Production, purification and evaluation of biodegrading potential of PHB depolymerase of Stenotrophomonas sp. RZS 7

There are numerous reports on PHB depolymerases produced by a wide variety of microorganisms isolated from various habitats, however, reports on PHB depolymerase isolated from plastic contaminated sites are scares. Thermophilic PHB polymerase produced by isolates obtained from plastic contaminated sites is expected to have better relevance for its application in plastic/bioplastic degradation. Although PHB has attracted commercial significance, the inefficient production and recovery methods, inefficient purification of PHB depolymerase and lack of ample knowledge on PHB degradation by PHB depolymerase have hampered its large scale commercialization. Therefore, to ensure the biodegradability of biopolymers, it becomes imperative to study the purification of the biodegrading enzyme system. We report the production, purification, and characterization of extracellular PHB depolymerase from Stenotrophomonas sp. RZS 7 isolated from a plastic contaminated site. The isolate produced extracellular poly-β-hydroxybutyrate (PHB) depolymerase in the mineral salt medium at 30oC during 4 days of incubation under shake flask condition. Purification of the enzyme was carried out by three different methods using PHB as a substrate. Purification of PHB depolymerase by ammonium salt precipitation, column chromatography, and solvent purification method was successfully carried out. Among the purification method tested, the enzyme was best purified by column chromatography on Octyl-Sepharose CL-4B column with maximum (0.7993 U mg-1 ml-1) purification yield. The molecular weight of purified PHB depolymerase (40 kDa) closely resembled with PHB depolymerase of Aureobacterium saperdae. Experiments on assessment of biodegradation of PHB in liquid culture medium and under natural soil conditions confirmed PHB biodegradation potential of Stenotrophomonas sp. RZS 7. The results obtained in FTIR analysis, HPLC study and GC-MS analysis confirmed the biodegradation attempt in liquid medium by Stenotrophomonas sp. RZS 7. Changes in surface morphology of PHB film in soil burial as observed in FE SEM analysis confirmed the biodegradation of PHB. The isolate was capable of degrading PHB and resulted in 87.74% degradation. Higher rate of degradation under natural soil condition is the result of activity of soil microbes that complemented the degradation by Stenotrophomonas sp. RZS 7.


Introduction
Poly-β-hydroxy alkanoates (PHAs) or Poly-β-hydroxybutyrate (PHB) are accumulated as a source of food and energy by a wide variety of bacteria growing under nitrogen-deficient but conditions and is mobilized during nutrient stress under the influence of PHB depolymerase [1][2][3]. PHA/PHB is considered as the best eco-friendly and renewable alternative to the synthetics petrochemical plastics because of its similar properties to synthetic plastic [4][5][6] besides being thermoplastic and biodegradable in nature. Because of such useful properties, it has attracted commercial interest for use as the best alternative to the hazardous synthetic petrochemical polymers and hence it has been successfully commercialized [7][8]. During last decades much research has been devoted towards distribution and occurrence of PHB degraders and studies on different PHB depolymerases. Jendrossek and Handrick [9] reported that PHB depolymerases are responsible for extracellular PHB degradation. Extracellular PHB depolymerases of Aspergillus fumigatus Pdf1 [10], A. Saperdae [11], Thermus thermophiles HB8 [12], Streptomyces bangladeshensis 77T-4 [13], Penicillium simplicissimum LAR13 [14], Acidovorax sp. TP4 [15], Emericellopsis minima W2 [16] has been isolated and purified.
There are many different degradation mechanisms that are combined synergistically in nature to degrade polymers [17]. These mechanisms include bio-deterioration, biofragmentation, assimilation, mineralization and enzymatic degradation through extracellular and intracellular depolymerases that hydrolyze the polymer to water soluble products. PHA depolymerases being water soluble have an ability to bind specifically to polyester surfaces.
Poly(HA)-degrading microorganisms secret specific poly(HA) depolymerases under starved conditions, which hydrolyze the polymer extracellularly to water soluble product and produce transparent clearing zones around the depolymerase secreting colonies (clear zone technique).
Enzymatic hydrolysis of P(3HB) results in 3HB dimer as the major product besides small amounts of 3HB monomer. These cleave mainly the second and third ester linkages from the hydroxyl terminus [18].
However, the organism isolated from the plastic contaminated site and having the ability to degrade PHB may be a potential source of dynamic PHB depolymerase. However, inefficient production and recovery process of PHB, inefficient purification of PHB depolymerase and lack of ample knowledge on PHB degradation by PHB depolymerase have hampered the large scale commercialization of PHB. Therefore, to ensure the biodegradability of biopolymers, it becomes imperative to study the purification of biodegrading enzyme system [16].
The present paper reports the production, purification, and characterization of extracellular PHB depolymerase of Stenotrophomonas sp. RZS 7 isolated from a plastic contaminated site.

PHB depolymerase assay
Following 4 day's incubation at 30 o C and 120 rpm, MM broth was centrifuged a 10,000 rpm for 15 min and the supernatant was assayed for PHB depolymerase [12]. For this purpose PHB granule (substrate for PHB depolymerase) were sonicated (20 kHz for 15 min) and suspended in 50 mM Tris-HCl buffer (pH 7.0) and 150 μg ml -1 of this suspension and 2 mM CaCl 2 was added in 50mM Tris-HCl buffer (pH 7.0) followed by the addition of culture supernatant (0.5 mL).
Enzyme activity was spectrophotometrically measured at 650 nm as a decrease in the PHB turbidity. One unit of PHB depolymerase was defined as the quantity of enzyme required to decrease the absorbance by 0.1 min -1 .

Purification of PHB depolymerase
Having confirmed the presence of PHB depolymerase in cell-free supernatant of MM broth, the broth was subjected for purification of the enzyme by three approaches as follows

Ammonium salt precipitation
The crude enzyme in the culture supernatant was precipitated by adding solid ammonium sulfate with continuous stirring at 4 o C for 1 h. The precipitate was dissolved in the Tris-HCl buffer (pH 7) supernatant and dissolved precipitate was transferred in a separate dialysis bag and allowed for overnight dialysis in chilled phosphate buffer [13]. The protein concentration of dialyzed supernatant, as well as dialyzed precipitate, was measured by the Lowry method [20]. PHB depolymerase activity of dialyzed supernatant and dialyzed precipitate was measured as described earlier.

Solvent purification method
The culture supernatant of the isolate was centrifuged at 10,000 rpm for 20 min. The residue obtained was dissolved in pre-chilled 1:1 acetone ethanol mixture, shaken well and kept in a water bath at 50 o C until all solvent is evaporated. The pellet obtained after evaporation was dissolved in Tris-HCl buffer (pH 7). PHB depolymerase activity of pellet, as well as supernatant, was carried out as described.

Column chromatography
The cell-free supernatant of the isolate was applied onto an Octyl-Sepharose CL-4B column preequilibrated with 50 mM glycine NaOH buffer (pH 9.0) and eluted with a gradient of 0 to 50% ethanol [12], the fractions were collected and PHB depolymerase activity of each fraction was determined as described.

Assessment of biodegradation of PHB
To evaluate the efficacy of degradation of biopolymer, following methods were used

Preparation of PHB film
Preparation of PHB film was done as per the method of Liang et al. [21]. For this purpose PHB powder (0.15 gm) was dissolved in 50 ml chloroform and vortexed for 10-15 min. The solution was poured into clean, dry Petri dishes and kept overnight at room temperature. PHB film was obtained following the evaporation of chloroform. This film was used for assessment of biodegradation.
PHB film was separately added as a sole carbon source into MSM and inoculated with Stenotrophomonas sp. RZS 7 and incubated at 30 o C for 10 days at 120 rpm and observed for degradation of PHB film. Qualitatively estimation involved the weight loss method.
Quantitatively estimation involved the measurement of change in optical density and crotonic acid concentration (μg mL -1 ) at different time interval [22] which is based on the conversion of PHB to crotonic acid when heated in presence of concentrated sulphuric acid. The absorbance was measured at 235 nm and the amount of crotonic acid was calculated from calibration curve prepared with standard crotonic acid in the range of 10-100 μg mL -1 .

PHB biodegradation under natural soil conditions
To check the biodegradability and durability of polymer sample under natural soil conditions, soil burial test method was carried out. Soil burial test methods are used to give an indication of the duration of polymer in a given soil under specific conditions. To study the biodegradation of PHB film, garden soil was taken in different trays (1800 gm per tray) and the polymeric film covered with net was incorporated into the tray with respective inoculum. The trays were covered with moist thick paper [23]. Degradation of PHB film by Stenotrophomonas sp. RZS 7 was observed in five different sets and was compared with negative control containing only sterile soil. The degradation of PHB film was expressed as percent reduction in the weight of PHB film using following formula % Weight loss = Initial weight of film -Weight of film after degradation = X100 Initial weight of film

Fourier-Transform Infrared Spectroscopy (FTIR) analysis
Following the desired incubation period for degradation, the inoculated and incubated liquid culture medium of Stenotrophomonas sp. RZS 7 was subjected for FTIR analysis by using FTIR (FTIR Model 8400S, Shimadzu, Japan,) using IR Solution version 1.40 software. FTIR spectra were recorded in the range of 4000 to 500 cm -1 [24]. The functional groups present in degradation pattern were determined by FTIR and compared with the control.

High Performance Liquid Chromatography (HPLC) analysis
For HPLC analysis of PHB film, the content compound was analyzed by HPLC (Younglin, South Korien, ACME-9000) [25]. For this purpose 20 μl of sample was injected into the C18 column (4.6 x 250 mm, particle size 5 µm) as stationary phase and allowed to run on mobile phase consisting of acetonitrile : water (30:70 v/v) at 254 nm with flow rate of 0.6 mL min -1 . The peaks were analyzed by using Autochro-3000 software.

Sample preparation
The liquid culture supernatant (2 mL) which was subjected for biodegradation by Stenotrophomonas sp. RZS 7 mixed with 2 ml chloroform and 2 ml of acidified methanol (acidified with 3% (v/v) H2SO4). For depolymerization and methanolysis of PHB the mixture was heated at 100 o C for 3 h. During heating the mixture was shaken occasionally and subsequently cooled to room temperature. After heating at 100 o C for 3 h, 1 ml of distilled water was added to the reaction mixture and the mixture was then vortexed for 2 min and allowed to stand for 10 min to separate the phases. At the bottom layer organic phase was obtained by glass Pasteur pipette and it was subjected for further analysis [26].

GC-MS analysis
For identification of monomers present in degraded sample, a coupled gas chromatography/mass spectrophotometry (GC-MS) was performed using gas chromatograph (Perkin Elmer, USA, Model Autosystem XL) with turbomass GC+ equipped with an SGE forte GC capillary column BP20 (30 m X 0.250 μ internal diameter X 0.25 μ) (Australia) and mass spectrophotometer. The mass spectra obtained were compared with the spectra of standard methyl esters of PHB available from National Institute of Standards and Technology (NIST) mass spectra library. The peak identities were first confirmed by matching relative retention time with respect to standard sample extracted at time zero.

GC-MS parameters
One μL of PHB degrading sample was injected by split injection with a split ratio of 10:1 using 10 μL syringe (SGE analytical, Australia). Helium was used as carrier gas at a flow rate of 1 mL min -1 . The oven temperature of column was programmed from 120 o C for 2 min, increased at a rate of 20 o C min -1 to 230 o C, and held at this temperature for 10 min. The temperature of injector and detector was 225 o C and 230 o C respectively.

Field Emission Scanning Electron Microscopy (FE SEM) analysis
The surface morphology of the PHB film was analyzed through Field Emission Scanning

Statistical analysis
All the experiments were performed in triplicate and the mean of three replicates was considered.
Each mean value was subjected to Student's t-test and values of P ≤ 0.05 were taken as statistically significant [27].

Results and Discussion
Production of PHB depolymerase  Table 2).
The protein concentration and PHB depolymerase specific activity and enzyme activity in pellet and supernatant were 0.219 mg mL -1 , 0.5114 U mg -1 mL -1 and 0.3881 UmL -1 respectively. This significant loss of enzyme activity may be due to precipitation resulting in denaturation of proteins by solvent system. Thus the solvent purification method proved insignificant vis-à-vis other purification methods. and Streptomyces bangladeshensis 77T-4 respectively using column chromatography.
Among all three purification methods, purification on Octyl sepharose CL-4B column gave good purification yield (Table 1), more enzyme activity and more specific activity. The protein concentration and PHB depolymerase specific activity and enzyme activity were 0.247 mg mL -1 , 0.7993 U mg -1 mL -1 and 0.309 U ml -1 respectively.

Determination of molecular weight of purified PHB depolymerase
The protein fraction of Stenotrophomonas sp. RZS 7 obtained from column chromatography having maximum PHB depolymerase enzyme activity showed single protein band in SDS PAGE having a molecular mass of approximately 40 kDa lying between molecular weight markers of 37.1 and 48.8. Sadocco et al. [11] have also reported the molecular mass of PHB depolymerase from Aureobacterium saperdae ranges from 42.7 kDa analyzed by SDS PAGE.

Qualitative measurement
A gradual reduction in the weight of PHB film was recorded after 8 days of incubation fragmentation in PHB film was observed whereas no changes observed in control sample of PHB film (Table 3). Varda et al. [32] have also reported similar findings in case of microbial degradation of starch based plastic by S. epidermidis.

Quantitative estimation
Isolate RZS7 caused the reduction in the amount of crotonic acid with increase in the biomass.
Maximum reduction in the amount of crotonic acid was recorded on 10th day of incubation at 37 o C ( Table 3). The reduction in crotonic acid concentration can be taken as the evidence of biodegradation of PHB film.  (Fig.1 and 2). The major functional groups in control sample

High Performance Liquid Chromatography (HPLC) analysis
The spectra of the original polymer sample before degradation revealed six peaks at 254 nm during 20 min flow at the rate of 0.6 mL min -1 (Fig. 3). While polymer sample degradation by Stenotrophomonas sp. RZS 7 revealed nine peaks having different retention time as compared to control (Fig. 4) indicating the formation of monomers. The sum of total area of peaks of degraded sample was less as compared to the sum of total area of peaks of control (undegraded) preparation. This reduction in the peak area confirmed the degradation of polymer.     (Table 4). McLafferty and Turecek (33) reported that the mass spectra of the methyl esters of PHB dominated by m/z = 74 represented the carbonyl end of the molecule and showed the characteristic of 3-hydroxyl functional groups. However the peak at m/z = 59 represented the hydroxyl end released when molecule was cleaved at the bond between C3 and C4.

Field Emission Scanning Electron Microscopy (FE SEM) analysis
Surface analysis of PHB film after soil burial experiment showed many differences in the morphology of PHB film as compared to control ( Figure 9A) and PHB film buried in soil ( Figure   9B). Biodegradation of PHB film caused erosion and roughening of surface of PHB film ( Figure   9C). Gautam and Kaur [35] have also reported the morphological changes in polyethylene surface after degradation compared with control following SEM analysis. Calabia and Tokiwa [36] analyzed the degradation of PHB by scanning electron microscopy, and observed the presence of hemispherical holes on the film surface after degradation and claimed the formation of holes due to colonization by strain Streptomyces sp. SC-17 on the surface of PHB film.