Thermophilic PHB depolymerase of Stenotrophomonas sp., an isolate from the plastic contaminated site is best purified on Octyl-Sepharose CL-4B

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/ml) purification yield. The molecular weight of purified PHB depolymerase (40 kDa) closely resembled with PHB depolymerase of Aureobacterium saperdae.


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)(4)(5)(6). 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 (7-8) 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. 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.
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
All the experiment was carried out using PHB powder. PHB was obtained from Sigma-Aldrich, Germany.

Source of bacterium
Stenotrophomonas sp. RZS 7 isolated from plastic contaminated site was previously identified (17) and used in the present study as a source of PHB depolymerase.

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 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.

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 (18). 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.  (14). Values were taken to be statistically significant at P ≤ 0.05 Values are the mean of three replicates Values were taken to be statistically significant at P ≤ 0.05 Values are the mean of three replicates