 kDa for the PhaZ5, PhaZ2, PhaZ1, and PhaZ4 encoded depolymerases, respectively 2.5 (Jendrossek et al., 1995). 1.5 Total protein (mg/ml) PHBV depolymerase 0.3 0.35 0.5 0.3 0 0.25 56 789 0.25 pH 0.2 0.2 0.15 2.5 0.15 0.1 0.1 1.5 0.05 0.05 0 0 0.5 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Fractions (3 ml) Glucose Fructose Lactose Sucrose FIG. 2 - Purification of PHBV depolymerase in crude extract of Carbon sources Streptoverticillium kashmirense AF1 by gel filtration on Sephadex G-75. Vo: 10 ml; Vt: 40 ml; flow rate: 15 ml/h; fraction volume: 3 ml; eluent: 50 mM; Tris- 2.5 HCl (pH 8.0). 1.5 1.8 1.6 0.5 1.4 1.2 0.8 Substrate concentration (%) 0.6 0.4 FIG. 1 - Effect of temperature (A), pH (B), carbon sources (C) 0.2 substrate concentration (D) on the poly(3-hydroxy- butyrate-co-3-hydoxyvalerate) depolymerase activi- 1 2 3 4 5 6 7 8 9 1011 1213 1415 1617 ty shown by Streptoverticillium kashmirense AF1 Fractions (3 ml) measured as zone of hydrolysis in mineral salt agar FIG. 3 - Specific activities (U/mg) of all the fractions obtained by after 4 weeks. gel chromatography of crude PHBV depolymerase. Zone diameter (cm) Zone diameter (cm) Zone diameter (cm) Zone diameter (cm) Specific activity (U/mg) Total protein (mg/ml) PHBV Depolymerase activity (U/ml) Ann. Microbiol., 57 (4), 583-588 (2007) 587 REFERENCES Abou-Zeid D.M., Müller R.J., Deckwer W.D. (2001). Degradation of natural and synthetic polyesters under anaerobic condi- tions. J. Biotech., 86: 113-126. 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Isolation and characterisation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) degrading actinomycetes and purification of PHBV depolymerase from newly isolatedStreptoverticillium kashmirense AF1

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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
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Abstract

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 1 1 1 1 Aamer Ali SHAH *, Fariha HASAN , Abdul HAMEED , Safia AHMED 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 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 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 CO 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 al., 1993). Klingbeil et al. (1996) and Manna et al. (1999) * Corresponding author. Phone: +92-51-90643065; have established the versatility of Streptomyces to degrade Fax: +92-51-9219888; E-mail: alishah_75@yahoo.com 584 A.A. Shah et al. P(3HB), P(3HB-co-19%3HV), P(3HB-co-97% 3HV) and 0.001, MnSO ·H O 0.001, agar 15, pH 7.0, (modified from 4 2 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 CO 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 OD 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 buffer, pH 8.0, was Polymer studied. PHBV containing 5% 3-hydroxyvalerate purified on Sephadex G-75 column. Each fraction (3 ml) (3HV) was obtained in the form of powder from Sigma- eluted from the column was analysed for total protein Aldrich Chemie, Germany. (absorbance at 280 nm), protein estimation (Lowry et al., 1951) and PHBV depolymerase activity (Kobayashi et al., Microorganism. The sewage sludge was collected from the 1999). The specific activity (enzyme activity/total protein, Sewage Treatment Plant, Rawalpindi, Pakistan, and was U/mg) of each fraction was also calculated. The fractions used as a source for isolating microorganisms having the with high specific activity were pooled, concentrated and ability to degrade plastics. An actinomycetes strain was iso- used for relative molecular mass determination by SDS- lated by enrichment technique from sewage sludge which PAGE on a 12% gel according to the method of Laemmli was able to degrade PHBV and was identified according to (1970). Gel was stained with Coomassie brilliant blue R- Bergey’s Manual of Determinative Bacteriology (Holt, 1993). 250 and calibrated with molecular mass markers for 14.4- 97.4 kDa. Inoculum preparation. Arginine glycerol salt medium containing (AGS) (g/l): arginine 1, glycerol 12.5, K HPO 1, Analysis of PHBV Biodegradation by Sturm Test. CO 2 4 2 KH PO 0.2, NaCl 1, CaCl 0.01, MgSO ·7H O 0.5, evolution as a result of PHBV biodegradation was deter- 2 4 2 4 2 FeSO ·6H O 0.01, CuSO ·5H O 0.001, ZnSO ·7H O, mined by Sturm Test. CO evolved as a result of degrada- 4 2 4 2 4 2 2 Ann. Microbiol., 57 (4), 583-588 (2007) 585 tion of polymeric chain was trapped in the absorption bot- due to inhibition of diffusion of extracellular enzymes. tles containing KOH. Barium chloride solution was added to Manna and Paul (2000) have reported that maximum zone the CO containing KOH bottles and as a result precipitates of inhibition was obtained after 12 days of incubation. With of barium carbonate (using CO released by breakdown of further incubation, there was no increase in the size of zone polymer) were formed. CO produced can be calculated diameter. gravimetrically by measuring amount (g/ml) of CO precip- itates evolved by addition of BaCl . Difference in the Optimisation of culture conditions for PHBV depoly- weights of precipitates both in test and control was merase production observed (Müller et al., 1992). Most of the PHBV degrading microorganisms grow better at either ambient or mesophilic temperatures, whereas, only a few species like Bacillus strain TT96 (Tansengco and RESULTS AND DISCUSSION Tokiwa, 1998) and Streptomyces strain MG (Tokiwa and Calabia, 2004) were reported to degrade it at higher tem- PHBV degrading microorganism peratures. There is little information on microbial degrada- The PHBV degrading actinomycetes strain was isolated, tion of PHBV at high temperatures (Takeda et al., 1998). In attached to PHBV film, buried in soil. The isolate was iden- the present study it was observed that the tified as Streptoverticillium kashmirense AF1 on the basis Streptoverticillium kashmirense AF1 was much proficient in of morphological and biochemical characteristics (Holt, degrading PHBV at 45 °C with zone diameter of about 2.2 1993). cm (p > 0.05) after 3 weeks of incubation (Fig. 1A). PHBV degrading microorganisms have earlier been iso- Increased microbial degradation of PHAs at 40 °C has also lated from various habitats by different researchers. Imam been reported by Mergaert et al. (1994). et al. (1999) have isolated a Pseudoalteromonas sp. strain Streptoverticillium kashmirense AF1 was able to NRRL B-30083 isolated from water. Few species of marine degrade the polymer with maximum zone of hydrolysis bacteria, including Acinetobacter johnsoniae, Comamonas (2.6 cm), at pH 8 (p > 0.05) followed by pH 7 (Fig. 1B). testosteroni, Flavobacterium johnsoniae, Vibrio ordalii and After purification and characterisation, it was confirmed Zoogloea ramigera (Mergaert et al., 1992; Mukai et al., that there were two types of PHBV depolymerases with dif- 1993), Pseudoalteromonas haloplanktis (Mergaert et al., ferent molecular sizes, produced under both alkaline and 1996) and Alcaligenes sp. (Nojiri and Saito, 1997) have acidic conditions. PHB depolymerase from municipal been identified as PHA-degraders. Mergaert et al. (1993) sewage sludge at different pH ranges has been isolated, at isolated various PHB and PHBV degrading bacterial strains pH 7 from Alcaligenes faecalis (Tanio et al., 1982), at pH 8 from soil, including; Bacillus strains, Streptomyces, from Pseudomonas lemoignei (Schirmer et al., 1993). Aspergillus fumigatus, Penicillium strains, Gram negative The optimum substrate concentration required for the bacteria including Acidovorax facilis and Variovorax para- production of PHBV depolymerase production was reported doxus. Khan et al. (2002) isolated PHBV degrading b- as 1% (w/v) as shown by clear zone of hydrolysis (2.4 cm) Proteobacteria closely related to Acidovorax, Comamonas, (p < 0.05) (Fig. 1C). The enzyme activity was decreased Aeromonas, Pseudomonas, g-Proteobacteria from sewage with further increase in polymer concentration due to sat- treatment plants. Abou-Zeid et al. (2001) also isolated few uration of extracellular depolymerases by the substrate in strains resembling Clostridium sp. from sewage sludge. the immediate vicinity of the microbial growth. Substrate Ghanem et al. (2005) isolated PHBV and PHB degrading concentration above the optimum level as a rule suppress- Nocardiopsis aegyptia from marine seashore environment. es the activity of the enzyme (Manna and Paul, 2000). The microbial degradation rate of poly(3-hydroxybutyrate- Jendrossek et al. (1993) have mentioned that most co-3-hydroxyvalerate) (PHBV) films in soil appears to be PHAs-degrading bacteria repress PHA depolymerase gene dependent on the microbial population and distribution, expression in the presence of a soluble carbon source that and the degradative ability of the PHBV-degrading microor- permits high growth rates. After exhaustion of the readily ganisms colonising the surface of incubated PHBV films available nutrients, the synthesis of PHA depolymerases is (Sang et al., 2000). Bacteria and actinomycetes required derepressed in many strains and halo formation begins much time to occupy the entire surface, due to their slow (Manna and Paul, 2000). In our study, maximum degrada- surface growth rate (Sang et al., 2002). Coleman et al. tion of PHBV was observed in the media supplemented with (1989) reported that in some soils fungi are the dominant easily consumable carbon source, such as lactose (2.7 cm) decomposers of organic matter, and increase their popula- (Fig. 1D). It is important to mention, that carbon catabolite tion abruptly for the utilisation of organic matter. repression (CCR) by lactose was observed for the organ- isms degrading polyhydroxyalkanoates, i.e. cells sense the Degradation of PHBV by Streptoverticillium kash- presence of a favourable carbon source, in this case lac- mirense AF1 tose, and transmit the information to the relevant control The degradation of PHBV by S. kashmirense AF1 on poly- units. Consequently, PHBV as a C-source is not depoly- mer containing agar plates was recorded on weekly basis; merised to act as a catabolite. The degradation efficiency there was increase in degradation with the increase in incu- obtained with similar organisms but different nutritional bation period. Maximum degradation, as evident by zone of environments may alter. Results of supplementation stud- hydrolysis, was observed within 30 days of incubation, ies have indicated that the synthesis of PHBV depoly- beyond which no further degradation was recorded. Such merases by the isolates is regulated by soluble carbon an increase in clear zone formation could be explained by sources. In addition, the degradation potential of the dif- the release of extracellular PHBV depolymerases by S. ferent isolated organisms was greatly influenced through kashmirense AF1, up to a period of 3-4 weeks. variation/optimisation of the growth medium, since differ- Furthermore, no increase was observed beyond 4 weeks, ent organisms exhibit different nutritional requirements 586 A.A. Shah et al. (Abou-Zeid et al., 2001). According to Manna and Paul Purification of PHBV depolymerase (2000) degradation of PHB by bacterial strains isolated Streptoverticillium kashmirense AF1 was used to produce from soil and sewage sludge was affected significantly enzyme under optimised conditions and was purified. when the PHB containing medium was supplemented with Protein was precipitated at 80% of ammonium sulphate. easily consumable carbon sources. The enzyme precipitates were dissolved in minimal quan- tity of buffer and were purified by gel permeation (Sephadex G-75). As a result two peaks having the depolymerase activity were obtained (Figs. 2, 3) and two 2.5 bands of 35 and 45 kDa, isolated through SDS-PAGE con- firmed the two peaks obtained by gel permeation (Fig. 4). The results suggested the presence of two different types 1.5 of depolymerases having different molecular weights. It can be stated that two depolymerases are encoded by two different genes, which can be further sequenced. Most PHA-degrading bacteria apparently contain only one 0.5 depolymerase, P. lemoignei has at least five, which differ slightly in their biochemical properties. PHA depolymeras- 30 37 40 45 50 es purified from recombinant Escherichia coli appeared as Temperature (°C) four bands on SDS-PAGE with molecular masses calculat- ed as 49, 46, 44 and 65 kDa for the PhaZ5, PhaZ2, PhaZ1, and PhaZ4 encoded depolymerases, respectively 2.5 (Jendrossek et al., 1995). 1.5 Total protein (mg/ml) PHBV depolymerase 0.3 0.35 0.5 0.3 0 0.25 56 789 0.25 pH 0.2 0.2 0.15 2.5 0.15 0.1 0.1 1.5 0.05 0.05 0 0 0.5 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Fractions (3 ml) Glucose Fructose Lactose Sucrose FIG. 2 - Purification of PHBV depolymerase in crude extract of Carbon sources Streptoverticillium kashmirense AF1 by gel filtration on Sephadex G-75. Vo: 10 ml; Vt: 40 ml; flow rate: 15 ml/h; fraction volume: 3 ml; eluent: 50 mM; Tris- 2.5 HCl (pH 8.0). 1.5 1.8 1.6 0.5 1.4 1.2 0.8 Substrate concentration (%) 0.6 0.4 FIG. 1 - Effect of temperature (A), pH (B), carbon sources (C) 0.2 substrate concentration (D) on the poly(3-hydroxy- butyrate-co-3-hydoxyvalerate) depolymerase activi- 1 2 3 4 5 6 7 8 9 1011 1213 1415 1617 ty shown by Streptoverticillium kashmirense AF1 Fractions (3 ml) measured as zone of hydrolysis in mineral salt agar FIG. 3 - Specific activities (U/mg) of all the fractions obtained by after 4 weeks. gel chromatography of crude PHBV depolymerase. Zone diameter (cm) Zone diameter (cm) Zone diameter (cm) Zone diameter (cm) Specific activity (U/mg) Total protein (mg/ml) PHBV Depolymerase activity (U/ml) Ann. Microbiol., 57 (4), 583-588 (2007) 587 REFERENCES Abou-Zeid D.M., Müller R.J., Deckwer W.D. (2001). Degradation of natural and synthetic polyesters under anaerobic condi- tions. J. Biotech., 86: 113-126. Briese B.H., Schmidt B., Jendrossek D. (1994). Pseudomonas lemoignei has five poly(hydroxyalkanoic acid) (PHA) depoly- merase genes: a comparative study of bacterial and eukary- otic PHA depolymerases. J. Environ. Polym. Degrad., 2: 75- Calabia B.P., Tokiwa Y.A. (2006). Novel PHB Depolymerase from a thermophilic Streptomyces sp. Biotechnol. Lett., 28: 383- Chowdhury A.A. (1963). Poly-b-hydroxybuttersa¨ure abbauende Bakterien und Exoenzyme. Arch. Mikrobiol., 47:167-200. Coleman C.C., Brussaard L., Beare M.H., Hendrix P.F., Hassink J., Henijnen C.E., Marinissen J.C.Y. (1989). Microbial-faunal interactions as they influence soil organic matter dynamics. In: Hattori T., Ishida Y., Maruyama Y., Morita R.Y., Uchida A., Eds, Recent Advances in Microbial Ecology, Proceedings of th the 5 international Symposium of Microbial Ecology, Japan Science Society Press, pp. 175-179. FIG. 4 - SDS-PAGE of the purified PHBV depolymerase from Streptoverticillium kashmirense AF1. 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Published: Nov 21, 2009

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