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Characterization of PHA Depolymerase in phenol degrading bacteria.

Abstract

Microbes play a major role for saving our environment by degrading chemical wastes, which are toxic either in their native form or modified to be toxic. Isolation of microbial strains able to degrade chemical compounds was start usually from polluted sources, such as soil. In our study aerobic strains isolated from garden soil using enrichment technique were purified and rescreened for their ability to degrade PHA (Polyhydroxyalkanoate). The most tow successful strains able to degrade both phenol and PHA were characterized by stander criteria as Alcaligenes xylosoxidans subsp. denitrificans and Citrobacter sp. PHA exodepolymerase enzymatic activities were characterized and the surface of the degradable polymer being scanned using electron microscope, which shows that bacterial cells tightly attached to the polymer surface during the degradation process causing surface deformation. We recommended using Alcaligenes xylosoxidans subsp. denitrificans and Citrobacter sp., for Phenol/PHA degradation and in other related bioremediation and biotechnological applications.

Keywords: Alcaligenes xylosoxidans subsp. denitrificans, Citrobacter sp., Phenol, Polyhydroxyalkanoate, depolymerase, electron microscope.

Introduction

Polyhydroxyalkanoate (PHA) is biopolymer accumulated as intracellular inclusion bodies under special nutritional conditions usually when nitrogen is limited and carbon is in excess. Lemoigne in 1923 was the first to characterize bacterial granules as PHB (Polyhydroxybutyrate) the arche type of PHA [1]. Later at 1983 De Smet et al., discovered PHAMCL (Polyhydroxyalkanoate Medium-Chain-Lenghth) in Pseudomonas oleovorans [2]. Huisman et al., 1989 confirmed that PHAMCL accumulation was a common feature of fluorescent pseudomonads [3]. Most of the PHA structure is known and PHA identified as a linear, head to tail polyester composed of 3-hydroxy fatty acid monomers. The carboxyl group of one monomer forms an ester bond with the hydroxyl group of another monomer.

The PHA inclusion bodies which can be used by bacteria as carbon source by internal enzyme called depolymerase. On the other hand when the bacterial cells died and the polymer relapsed to outer environment, another class of depolymerase, the extracellular depolymerase start to degrade the polymer outside the cell to use it as a sole carbon source. These two processes happened in nature spontaneously when the conditions are suitable.

Aerobic and anaerobic PHA-degrading microbes were isolated from soil, compost, sludge, fresh and marine water and from various other ecosystems [4-6]. The bacterial origins of the PHA make these polyesters a natural material, and therefore many microorganisms have the ability to degrade the macromolecules.

PHA show material properties that are similar to some common plastic such as Polypropylene [7].

The molecular mass of PHA depends on the substrate and the type of the producing strains, but is generally in order of 5x[10.sup.4] to 1x[10.sup.6]Da. While depolymerases have been studied for about 40 years, the first structural gene of a PHA depolymerase (Alcaliginus. faecalis, phaZAf) was cloned and sequenced in 1989 by Saito et al., [8].

When the polymer extracted from the cell, the granule surface either damaged or lost and the polymer chain tend to adapt ordered helical conformation and develop a crystalline phase.

PHA depolymerases known to be stable under various conditions such as temperature and pH, their molecular rang below 100 KDa which have their high activity usually at the alkaline range (7.5-9.8) but some can have pH optima at 5.5 or 6.0, most depolymerases are inhibited by serine hydrolase like acylsulphonyl compounds [9].

Some species of bacteria contain more than one depolymerese gene, like pseudomonads.

Depolymerases are specific for either PHASCL (Polyhydroxyalkanoate short-chain-length) or PHAMCL and for the polymers consisting of monomer composition in the (R) configuration [10].

The degrading of toxic compounds is an important feature of microbes, such as phenol which present as natural or artificial mono-aromatic compounds in various environmental sites [11]. Phenol and its derivatives are counted as one of the most dominant pollutant in rivers, industrial effluents from many applications like wine-distillery, olive oil extraction, green olive debittering, cork preparation, wood debarking, detoxification of coffee husk, and land filled run of wastes [12-17].

Many strategies for phenol wastes treatment have been introduced using microbes able to utilize phenol as a sole carbon source. The treatment describes using microbes as a seeding stock, immobilizing microbial cells, semicontinuous culture and using either entrapped or adsorbed cell as the biological active catalyst in different types of bioreactors [18-20]

Phenol and substituted phenols such as cresol and chlorinated phenols are byproducts in number of industrial processes such as petroleum refining petrochemical operations, coal cooking, coal gasification and liquefaction and steel productions [11]. Microbial degradation of phenol and phenolic compounds is the best solution where phenol utilized by microbes as a source of carbon and degrades to save C[O.sub.2] [21]. The aim of this work is to isolate and characterize microbial strains able to degrade both Phenol and PHA. The strategy is based on screening bacterial isolates from soil enriched with phenol and selects the best strains able to degrade PHA.

Material and Methods

Bacterial strains, plasmid and growth conditions

E. coli XL1 blue [22-23] was used in this study as a recombinant strain for expression of pAAE1 a pGemT-Easy plasmid containing coding region of phaC gene from P. aeruginosa A6 downstream of lac promoter. For general cultivation of microbial strains Luria-Bertani (LB)-Medium [24] was used. For enrichment of phenol degrading bacteria from soil a medium described by Salem and Al-Barakati (2004) [25] was used and consists of NH4NO3, 1 g [l.sup.-1]; (NH4)2SO4, 0.5 g [l.sup.-1]; MgSO4.7H20, 0.5 g [l.sup.-1]; KH2PO4, 0.5 g [l.sup.-1]; K2HPO4, 1 g [l.sup.-1]; NaCl, 0.5 g [l.sup.-1]; CaCl2, 0.02 g [l.sup.-1]; trace element solution 2 ml [l.sup.-1] (Schlegel et al., 1961) [26] ; pH 7.0 containing phenol 0.5 g [l.sup.-1] as the sole carbon source. For solid media 17.0 g [l.sup.-1] agar was added to the media. For screening PHA degrading bacteria the same media is used but PHA strips were used instead of phenol as a carbon source. For PHA production E. coli XL1 blue harboring pAAE1 plasmid was cultivated in 3L LB medium supplemented with 1% dodecanoate and IPTG at 30 [degrees]C for 24 hr at shaker incubator 180 r/min.

Enrichment methods

The bacterial strains were obtained from soil and collected from different locations using an enrichment technique (soil samples were daily supplemented with a defined concentration of phenol for 4-5 weeks). The above described minimal medium was used containing phenol 0.5 [g1.sup.-1] as the sole carbon source.

Isolation of phenol degrading bacteria

The agar medium containing phenol as sole carbon source was inoculated with 0.1 ml of the diluted soil. The plates were incubated at 30 [degrees]C for 48 hr. Phenol degrading colonies showed good growth. Further purification by streaking on a solid medium of the same composition was performed, and then isolated on phenol-agar slants.

Phenol assay

The method described by Korenman et al., (1998) was used [27]. The method is based on rapid condensation with 4-aminoantipyrine followed by the oxidation with potassium ferricyanide under alkaline conditions. This gives a red colored product, which is measured by using spectrophotometer at 492 nm and the result compared with standard curve prepared with different phenol concentration.

Bacterial strain growth curve

Degradation of phenol was carried out in 250 ml Erlenmeyer flasks, each containing 100 ml of basal medium of the same composition as that used for isolation. Each flask was inoculated with 2 ml of a pure bacterial cell suspension represent one strain isolated from enriched methods and cultivated in phenol agar as described above (about 60 mg Cell Dry Wight) and prepared from 48 hr old slants of the test organisms. The flasks were incubated at 30[degrees]C in an incubator shaker (180 r/m). At time intervals the residual phenol concentration, pH and bacterial growth were determined until complete consumption of phenol.

Preparation of PHA

The PHA was produced by E. coli XL1 blue harboring pAAE1 plasmid which containing [phaC.sub.PaA6] synthase after 24 hr cultivation in accumulating condition as described above. The cells isolated by centrifugation at 10000 rpm for 10 min and washed three times by 0.9% NaCl solution then recentrifuged and lyophilized. The PHA obtained from the lyophilized cells by extraction with chloroform in a Soxhlet apparatus. The extraction was performed by recycling chloroform through the lyophilized cells for 24-48 h. The polyester dissolved in chloroform was precipitated by the addition of 10 vol., of methanol to the polymer-containing chloroform solution and subsequently separated from the solvent by filtration or centrifugation. Remaining solvents were removed by exposure of the polyester to a stream of air. For further purification, PHA was again dissolved in chloroform and precipitated with methanol as described above in order to obtain highly purified PHA.

GC analysis of PHA

The purified polymer was analyzed by gas chromatography (GC) as described previously by Timm and Steinbuchel, 1990 [28].

Media for screening of PHA degrading bacteria

For screening of PHA degrading bacteria from those isolates which able to degrade phenol, a liquid minimal salt media was used as mentioned above and PHA stripes (10 mg) polymer were used as a sole carbon source.

Isolation of PHA degrading bacteria

To sterilize the polymer stripes without deformation of its surface the stripes were incubating in LB media overnight followed by fast freezing in -80 [degrees]C for 3 hr followed by incubation in cold ethanol for 30 min, and then washed with sterile distilled water.

After sterilization each stripes added to a 20 ml flask contain Salem and Al-Barakati minimal medium [26] and incubated at 30 [degrees]C for 48 hr on shaker at 180 rpm. Contaminated flasks were omitted then the uncontaminated flasks which contain the PHA stripes were inoculated with 0.1 ml fresh culture of different phenol degrading isolates separately. The flasks were reincubated at 30 [degrees]C for one week on shaker incubator at 180 r/m.

The growth was observed during the incubation period and determined optically at [OD.sub.600]. The most active strains were re-examined to prove the degradation efficiency. For more conformation the strains were cultivated again in media containing PHA as above and the PHA degradation rate was determined.

Selection of the best isolates

The best isolates which show fast growth and degrading rate were selected and reconfirmed for their ability to degrade both phenol and PHA. The strains preserved in -80[degrees]C in DMSO stock solution for further investigations.

Preparation of polymer suspensions for enzyme activity

The homopolyesters PHA was dissolved in chloroform solution in a final concentration of 2 g/liter. Four volumes of [PHA.sub.MCL]-Chloroform solution was added slowly under stirring into 1 volume of cool distilled water. Then the organic solvent was removed by using a rotary evaporator, and a milky PHA suspension in water was obtained [29].

Assay of PHA depolymerase

The bacterial cells which grown in minimal media contain PHA as above were removed by centrifugation at 10000 rpm for 10 min and the concentration of the protein was determined by Bradford method, using bovine serum albumin as a standard [30]. PHA depolymerase activities were measured as described by Jendrossek et al., (1995), photometrically by the decrease of the optical density at 650 nm in 1ml cuvettes containing 4, 10 and 20 [micro]g of PHA granules in 50 mM Tris-HCl (pH 8.0) and 1 mM Ca[Cl.sub.2] at 37[degrees]C. The apparent extinction coefficients for PHA were 2.74, 7.3 and 8.2 [micro]l [ng.sup.-1] [cm.sup.-1] [29]. All measurements were performed with one preparation. The effect of PMSF as an inhibitor for PHA depolymerse was determined using 0.0, 0.2, 0.4, 0.6, 0.8 and 0.1 mM PMSF in the same assay conditions as above. One unit of depolymerase activity was defined as the hydrolysis of 1[micro]g of polyester per min as described by Jendrossek et al., 1995 [29].

Kinetic

Kinetic parameters for the degradation of PHA were determined and the apparent kinetic parameters were estimated from Lineweaver-Burk plots; Km and Vmax were calculated for each enzyme and summarized in the result section.

Identification of microbial strain

Bacterial stains are identified with help of API 20 NE system (BioMereux Vitek, Inc., Hazelwood Mo.) The system consists of a disposable plastic strip with 20 couples containing dehydrate reagent. Isolates were picked up with a sterile loop from 7 hr plates and were added to the media provided to construct it a suspension. The density of suspension was standardized according to the 2-3 Macfarlane standards. Suspensions were used to fill the capsules of the test strips as directed by the manufacturer. After incubation, at 30 [degrees]C the growth in each well was recorded at 24 hr. A profile number based upon the reactions observed was generated. Identifications were made by reference to the API analytical profile Index. The result was compared with the API reference identification.

Sample preparation for electron microscopy

To study the depolymerase activity effect on the polymer surface, electron microscope was used to scan the surface of the treated PHA polymer where, 1 [cm.sup.2] x 1 mm PHA strips were fixed in the surface of flat glass slid and washed gently by distilled water for 3 sec then allowed to dry at 37 [degrees]C. The dry polymer surface then coated with approximately 15 nm gold (SPI-Module [TM] sputter Coater).

Scanning of the polymer surface

The golden coated sample then subjected to scan by analytical scanning electron microscope (Jeal JSM-6360LA) with secondary element at 20 KV acceleration voltages and at room temperature. The digital image were adjusted and saved.

Result

Growth determination during phenol degradation

Determination of growth or cell leakage was performed at [O.D.sub.546] spectrophotometericaly. The dry cells weight, change in pH and the phenol concentrations were determined, where the phenol concentration were reversible to the growth, while pH were decreased during the degradation process. The measurement was found to be linear with substrate concentrations between 0.3-1 g/L for A. xylosoxidans subsp. denitrificans, and between 0.4-1 g/L for C. sp. The growth parameter during the phenol degradation and the change in the pH were summarised in Figure 1 and 2 respectively. About 50% of phenol was degraded at the middle of log phase while complete degradation was observed before the end of stationery phase.

Polymer composition

The extracted PHA polymer after complete methylation was analyzed using GC and the monomeric constituent of the used PHA polymer were determined and the polymer shown to be consists of 55.8 % 3-HD (3-Hydroxyoctanoate) as a major constituent and 25.8 % 3-HO (3-Hydroxyoctanoate), 10.2 % 3-HHX (3-Hydroxyhexahexanoate) and 8.1 % 3-HDD (3-dodecanoate).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Biochemical identification

According to the API reference identification, the result showed correct identifications of the isolates without the need to supplement extra tests. The isolate identified as A. xylosoxidans subsp. denitrificans and C. sp. with very good identification.

Polymer surface scanning

While the polymer used in the growth of the bacterial strains and in depolymerase enzyme preparation was in solid form as strips, the surface of the strips were scanned during the degradation process by A. xylosoxidans subsp. denitrificans and C. sp., using electron microscope. The deformation of the polymer surface was observed as in Figure 3 and 4 respectively. While the polymer surface was gently washed during the preparation steps, bacterial strains still attached to their surface which indicates a presence of direct contact during the degradation process beside the depolymerase activity.

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

In vitro PhaC depolymerase enzyme assay

PHA depolymerase activities for Alcaligenes xylosoxidans subsp. denitrificans and Citrobacter sp., in presence of different PMSF (phenylmethanesulphonylfluoride) concentration were determined spectrophotometrically by monitoring the decrease in the absorbance of the polymer at 650 nm in the presence of PHA depolymerase [29]. The decrease in absorbance which is related to the polymer degradation at different assay conditions were measured and the Vmax and Km value were calculated using Lineweaver-Burk plots and summarized as in Table 1 and 2.

Discussion

Toxic compounds were accumulated in our environment as a result of Human daily activities with agricultural and industrial different applications [12-17]. Plants contain several kinds of phenolic compounds which released to the nature. A spontaneous degradation for those compounds happed by many kinds of reactions including aerobic and anaerobic microbial degradation. The phenolic compounds problem becomes serious when a biased accumulation happened usually due to our live wastes which contaminate the ecosystem. This extra accumulation of phenolic compounds in nature leads to change the biodiversity of the microbial strains which may lead to unusual problems. Screening for microbial strains able to degrade phenolic compounds could help in save our environment from such toxic compounds. In our study we looking for strains not only able to degrade phenol but also could degrade bioplastic (PHA) which cause no detectable toxicity to the nature and utilized by many microbes after degradation as a carbon source. The idea comes from that, microbial strains able to degrade toxic compounds are more environmentally adapted and can be used in unusual condition. If we plan to degrade PHA wastes we expect that these wastes will not be alone but will be surrounded with many other chemical and biological wastes which could inhibit the microbial degradation process. In more deep look monooxygenase is responsible for the monohydroxylation of phenol aromatic ring at position ortho to the pre-existing hydroxyle group result in catechol which catalyzed by specific dioxygenase between the tow hydroxyl groups initiating the ortho-pathway that leads to succinyle-CoA and acetyl-CoA as in Acinitobacter spp., or at the adjacent bond to initiate the meta- pathway that lead to pyruvate and acetaldehyde as in Pseudomonas spp [31-32]. The presence of those powerful degrading enzymes facilitates the ability of microbial strains to degrade different kind of phenolic compounds and other related chemical structure. The presence of depolymerase which is more simple in their function than the previous enzymes can give the microbial strains another source of carbon. While we are looking for powerful strains able to degrade phenole and PHAs, and due to that the phenolic compounds degradation is more complicated than that of PHA, we start to screen for phenol degrading bacteria using enrichment technique. The most successful strains which have been isolated and purified each alone and reexamined for their ability to degrade phenol have been rescreend for their ability to degrade PHA in a selected media where PHA was the only carbon source. Moreover PHA have been used in a form of strips which become more complicated for microbes but represent our target to isolate microbes can be used directly in the field for bioremediation. Two strains were successfully grow on PHA strips faster than the other strains were selected and identified by stander criteria as A. xylosoxidans subsp. denitrificans and C. sp. The depolymerase activities have been characterized using methods described early by Jendrossek et al., 1995] [29]. We use this method where it is more clear and easily to use than other described methods even depolymerase activity could be observed by naked eye. Kinetic studies were performed to evaluate the depolymerases in presence of PMSF as a specific inhibitors and the result as shown in table 1 and 2 represent the Vmax and Km value which show the different in the depolymerase activities in presence or absence of inhibitors which is a strong indicator about the inhibition of the depolymerase during the reaction condition by blocking the enzyme active site. The electron microscope have been used to visualize the effect of A. xylosoxidans subsp. denitrificans and C. sp. on the polymer surface where it clearly demonstrate the change in the polymer surface due to the depolymerase activity.

In conclusion we succeeded to isolate microbial stains able to degrade both of phenol and PHA aiming to use them in bioremediation different applications. We used simple strategy for screening and measuring either the degradation process or the enzyme activity of PHA depolymerase. We recommended using our strategy for screening microbial strains able to degrade toxic compounds like phenol, examine their ability to degrade the less or non-toxic compounds, like PHA aiming to enhance the overall degradation and biotechnological related processes.

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Amara A. Amro (1) * and Salem R. Soheir (2)

(1) Protein Dept., Genetic Engineering and Biotechnology Research Institute, Mubarak City for Scientific Research and Technology Applications, Alexandria, Egypt

(2) Faculty of Education, Alexandria University, Egypt

* Corresponding author E-mail: amroamara@web.de
Table 1: Vmax and Km for Alcaligenes xylosoxidans subsp.
denitrificans

 mM of PMSF

 0.0 0.02 0.04 0.06 0.08 0.1
Vmax 86.22 48.22 15.74 15.35 13.33 10.31
Km 6.35 3.77 0.93 13.33 0.087 1.93

Table 2: Vmax and Km for Citrobacter sp.

 mM of PMSF

 0.0 0.02 0.04 0.06 0.08 0.1
Vmax 38.51 35.78 3.41 1.77 1.76 1.71
Km 0.58 0.3 0.21 2.07 2.074 0.86
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Title Annotation:polyhydroxyalkanoate
Author:Amro, Amara A.; Soheir, Salem R.
Publication:International Journal of Biotechnology & Biochemistry
Geographic Code:7EGYP
Date:Jan 1, 2007
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