Production, purification and evaluation of biodegradation potential of PHB depolymerase of Stenotrophomonas sp. RZS7.

There are numerous reports on poly-β-hydroxybutyrate (PHB) depolymerases produced by various microorganisms isolated from various habitats, however, reports on PHB depolymerase production by an isolate from plastic rich sites scares. 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. RZS7 isolated from a dumping yard rich in plastic waste. The isolate produced extracellular PHB depolymerase in the mineral salt medium (MSM) at 30°C during 4 days of incubation under shaking. The enzyme was purified by three methods namely ammonium salt precipitation, column chromatography, and solvent purification. Among these purification methods, the enzyme was best purified by column chromatography on the Octyl-Sepharose CL-4B column giving optimum yield (0.7993 Umg-1mL-1). The molecular weight of purified PHB depolymerase was 40 kDa. Studies on the assessment of biodegradation of PHB in liquid culture medium and under natural soil conditions confirmed PHB biodegradation potential of Stenotrophomonas sp. RZS7. The results obtained in Fourier-Transform Infrared (FTIR) analysis, High-Performance Liquid Chromatography (HPLC) study and Gas Chromatography Mass-Spectrometry (GC-MS) analysis confirmed the biodegradation of PHB in liquid medium by Stenotrophomonas sp. RZS7. Changes in surface morphology of PHB film in soil burial as observed in Field Emission Scanning Electron Microscopy (FESEM) analysis confirmed the biodegradation of PHB under natural soil environment. The isolate was capable of degrading PHB and it resulted in 87.74% biodegradation. A higher rate of degradation under the natural soil condition is the result of the activity of soil microbes that complemented the biodegradation of PHB by Stenotrophomonas sp. RZS7.

Introduction

Poly-β-hydroxy alkanoates (PHAs) or PHB are accumulated as a source of food and energy by a wide variety of bacteria growing under nitrogen stress but carbon-rich conditions isutilized during starvation by the activity of PHB depolymerase [1-3]. PHAs/PHB are considered as the best environment-friendly and renewable option to the synthetics petrochemical plastics because of its similar properties to synthetic plastic [4-6] 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 the last decades much research has been devoted towards the 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], Aureobacterium saperdae [11], Thermus thermophiles HB8 [12], Streptomyces bangladeshensis 77T-4 [13], Penicillium simplicissimum LAR13 [14], Acidovorax sp. TP4 [15], Streptomyces KJ-72 [16] have been isolated and purified.

There are many different degradation mechanisms that are combined synergistically in nature to degrade polymers [17]. These mechanisms include biodeterioration, biofragmentation, assimilation, mineralization, and enzymatic degradation through extracellular and intracellular depolymerases that break the polymer to water-soluble products. [17]. PHB depolymerases produced by PHA degrading microorganism excrete under starved conditions, which degrade the polymer to water-soluble product forming clearing zones around the PHB depolymerase producing colonies. The action of PHB depolymerase breaks the ester bonds in PHB to produces PHB dimer and 3HB monomer. [18].

The bacterial isolate obtained from the plastic contaminated habitat that possess the ability to degrade PHB may be a potential source of commercially valuable PHB depolymerase, provided that the production, recovery, and purification protocols are properly optimized for large scale production of PHB depolymerase. Therefore, to ensure the biodegradability of biopolymers, it becomes imperative to study the purification of PHB biodegrading enzyme system [16]. PHB depolymerase of isolates obtained from plastic rich habitat is expected to have better relevance for its application in plastic/ bioplastic degradation.

The present paper reports the production, purification, and characterization of extracellular PHB depolymerase of Stenotrophomonas sp. RZS7 isolated from a plastic contaminated site in North Maharashtra region of India.

Materials and methods

PHB

PHB procured from Sigma-Aldrich, Germany was used as a standard for all experiments.

Source of cultures

Cultures of fungi; Aspergillus niger NCIM1025, A. flavus NCIM650, Alternaria alternate ARI715, and Cercospora arachichola were procured from NCIM, NCL, Pune, Maharashtra, India. Alcaligenes sp RZS4 and Pseudomonas sp. RZS1 and Streptomycetes sp. were obtained from the culture depository of the Department. For present study, Stenotrophomonas sp. RZS7 that was previously identified [19] was used as PHB depolymerase producer.

Production of PHB depolymerase

Production of PHB depolymerase was carried out by shake flask method by growing Stenotrophomonas sp. RZS7 in MSM containing (gL-1) PHB, 0.015 K2HPO4, 0.07 g; KH2PO4, 0.07 g; MgSO4, 0.07 g; NH4Cl, 0.1 g; NaNO3, 0.1 g; NaCl, 0.5 mg; FeSO4, 0.2 mg, ZnSO4, 0.7 mg [14] at 120 rpm for 4 days at 30°C.

PHB depolymerase assay

Following the incubation, MSM was centrifuged a 10,000 rpm for 15 min and the supernatant was used for PHB depolymerase assay [12]. The reaction mixture contained 150 μg ml-1 PHB granules, (substrate for PHB depolymerase) and 2 mM CaCl2 50 mM Tris-HCl buffer (pH 7.0) and 0.5 mL of supernatant from MSM. 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

The cell-free supernatant of, MSM was subjected for purification of the enzyme as follows -

Ammonium salt precipitation

The cell-free supernatant was precipitated by adding increasing concentration (10–80% w/v) of solid ammonium sulfate with continuous stirring at 4°C for 1 h. The precipitate was dissolved in the Tris-HCl buffer (pH 7), supernatant and the dissolved precipitate was transferred in a separate dialysis bag and allowed for overnight dialysis in chilled phosphate buffer (pH 7.0) [13]. The protein content of dialyzed supernatant and dialyzed precipitate was measured as per Lowry et. al. method [20]. PHB depolymerase activity of dialyzed supernatant and dialyzed precipitate was measured [12].

Solvent purification method

The cell free supernatant 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°C until all solvent is evaporated. The pellet obtained after evaporation was dissolved in Tris-HCl buffer (pH 7). PHB depolymerase activity of pellet and supernatant was measured [12].

Column chromatography

The cell-free supernatant obtained from MSM was loaded onto an Octyl-Sepharose CL-4B column pre-equilibrated with 50 mM glycine NaOH buffer (pH 9.0) followed by elution with 50% ethanol [12]. The eluting fractions were separately collected and subjected for PHB depolymerase [12].

Determination of molecular weight of purified PHB depolymerase

Determination of the molecular weight of purified PHB depolymerase was performed by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Purified fractions and standard protein marker (Ge-Nei, Bangaluru, India) were electrophoresed at 50 mv for 2 h. The separated bands were stained with Coomassie Brilliant Blue R-250 and visualized under UV transilluminator. The separated bands of test protein were compared with standard protein markers. The proteins content of bands was estimated by as per Lowry et al. [20].

Assessment of biodegradation of PHB

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

PHB biodegradation under laboratory conditions

Preparation of PHB film was done as per Linag et al. [21].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 at30°C. PHB film was obtained following the evaporation of chloroform. This film was used for the assessment of biodegradation.

PHB film was separately added as a sole carbon source into MSM and inoculated with Stenotrophomonas sp. RZS7 and incubated at 30°C for 10 days at 120 rpm and observed for degradation of PHB film. Qualitative estimation involved the weight loss method. Quantitative estimation involved the measurement of change in optical density and crotonic acid concentration (μgmL-1) at a different time intervals at 235 nm, [22]. The amount of crotonic acid was calculated from the calibration curve prepared with standard crotonic acid in the range of 10–100 μgmL-1.

PHB biodegradation under natural soil conditions

Soil burial test gives an indication of the biodegradation of the polymer in a given soil under specific conditions. To check the biodegradability and durability of polymer sample under natural soil conditions, soil burial test was carried out. For this purpose garden soil was taken in different trays (1800 gm per tray) and the PHB film covered with the net was incorporated into the tray with Stenotrophomonas sp. RZS7 and trays were covered with moist thick paper [23]. Degradation of PHB film by Stenotrophomonas sp. RZS7 was observed in five different sets and was compared with negative control containing only sterile soil. The degradation of PHB film was measured in terms of weight loss and was expressed as percent reduction in the weight of PHB film

Fourier-transform infra red (FTIR) spectroscopy analysis

Following the desired incubation period for degradation, the inoculated and incubated liquid culture medium of Stenotrophomonas sp. RZS7 was subjected to FTIR analysis on FTIR (FTIR Model 8400S, Shimadzu, Japan,) by using IR solution version 1.40 software. FTIR spectra were read between 4000 to 500 cm-1 [24]. The functional groups present in degradation patterns were determined by FTIR and compared with the control.

High-performance liquid chromatography (HPLC) analysis

HPLC analysis of the preparation was analyzed on HPLC (Younglin, South Koria, ACME-9000) [25]. For this purpose, 20 μL of sample was injected into the C18 column as stationary phase and allowed to run 0.6 mL min-1 on a mobile phase of acetonitrile: water (30:70 v/v) at 254 nm. The peaks were analyzed by using Autochro-3000 software.

Gas chromatography-mass spectroscopic (GC-MS) analysis

Sample preparation

The cell-free supernatant (2 mL) from a liquid medium was mixed with 2 mL chloroform and 2 mL of acidified methanol. For depolymerization and methanolysis of PHB, the mixture was heated at 100°C for 3 h with occasional shaking followed by subsequently cooling at 30°C followed by heating at 100°C for 3 h and adding 1 mL of distilled water. The whole content was vortexed for 2 min and allowed to stand for 10 min to separate the phases. At the bottom layer, organic phase was obtained by a glass Pasteur pipette and it was subjected for spectral analysis [26].

GC-MS analysis

Monomers present in the degraded sample were identified by GC-MS (Model Autosystem XL, Perkin Elmer, USA) equipped with an SGE forte GC capillary column BP20 (Australia) and mass spectrophotometer. The mass spectra obtained were compared with the spectra of standard methyl esters of PHB available from mass spectra library of the National Institute of Standards and Technology (NIST), USA. The peak identities were initially identified based on their matching of relative retention time with respect to standard sample extracted at time zero.

GC-MS parameters

One μL of sample was injected with a split ratio of 10:1. The rate of flow of carrier gas (helium) was 1 mL min-1. The oven temperature of the column was programmed from 120°C for 2 min, increased at a rate of 20°C min-1 to 230°C, and held at this temperature for 10 min. The temperature of the injector and detector was 225°C and 230°C respectively.

Field emission scanning electron microscopy (FE SEM) analysis

FE SEM was used to study the surface morphology and structural changes in PHB film following the biodegradation in soil burial test. PHB film from soil burial sample was thoroughly washed with sterile distilled water and mounted on stub using carbon tape it was made conducting by gold coating in vacuum by evaporation. The polymer film was dried in an Infrared (IR) lamp. The images of the treated test samples and untreated test samples were compared.

Statistical analysis

Three replicates of each experiment were run and the average value of triplicates was recorded. The data was analyzed by Student’s t-test and values of P ≤ 0.05 were taken as statistically significant [27].

Results and discussion

After 4 days of incubation at 30°C at 120 rpm in MSM, Stenotrophomonas sp. RZS7 yielded 0.721 U mL-1 PHB depolymerase. Similar enzyme activities have been reported by other researchers [21,28]

Protein precipitation was increasing with increase in the concentrations of ammonium sulfate in the cell-free supernatant of Stenotrophomonas sp. RZS7, maximum proteins were precipitated with 70% ammonium sulfate concentration (Table 1). The protein concentration, specific activity and enzyme activity of PHB depolymerase in the dialyzed precipitate of Stenotrophomonas sp. RZS7 were 0.219 mgmL-1, 0.7031 Umg-1mL-1 and 0.154 UmL-1 respectively. Zhou et al. [28] have precipitated PHB depolymerase of Escherichia coli, and Penicillium sp. DS9701-D2 in 70 and 75% of ammonium sulfate respectively. Shivkumar et al. [29] have purified PHB depolymerase of Penicillium citrinum S2 in 80% of ammonium sulfate.

Table 1

Purification profile of PHB depolymerase by various methods.

Purification method	Enzyme activity (Umg-1mL-1)
Salt precipitation	0.7031 (0.090)
Solvent purification	0.1120 (0.022)
Column chromatography	0.7993 (0.099)

Values are statistically significant at P ≤ 0.05

Values are the mean of triplicates

Values in the parenthesis are standard deviation

Solvent purification of PHB depolymerase of Stenotrophomonas sp. RZS7 retained only 51.14% and 38.81% enzyme activity in pellet and supernatant (Table 1).

The protein concentration, specific activity and enzyme activity of PHB depolymerase in pellet and supernatant were 0.219 mgmL-1, 0.5114 Umg-1mL-1, and 0.3881 UmL-1 respectively. The loss of enzyme activity in solvent system is due to the denaturation of proteins caused by the solvent system. Thus the solvent purification method proved an ineffective for purification of PHB depolymerase of Stenotrophomonas sp. RZS7.

A total of five fractions were obtained from Octyl-Sepharose CL-4B. Among all the fractions analyzed for PHB depolymerase enzyme activity, the 3rd fraction showed maximum enzyme activity (Table 2). PHBV depolymerase of Bacillus sp. AF3 and Streptoverticillium kashmirense AF1 have also been purified on Sephadex G-75 [30]. Kim et al. [31] have also purified PHB depolymerase of Emericellopsis minima W2 and Streptomyces sp. KJ-72 on Sephadex G-100 and Sephadex G-150 respectively. Papaneophytou et al. [12] and Hsu et al. [13] have isolated and purified extracellular PHB depolymerases of Thermus thermophiles HB8 and Streptomyces bangladeshensis 77T-4 respectively using column chromatography.

Table 2

Purification of PHB depolymerase of Stenotrophomonas sp. RZS7 on Octyl sepharose column.

Fraction	Total activity (UmL-1)	Total protein (mgmL-1)	Specific activity (U mg-1mL-1)	% activity
1	0.027 (0.027)	0.031 (0.010)	0.0782 (0.032)	11.13 (0.012)
2	0.101 (0.011)	0.153 (0.037)	0.3781 (0.041)	48.31 (0.039)
3	0.247 (0.015)	0.309 (0.074)	0.7993 (0.090)	79.93 (0.092)
4	0.017 (0.001)	0.015 (0.009)	0.0106 (0.011)	04.01 (0.012)
5	0.003 (0.001)	0.006 (0.005)	0.0011 (0.008)	00.10 (0.002)

Values were statistically significant at P ≤ 0.05

Values in the parenthesis are standard deviation

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, specific activity and enzyme activity of PHB depolymerase were 0.309 UmL-1, 0.7993 Umg-1 mL-1 and 0.247 mgmL-1 respectively.

The protein fraction of Stenotrophomonas sp. RZS7 obtained from the octyl sepharose CL-4B column that exhibited maximum enzyme activity revealed a single protein band in SDS PAGE having a molecular mass of about 40 kDa Sadocco et al. [11] have also reported the molecular mass of 42.7 kDa PHB depolymerase in Aureobacterium saperdae.

Among the various reference cultures taken from our own departmental culture depository; all fungal and bacterial cultures were unable to degrade PHB when growing in PHB containing MSM. However, only Streptomycetes sp. was found to degrade PHB on 10th day of incubation but the extent of PHB biodegradation in this case was less. Hence for further biodegradation studies Stenotrophomonas sp. RZS7 was used.

Qualitative measurement

A gradual reduction in the weight of PHB film was recorded after 8 days of incubation fragmentation in PHB film was observed while no changes occurred in a control sample of PHB film (Table 3). Varda et al. [32] have recorded similar loss of weight of starch-based plastic by S. epidermidis.

Table 3

Qualitative measurement of biodegradation of PHB film.

Weight of PHB film	Weight loss of PHB film (g)	OD	Crotonic acid* (μgmL-1)
Initial weight	0.159 (0.031)	0.000	1000 0.030)
After 2 days	0.148 (0.029)	0.014	813 (0.026)
After 4 days	0.109 (0.021)	0.019	794 (0.019)
After 6 days	0.099 (0.017)	0.035	705 (0.018)
After 8 days	Fragmentation	0.050	686 (0.015)

Values were statistically significant at P ≤ 0.05

Values in the parenthesis are standard deviation

*Crotonic acid was as PHB standard for spectrophotometric estimation of PHB as when PHB is heated with concentrated sulphuric acid it gets converted into crotonic acid.

Quantitative estimation

Isolate RZS7 caused the reduction in the amount of crotonic acid with an increase in the biomass. The maximum reduction in the amount of crotonic acid was recorded on 10th day of incubation at 37°C (Table 3). The reduction in crotonic acid concentration can be taken as the evidence of biodegradation of PHB film.

Fourier transform infrared (FTIR) spectroscopy analysis

FTIR analysis of liquid culture medium containing PHB film and inoculated with Stenotrophomonas sp. RZS7 showed changes in the functional groups and significant shift of wavenumbers, indicating the biodegradation of polymer. It was observed that in case of control preparation (without inoculum) FTIR chromatogram peaks appearing at 3020.63 cm-1 changed to 3018.70, 2926.11, 2856.67 cm-1, 2399.53 cm-1 changed to 2360.95, 2333.94 cm-1, 2088.98 cm-1 changed to 2079.33 cm-1, 1641.48 cm-1 shifted to 1629.90 cm-1 and 1631.83 cm-1 shifted towards 1529.60, 1427.37, 1381.08 cm-1 (Figs 1 and 2). The major functional groups in control sample were found at wavelength frequency 1641.48 and 1631.83 denoted C = O stretch in ketone, frequency 3020.63 denoted C-H which stretch in alkane, wavelength frequency 1014.59 denoted C-O which stretch in ester and 756.12 denoted C-H which is rock alkane. However the major functional group frequency changes observed in degrading sample i.e. 1629.90 denoted C = O which stretch in ketone, 1215.19 which denote alkyl halide, 2399.53,2360.95 and 2333.94 denoted H-C = O group, 927.79 denoted C-O which stretch in ester, 769.62 denoted C-H which is rock alkane. These changes in wavelength frequency of major functional groups in degrading samples as compared to control give preliminary proof of biodegradation of polymer by Stenotrophomonas sp. RZS7.

Fig 1

FTIR chromatogram of PHB (Control) read between 4000 to 500 cm-1 and analyzed with 1.40 software.

Fig 2

FTIR chromatogram of PHB after degradation by RZS7 read between 4000 to 500 cm-1 and analyzed with 1.40 software. The functional groups present in the spectra were compared with the functional groups in control preparation.

High performance liquid chromatography (HPLC) analysis

The spectra of the original polymer sample before degradation revealed six peaks at 254 nm (Fig 3). While polymer sample degradation by Stenotrophomonas sp. RZS7 revealed nine peaks having different retention time as compared to control (Fig 4) indicating the formation of monomers. The sum of the total area of peaks of the degraded sample was less than the sum of the total area of peaks of control (undegraded) preparation. This reduction in the peak area confirmed the degradation of the polymer.

Fig 3

HPLC spectra of polymer before degradation (Control) recorded at 254 nm).

Spectral analysis was carried out at 254 nm with autochro-3000 software.

Fig 4

HPLC spectra of polymer after degradation recorded at 254 nm.

The peaks were analyzed by using Autochro-3000 software and compared with the peaks of control.

The degradation pattern on the basis of removal or decrease in the retention time of the peaks and their comparison with the retention time of the peaks of reference demonstrated the disappearance of peaks indicating biodegradation of PHB components by Stenotrophomonas sp. RZS7 (Fig 5 and Fig 6). A significant decrease in area height and percentage in the degraded sample was clear over the control (undegraded) sample. The total area of peaks of degraded sample was less as compared to the total area of peaks of control (undegraded) preparation. This reduction in the peak area confirmed the conversion of PHB due to its biodegradation into monomers.

Fig 5

Chromatogram of polymer sample before biodegradation (Standard).

GC MS analysis was performed on gas chromatograph equipped with capillary column BP20 and mass spectrophotometer. The mass spectra obtained were recorded.

Fig 6

Chromatogram of polymer sample after biodegradation.

GC MS analysis was performed by using gas chromatograph with turbomass GC capillary column BP20 and mass spectrophotometer. The mass spectra obtained were compared and identified with the library of spectra of standard methyl esters of PHB. The peaks were identified by matching relative retention time with respect to standard sample extracted at time zero.

The fragmentation patterns with GC-MS and the identities of peaks in the mass spectra were correlated to the carbonyl and hydroxyl ends of the represented hydroxyl alkanoates ethyl esters of PHB after degradation. Tridecanoic acid 12-methyl-methyl ester and heptacosanoic acid methyl ester in standard polymer sample were completely degraded whereas Decanoic acid methyl ester, undecanoic acid methyl ester, and nonanoic acid methyl ester were detected in the degraded sample (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 indicated the release of the hydroxyl end during the cleavage of the molecule between C3 and C4.

Table 4

GC MS analysis of methanolysed polymer before degradation (Standard) and after degradation.

Retention time	Methyl ester PHB monomers (before degradation)	MW	Methyl ester PHB monomers (after degradation)	MW
	Pentadecanoic acid, 14-methyl-, methyl ester Hexadecanoic acid, methyl ester	270	Tridecanoic acid, methyl ester	228
	Tridecanoic acid, methyl ester	228	Pentadecanoic acid, 14-methyl-, methyl esterHexadecanoic acid, methyl ester	270
	Hexadecanoic acid, 15-methyl-, methyl esterHeptadecanoic acid, methyl ester	284	Hexadecanoic acid, 15-methyl-, methyl esterHeptadecanoic acid, methyl ester	284
	Heneicosanoic acid, methyl ester	340	Decanoic acid, methyl ester	186
	Docosanoic acid, methyl ester	354	Dodecanoic acid, methyl ester	214
	Eicosanoic acid, methyl ester	326	Undecanoic acid, methyl ester	200
	Hexacosanoic acid, methyl ester	410	Methyl tetradecanoate	242
	Methyl tetradecanoateTridecanoic acid, 12-methyl-, methyl ester	242	Pentadecanoic acid, methyl ester	256
	Octadecanoic acid, methyl ester	298	Eicosanoic acid, methyl ester	326
	Dodecanoic acid, methyl ester	214	Heneicosanoic acid, methyl ester	340
	Pentadecanoic acid, methyl ester	256	Octadecanoic acid, methyl ester	298
	Heptacosanoic acid, methyl ester	424	Docosanoic acid, methyl ester	354
			Hexacosanoic acid, methyl ester	410
			Nonanoic acid, methyl ester	172

The mass spectra of standard polymer sample and the characteristics signals were recorded at m/z = 29, 39, 41, 43, 45, 55, 57, 59, 74, 75, 87, 101, 111, 131, 143, 157, 171, 185, 199, 219, 227, 239, 265, 270, 271, 314, 415. However in polymer degrading sample m/z = 29, 32, 41, 43, 44, 55, 57, 69, 74, 75, 87, 97, 111, 129, 143, 145, 169, 176, 199, 220, 227, 270 (Fig 7) which can be attributed to oligomers of PHB (Fig 8). Wennan et al. [34] reported that m/z = 29, 32, 41, 43, 55, 69, 75, 87, 97, 111, 143 denotes Pentadecanoic acid methyl ester, m/z = 29, 41, 55, 57, 69, 74, 75, 87, 97 denotes Decanoic acid, methyl ester, m/z = 41, 43, 55, 74 denotes Dodecanoic acid methyl ester, m/z = 74 denotes Hexadecanoic acid methyl ester, m/z = 43, 55, 74 denotes Octadecanoic acid methyl ester.

Fig 7

GC-MS spectra of polymer sample before degradation showing mass spectra of m/z = 29, 39, 41, 43, 45, 55, 57, 59, 74, 75, 87, 101, 111, 131, 143, 157, 171, 185, 199, 219, 227, 239, 265, 270, 271, 314, 415 indicating the presence of intact polymer of PHB.

Fig 8

GC-MS spectra of polymer sample after degradation showing mass spectra of m/z 29, 32, 41, 43, 44, 55, 57, 69, 74, 75, 87, 97, 111, 129, 143, 145, 169, 176, 199, 220, 227, 270 indicating the presence of oligomers of PHB.

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 (Fig 9A) and PHB film buried in the soil (Fig 9B). Biodegradation of PHB film caused erosion and roughening of the surface of PHB film (Fig 9C). Gautam and Kaur [35] have also reported the morphological changes in polyethylene surface after degradation as compared with control. Calabia and Tokiwa [36] analyzed the degradation of PHB by SEM and observed the presence of holes on the surface of PHB film due to the growth of Streptomyces sp. SC-17 on the surface of PHB film causing biodegradation of PH B.

Fig 9

Morphological changes in PHB film after soil burial test observed under FE SEM; (A) Control set, (B) PHB film buried in presence of natural soil, (C) PHB film degraded by Stenotrophomonas sp. RZS7.

Conclusion

The present attempt provided PHB degrading Stenotrophomonas sp. RZS 7 which utilized PHB as a sole source of carbon under the influence of extracellular PHB depolymerase. The enzyme acted as a key enzyme responsible for the biodegradation of PHB. Purification of PHB depolymerase by solvent purification resulted in protein precipitation and denaturation of the enzyme. Column chromatography appeared as an efficient and best purification method as it yielded maximum protein, maximum enzyme activity, and maximum specific activity. The molecular weight of purified PHB depolymerase of Stenotrophomonas sp. RZS7 (40 kDa) matched with a molecular weight of Aureobacterium saperdae.

Biodegradation of PHB in liquid culture medium and under natural soil conditions confirmed PHB biodegradation potential of Stenotrophomonas sp. RZS7. The results obtained in FTIR analysis, HPLC study and GC-MS analysis confirmed the biodegradation ability of Stenotrophomonas sp. RZS7 in liquid medium. Changes in surface morphology of PHB film in the soil burial as observed in FE SEM analysis confirmed the ability of isolate to degrade PHB under natural soil conditions. Higher rate of degradation under natural soil condition is the result of activity of soil microbes that complemented the degradation by Stenotrophomonas sp. RZS7.

3 Sep 2019

PONE-D-19-19023

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

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: the author failed to explain concentration of am.sulphate used for Isolation of protein. doing SDS PAGE is outdated and not enough for protein work. technically 2D PAGE and MALDI is required. Results are not statistically expressed(SD values, Vlaue, Pvalue are missing).

Reviewer #2: Dear Editor and authors:

Best regards, I appreciated the research idea where it concerning by the biodegradation of certain polymeric waste by microbial enzyme. Since, there is no perfect work, so I have some comments on it:

1. Biodegrading word in the title should be "biodegradation".

2. The 9 lines in the beginning of the abstract should be transfer to the introduction section.

3. All the paragraphs have not included the start space.

4. The materials and methods section included lot of expressing mistakes which I corrected them in my comments in the attached file.

5. In the results; Tables did not express the results and data mentioned in the text as follow:

• Table 1 not express the results of Ammonium salt precipitation method as mentioned in the text, but it expressed the results of Octyl sepharose column method. Also, what is these values contained between brackets in the table?

• Data in Table 2 are differed from data mentioned in the text for all purification methods.

6. The phrase" The dialyzed precipitate of Stenotrophomonas sp. RZS 7 obtained after ammonium salt precipitation when applied onto an Octyl-Sepharose CL-4B column……" was mentioned under title of Column chromatography ; this really confusing where, are the three purification methods separated from each others? Or they are complementary?

7. According to materials and methods: Crotonic acid was produced as a result of degradation of PHB film so there is a reverse relation between the amount and concentrations of them which is differed with results in table 3 which recorded decreasing in conc. of crotonic acid by degradation of PHB film!!!!!! Logically It must be increase. This point needs clarification for the reader.

8. Fig 1 and Fig 2 are the same!!!!!! Insert fig 2 which reflect the data mentioned in the text.

9. Correct titles in table 4 where all are before degradation, while it should be before and after.

10. Put A, B,C on photos of Fig 9

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Samah El-Debaiky

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: PONE-D-19-19023_reviewer.doc

Click here for additional data file.

7 Sep 2019

Reviewer #1:

Que. The author failed to explain concentration of am.sulphate used for Isolation of protein. doing SDS PAGE is outdated and not enough for protein work. technically 2D PAGE and MALDI is required. Results are not statistically expressed (SD values, Vlaue, Pvalue are missing).

Ans : Concentration of ammonium per sulphate is now included in SDS-PAGE under Materials and Methods part. SDS-PAGE is still used as one of the basic methods for determining the molecular weight. Data in results is statistically analyzed and the values in Tables are the average of triplicates. Values were taken to be statistically significant at P ≤ 0.05, and values of standard deviation (SD) are already given in the parenthesis.

Reviewer #2:

1. Biodegrading word in the title should be "biodegradation".

Ans : Revised in the title

2. The 9 lines in the beginning of the abstract should be transfer to the introduction section.

Ans : Sentence from line 3 to 5 is now shifted to introduction. Rest of the text is more relevant in Abstract and hence not shifted

3. All the paragraphs have not included the start space.

Ans : Now every paragraph has given 1Tab start spacing

4. The materials and methods section included lot of expressing mistakes which I corrected them in my comments in the attached file.

Ans : All the typos and mistakes in Materials and Methods have been rectified.

5. In the results; Tables did not express the results and data mentioned in the text as follow:

• Table 1 not express the results of Ammonium salt precipitation method as mentioned in the text, but it expressed the results of Octyl sepharose column method. Also, what is these values contained between brackets in the table?

Ans : Tables are now placed at proper place and Table Numbers have been revised. The values in the brackets are Standard Deviation (SD) value.

• Data in Table 2 are differed from data mentioned in the text for all purification methods.

Ans : Tables are now placed at proper place and Table Numbers have been revised. The values in the brackets are Standard Deviation (SD) value.

6. The phrase" The dialyzed precipitate of Stenotrophomonas sp. RZS 7 obtained after ammonium salt precipitation when applied onto an Octyl-Sepharose CL-4B column……" was mentioned under title of Column chromatography ; this really confusing where, are the three purification methods separated from each others? Or they are complementary?

Ans : All three methods were separate. Dialyzed precipitate of ammonium sulphate method was used for column chromatography. Octyl-Sepharose is a resin used in CL-4B column under column chromatography

7. According to materials and methods: Crotonic acid was produced as a result of degradation of PHB film so there is a reverse relation between the amount and concentrations of them which is differed with results in table 3 which recorded decreasing in conc. of crotonic acid by degradation of PHB film!!!!!! Logically It must be increase. This point needs clarification for the reader.

Ans : Crotonic acid is not the end product of PHB degradation. It is used as standard for spectrophotometric estimation of PHB. As per Law and Slepecky method [22] when PHB is heated with concentrated sulphuric acid it gets converted into crotonic acid. Clarification is now given at the footnote of Table 3

8. Fig 1 and Fig 2 are the same!!!!!! Insert fig 2 which reflect the data mentioned in the text.

Ans : Fig 2 is now replaced with another figure

9. Correct titles in table 4 where all are before degradation, while it should be before and after.

Ans : Corrected

10. Put A, B,C on photos of Fig 9

Ans : Revised

Submitted filename: PONE-D-19-19023 Response to Reviewers.doc

Click here for additional data file.

30 Oct 2019

PONE-D-19-19023R1

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

PLOS ONE

Dear Sayyed,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Reviewers suggested few minor changes in the manuscript. Kindly go through it and do the necessary changes accordigly.

We would appreciate receiving your revised manuscript by 05.11.2019. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Please include the following items when submitting your revised manuscript:

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Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Pradeep Kumar

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: Dear editor and authors,

Have a good day, my regards to all of you. The authors stand on all the comments except 4 points: 1) line 3 in abstract contains double dots. 2)under title PHB depolymerase assay in methods: correct "a 10,000 rpm" to "at 10,000 rpm". 3) under each table: change "Figures in the parenthesis " to " Values in the parenthesis ". 4) Equation in page 9: change "= X100" to "X100"

Reviewer #3: The submitted paper was well-written, clearly followed an interesting field of research to update the mind of respected academic readers about one of the most important natural enzymes that deal with environmental issues. Generally, authors have done their best to provide high-quality data for both journal and reviewers. The highlighted points withing this paper has enough worth to publish with PLOS ONE journal. Meanwhile, I highly recommend the respected authors to add the figure of their protein purification steps gel to their paper. Due to having critical and functional roles among bacterial species, it is quite quintessential the authors discuss the possible structure of this protein by considering other closest species to highlight valued points for further studies. Also, figure 1 has not enough quality to put it within the MS body text. It is better the authors try to annex it to their paper as supplementary record. Figures 5-7 also have a low quality. Please change their resolution option through an image editor software.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: Yes

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

31 Oct 2019

PONE-D-19-19023R1

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

Reviewer #2:

The authors stand on all the comments except 4 points:

1) line 3 in abstract contains double dots.

Ans: One extra dot is now removed

2) Under title PHB depolymerase assay in methods: correct "a 10,000 rpm" to "at 10,000 rpm".

Ans: Revised as “at 10,000 rpm”

3) Under each table: change "Figures in the parenthesis " to " Values in the parenthesis ".

Ans : "Figures in the parenthesis is now revised as " Values in the parenthesis " under all tables.

4) Equation in page 9: change "= X100" to "X100"

Ans : Revised as X100

Reviewer #3:

The submitted paper was well-written, clearly followed an interesting field of research to update the mind of respected academic readers about one of the most important natural enzymes that deal with environmental issues. Generally, authors have done their best to provide high-quality data for both journal and reviewers. The highlighted points withing this paper has enough worth to publish with PLOS ONE journal. Meanwhile, I highly recommend the respected authors to add the figure of their protein purification steps gel to their paper. Due to having critical and functional roles among bacterial species, it is quite quintessential the authors discuss the possible structure of this protein by considering other closest species to highlight valued points for further studies.

Ans : This figure has already been published, placing here it will be a duplication

Also, figure 1 has not enough quality to put it within the MS body text. It is better the authors try to annex it to their paper as supplementary record.

Ans : Fig 1 is the FTIR spectra of PHB (Standard) and is required for comparison with Fig 2 FTIR spectra of PHB (Sample). SO it is retained in the paper.

Figures 5-7 also have a low quality. Please change their resolution option through image editor software.

Ans : Quality of Figure 5-7 have been improved.

Submitted filename: PONE-D-19-19023 Response to Reviewers.doc

Click here for additional data file.

13 Nov 2019

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

PONE-D-19-19023R2

Dear Dr. Sayyed,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Pradeep Kumar

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: (No Response)

Reviewer #3: All comments were addressed correctly as I expected. After revision process, the manuscript seems have a good structure and well revised. The quality of figures has improved, and English text right now sounds so fluent. To me, publishing this work as a valued article in PONE has no problem. The respected editorial team, however, will determine the destiny of this paper either for publishing or rejecting its content.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: Yes: Dr. Samah El-Debaiky

Reviewer #3: No

10 Dec 2019

PONE-D-19-19023R2

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

Dear Dr. Sayyed:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Pradeep Kumar

Academic Editor

PLOS ONE