Production, purification, and kinetics of the poly-β ... · 2 26 Abstract 27...

1

1 Production, purification, and kinetics of the poly-β-hydroxybutyrate

2 depolymerase from Microbacterium paraoxydans RZS6: A novel

3 biopolymer-degrading organism isolated from a dumping yard

4

5 RZ Sayyed1*, SJ Wani1, Abdullah A. Alyousef2, Abdulaziz Alqasim2, Asad Syed3

6

7 1Department of Microbiology, PSGVP Mandal’s, Arts, Science, and Commerce College,

8 Shahada 425 409, Maharashtra, India

9 2Microbiology Research Group, Department of Clinical Laboratory Sciences, College of

10 Applied Medical Sciences, King Saud University, P.O.Box 10219, Riyadh 11433, Saudi

11 Arabia

12 3Department of Botany and Microbiology, College of Science, King Saud University,

13 Riyadh, 11451, Saudi Arabia

14

15

16

17

18

19

20 *Corresponding author:

21 RZ Sayyed

22 Department of Microbiology, PSGVP Mandal’s, Arts, Science, and Commerce College,

23 Shahada 425 409, Maharashtra, India

24 E-mail: [email protected]

.CC-BY 4.0 International licenseavailable under awas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

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2

26 Abstract

27 Poly-β-hydroxybutyrate (PHB) depolymerase can decompose biodegradable polymers and

28 therefore has great commercial significance in the bioplastic sector. However, few reports

29 have described PHB depolymerases based on isolates obtained from plastic-contaminated

30 sites that reflect the potential of the source organism. In this study, we evaluated

31 Microbacterium paraoxydans RZS6 as a producer of extracellular PHB depolymerase

32 isolated from a plastic-contaminated site in the municipal area of Shahada, Maharashtra,

33 India, for the first time. The isolate was identified using the polyphasic approach, i.e., 16S

34 rRNA gene sequencing, gas chromatographic analysis of fatty acid methyl esters, and

35 BIOLOG identification, and was found to hydrolyze PHB on minimal salt medium

36 containing PHB as the only source of carbon. Both isolates produced PHB depolymerase at

37 30°C within 2 days and at 45°C within 4 days. The enzyme was purified most efficiently

38 using an octyl-sepharose CL-4B column, with the highest purification yield of 6.675

39 U/mg/mL. The enzyme required Ce2+ and Mg2+ ions but was inhibited by Fe2+ ions and

40 mercaptoethanol. Moreover, enzyme kinetic analysis revealed that the enzyme was a

41 metalloenzyme requiring Mg2+ ions, with optimum enzyme activity at 45°C (thermophilic)

42 and under neutrophilic conditions (optimum pH = 7). The presence of Fe2+ ions (1 mM) and

43 mercaptoethanol (1000 ppm) completely inhibited the enzyme activity. The molecular weight

44 of the enzyme (40 kDa), as estimated by sodium dodecyl sulfate-polyacrylamide gel

45 electrophoresis, closely resembled that of PHB depolymerase from Aureobacterium

46 saperdae. Scale-up from the shake-flask level to a laboratory-scale bioreactor further

47 enhanced the enzyme yield. Our findings highlighted the applicability of M. paraoxydans as a

48 producer of extracellular PHB depolymerase isolated from a plastic-contaminated site in the

49 municipal area of Shahada, Maharashtra, India.

50



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51 Keywords: Microbacterium paraoxydans RZS6, poly-β-hydroxybutyrate depolymerase,

52 production, purification, enzyme kinetics.

53 Introduction

54 Poly-β-hydroxy alkanoates (PHAs) and poly-β-hydroxybutyrate (PHB) are stored as food and

55 energy sources in bacteria under a carbon-rich environment and are catabolized during

56 nutrient stress conditions under the influence of PHB depolymerase. PHB is a biocompatible,

57 thermoplastic, nontoxic, completely biodegradable molecule exhibiting the properties of

58 synthetic plastics. Moreover, PHB is easily degraded by PHB depolymerases and hence is an

59 eco-friendly alternative to recalcitrant synthetic plastics (1-5)(Sayyed and Chincholkar 2009;

60 Gangure et al. 2017; Wani et al. 2016; Wani and Sayyed 2016; Soam et al. 2012).

61 PHB is degraded under natural conditions by the actions of PHB depolymerases produced

62 by a wide variety of microorganisms (6, 7) (Papaneophytou et al. 2009; Hsu et al. 2012).

63 Although PHB has commercial applications, identification of potent PHB degraders from

64 relevant habitats and evaluation of their production, scale-up purification, and enzyme kinetic

65 must be carried out. Reports on bacterial PHB depolymerases isolated from plastic-

66 contaminated sites, which reflect the biodegradation potential of the enzyme, are scarce.

67 Organisms isolated from plastic-contaminated sites that are capable of degrading PHB may

68 serve as potential sources of efficient PHB depolymerases.

69 Accordingly, in this study, we aimed to isolate PHB-degrading bacteria from plastic-rich

70 dumping yards. We describe the isolation and polyphasic identification of a PHB

71 depolymerase-producing Microbacterium paraoxydans RZS6 isolated from plastic-

72 contaminated sites and then the production, purification, characterization, and enzyme

73 kinetics of the identified PHB depolymerase.

74



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75 Materials and Methods

76 Chemicals and glassware

77 All the chemicals used in this study were of analytical research grade. PHB was purchased

78 from Sigma-Aldrich (Germany); all other chemicals were purchased from Hi-Media

79 Laboratories (Mumbai, India). The glassware was cleaned using 6N HCl and K2Cr2O7, rinsed

80 with double-distilled water and dried in an oven.

81 Isolation and screening of PHB depolymerase-producing bacteria

82 Sample collection, isolation, and screening of the PHB depolymerase-producing bacteria

83 were performed as described by Wani et al. (2016) (3). M. paraoxydans RZS6 and

84 Stenotrophomonas maltophilia RZS7 were isolated from plastic-contaminated sites located at

85 latitude 21° 30′ 47.09″ N and longitude 74° 28′ 40.47″ E. These sampling sites were chosen

86 purposefully due to the higher probability of finding microflora that would be metabolically

87 very active in biopolymer degradation.

88 Selection of potent isolates

89 Isolates producing the zone of PHB hydrolysis were grown on MSM containing different

90 concentrations of PHB (0.1–0.4%) at 30°C for 10 days (8) (Pushpita et al. 2006). The

91 degradation of PHB was detected by observing the time profile of the growth of the isolates,

92 and by observing the formation of the zone of PHB clearance surrounding the colonies. The

93 level of PHB degradation was measured from the diameter of the zone of PHB hydrolysis.

94 Temperature profile of the potent isolates

95 In order to assess the thermostability of the PHB depolymerase, the isolate was subjected to

96 PHB degradation assays for a period of 10 days at 28°C, 37°C, or 45°C in MSM containing

97 different concentrations of PHB (0.1%, 0.2%, 0.3%, and 0.4%). The influence of temperature

98 on PHB degradation was assessed by measuring the zone of PHB hydrolysis on each plate.

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100 Polyphasic identification of the isolates

101 Isolates showing the highest potential for PHB degradation on the PHB-agar were considered

102 potent PHB depolymerase producers and were subjected to polyphasic identification.

103 Preliminary identification

104 Colonies of PHB-degrading isolates on nutrient agar (NA) medium were characterized using

105 the Gram-staining method, morphological characteristic analysis, and taxonomic

106 characterization by biochemical kits (Hi-Media, Mumbai, India). Isolates were identified

107 according to Bergey’s Manual of Determinative Bacteriology (9) (Brenner et al. 2005).

108 16S rRNA gene sequencing

109 Sequencing of 16S rRNA genes of the isolate RZS6 was performed as per the method of (10)

110 Gangurde et al. (2013). DNA of the isolate was extracted according to the methods of (11)

111 Sambrook and Russel (2001) using a HiPurA Plant Genomic DNA Miniprep purification spin

112 kit. Amplification of the 16S rRNA genes was performed using the following primers: 27f

113 (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492r (3′-ACGGCTACCTTGTTACGACTT-

114 5′) (12) (Pediyar et al. 2002). The amplified sequences were analyzed using gapped BLASTn

115 (http://www.ncbi.nlm.nih.gov), and the evolutionary relationship was computed using the

116 neighbor-joining method in Clustal W software (13) (Thompson et al. 1997). Phylogenetic

117 trees were constructed according to the methods described by Tamura and Kumar (2004)

118 (14). The 16S rRNA gene sequences of the isolate were submitted to GenBank.

119 Fatty acid methyl ester (FAME) analysis

120

121 Fatty acids from whole cells of the isolate RZS6 derivatized to methyl esters were analyzed

122 by gas chromatography using the Sherlock Microbial Identification System (MIDI, Inc.,

123 Newark, DE, USA) (15) (Gulati et al. 2008). Identification and quantification of fatty acids

124 were carried out by comparing the retention time and peak area of the samples with those of



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125 standard fatty acids. Differences in the fatty acid profiles were computed using the Sherlock

126 bacterial fatty acid ITSA1 aerobe reference library (16) (Sasser 2006).

127 Phenotypic fingerprinting

128 Phenotypic fingerprinting of isolate RZS6 was carried out using a GEN III MicroPlate test

129 panel with 95 carbon source utilization assays (17) (Shaikh et al. 2014) and a Microbial

130 Identification System, 1998 (Biolog Inc., CA, USA) with Micro Log version 4.2 database

131 software (Biolog Microstation System; Biolog Inc.).

132 Production and activity assay of PHB depolymerase

133 Growth kinetics and PHB depolymerase activity

134 Growth curve experiments were performed to evaluate the ability of the isolate to mineralize

135 the substrate (PHB), as described by Maria and Zauscher (2002) (18). The growth rate of M.

136 paraoxydans RZS6 in PHB MM was monitored over time at 620 nm in the presence of PHB

137 as a substrate by withdrawing sample after every 12 h. The PHB depolymerase activity of the

138 isolates was estimated as described by Papaneophytou et al. (2009) (6).

139 Production of PHB depolymerase

140 The production of PHB depolymerase was evaluated under shake-flask conditions by

141 separately growing M. paraoxydans RZS6 at 30°C and 120 rpm for 4 days in MSM

142 containing PHB (0.15%) (19) (Han and Kim 2002),

143 PHB depolymerase assay

144 After incubation, the MSM broth culture was centrifuged at 5000g for 15 min., and the PHB

145 depolymerase activity of the supernatant was assayed as described by Papaneophytou et al.

146 (2009) (6). M. paraoxydans RZS6 (5 × 106 cells/mL) was grown in two reaction mixtures,

147 each consisting of 50 mM Tris-HCl buffer (pH 7.0), 150 μg/mL PHB (prepared by sonication

148 at 20 kHz for 15 min), and 0.5 mL of 2 mM CaCl2 at 30°C for 10 min. The PHB

149 depolymerase activity was assayed as a decrease in the PHB turbidity at 650 nm. One unit of



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150 PHB depolymerase activity was defined as the quantity of enzyme required to cause a 0.1

151 decrease in absorbance at 650 nm per min.

152 Purification of PHB depolymerase

153 Having confirmed the presence of a PHB depolymerase in the cell-free supernatant of the

154 MSM broth culture, the supernatant was subjected to purification using three approaches, as

155 described below.

156 Ammonium sulfate precipitation

157 The crude PHB depolymerase in the supernatant was precipitated by the gradual addition of

158 increasing concentrations (10–40% w/v) of an ammonium salt. The obtained precipitate was

159 dialyzed overnight (7) (Hsu et al. 2012), followed by estimation of the protein concentration

160 and enzyme activity.

161 Solvent purification method

162 The culture supernatant of the isolate was centrifuged at 10,000 rpm for 20 min, and residues

163 were dissolved in a pre-chilled 1:1 mixture of acetone and ethanol and kept in a water bath at

164 50°C to allow the evaporation of the solvent. The obtained pellet was dissolved in Tris-HCl

165 buffer (pH 7), and the protein content and enzyme activity from the supernatant and pellet

166 were assayed.

167 Column chromatography

168 The culture supernatant of M. paraoxydans RZS6 was loaded on to an octyl-sepharose CL-4B

169 column charged with glycine-NaOH buffer (pH 9.0) and eluted using a 0–50% gradient of

170 ethanol (20) (Kim et al. 2002). The fractions were collected and subjected to estimation of

171 protein content and enzyme activity.

172 Determination of molecular weight

173 The molecular weight of the purified PHB depolymerase of the isolate was determined using

174 sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with standard



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175 molecular weight markers, such as phosphorylase B (82.2 kDa), bovine serum albumin (64.2

176 kDa), egg albumin (48.8 kDa), carbonic anhydrase (37.1 kDa), trypsin inhibitor (25.9 kDa),

177 lysozyme (19.4 kDa), lysozyme (14.8 kDa), and lysozyme (6.0 kDa). The protein

178 concentration of the purified band was measured using bovine serum albumin as a standard

179 (21, 19) (Lowry et al. 1951; Han and Kim 2002).

180 Enzyme kinetics

181 Effects of temperature on enzyme activity and determination of the thermostability of the

182 enzyme

183 To determine the temperature optima and sensitivity of the PHB depolymerase, log culture of

184 M. paraoxydans RZS6 (5 × 106 cells/mL) was grown in the reaction mixture at different

185 temperatures ranging from 5 to 70°C for 10 min, and the enzyme activity was then measured

186 as described above.

187 Effects of pH on enzyme activity and determination of the pH stability of the enzyme

188 The effects of pH on enzyme activity and the pH stability of the enzyme were determined in

189 reaction mixtures having varying pH values in the range of 2 to 13.

190 Effects of metal ions on the enzyme

191 In order to ascertain the metal requirement and type of metal required for the activity of the

192 PHB depolymerase, M. paraoxydans RZS6 was separately grown in various reaction

193 mixtures, each containing one type of metal ion, e.g., Ca2+, Mg2+, Mn2+, Cu2+, Co2+, Hg2+,

194 Zn2+, and Fe2+ (1 mM), grown at 30°C for 10 min. Enzyme activity was then measured.

195 Effects of different chemicals on the enzyme

196 In order to determine the effects of solvents, namely, methanol (10%, v/v), ethanol (10%,

197 v/v), acetone (10%, v/v), mercaptoethanol (1%, v/v), Tween-20 (1%, v/v), Tween-80 (1%,

198 v/v), ethylenediaminetetraacetic acid (EDTA; 1 mM), NaCl (1 mM), KCl (1 mM), and

199 NaNO3 (1 mM), on the activity of the PHB depolymerase. The solvents and chemicals were



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200 added individually into each reaction mixture, followed by inoculation with the isolate RZS6,

201 incubation at 30°C for 10 min, and measurement of enzyme activity.

202 Scale up of the optimized process to a laboratory-scale bioreactor

203 To evaluate the performance of the organism in the bioreactor and to confirm the validity of

204 the optimized shake-flask studies, the process was scaled-up to a fully-automated bioreactor

205 of 5-L capacity (Model LF-5; Murhopye Scientific Co., Mysore, India). The bioreactor was

206 sterilized, along with the above-optimized medium (working volume 3 L), at 121°C for 20

207 min; the reactor was cooled and then inoculated individually with 3% (v/v) inoculum of M.

208 paraoxydans RZS6. The samples were withdrawn after 12 h and subjected to estimation of

209 protein concentrations (21) (Lowry et al. 1951) and enzyme activities.

210 Statistical analysis

211 All the experiments were performed in triplicate and the mean of three replicates was

212 considered. Each mean value was subjected to Student’s t-test and. Values of P ≤ 0.05 were

213 taken as statistically significant (22) (Parker 1979).

214 Results and Discussion

215 Isolation and screening of PHB depolymerase-producing bacteria

216 In total, 39 isolates were obtained from the respective plastic-contaminated sites; among

217 these 39 isolates, seven isolates grew well and produced varying degrees of the zone of PHB

218 hydrolysis on minimal medium containing PHB as the only carbon source. The isolate RZS6

219 produced the largest zone of PHB hydrolysis (27.9 mm) and was therefore selected as the

220 best PHB depolymerase producer. The hydrolysis of PHB reflected the ability of the isolate

221 to produce PHB depolymerase.

222 Although several bacteria are known to secrete PHB depolymerase, which degrades

223 PHB, our findings demonstrated, for the first time, the production of a PHB depolymerase

224 from M. paraoxydans RZS6. The PHB depolymerase produced from these isolates (which



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225 were obtained from a plastic-contaminated environment) may be relevant for applications in

226 plastic/bioplastic degradation. Mergaert et al. (1993) (23) also isolated 295 strains that

227 degraded PHB and P (3HB-co-3HV) copolymer on MSM having PHB. Elbanna et al. (2004)

228 (24) reported Schlegelella thermodepolymerans and Pseudomonas indica K2 as PHA

229 degraders. Additionally, Gangurde et al. (2017) (2) also reported PHB biodegradation by soil

230 micro-flora.

231 Selection of potent isolates

232 PHB-degrading isolates exhibited a range of PHB biodegradation abilities in MSM

233 containing varying concentrations of PHB. PHB degradation was dependent on the amount of

234 PHB in the medium. Optimum biodegradation of PHB was recorded with 0.2% PHB (25)

235 (Minna 2002). Augusta et al. (1993) (26) reported that the diffusion rate of the enzyme, level

236 of enzyme activity, and incubation conditions affect the degradation rate. Moreover, Kim et

237 al. (2003) (27) also reported similar observations with Aspergillus sp. strain NA-25.

238 Temperature profile of the potent isolates

239 The isolate RZS6 exhibited optimum PHB degradation at 30°C with 0.2% PHB as a

240 substrate. Importantly, the temperature profile of PHB degradation is dependent on the

241 activity of the enzyme and therefore changes with the producing organism. Certain PHB

242 depolymerases function in the mesophilic range of temperatures, whereas others are

243 thermotolerant or thermophilic in nature (6) (Papaneophytou et al. 2009). Wang et al. (2012)

244 (28) have reported similar observations on a poly-depolymerase (3-hydroxybutyrate-co-3-

245 hydroxy valerate) from Acidovorax sp. HB01.

246 Polyphasic identification of potent PHB depolymerase producers

247 Preliminary identification

248 The phenotypic characteristics of the potent PHB-degrading isolate RZS6 were similar to

249 those of Microbacterium sp.



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250 16S rRNA gene sequencing

251 A BLAST search of the 16S rRNA gene sequences of the isolates with the 16S rRNA gene

252 sequences of the NCBI GenBank database revealed the highest similarity and homology of

253 the isolate RZS6 with M. paraoxydans (Fig 1). Thus, we identified the isolate as M.

254 paraoxydans; the 16S rRNA gene sequence of the isolate was submitted to NCBI GenBank

255 (http://www.ncbi.nlm.nib.gov/) under the name M. paraoxydans RZS6 (accession

256 no. KP862607).

257 Fig 1. Phylogenetic analysis of M. paraoxydans RZS6 and related species by the neighbor-

258 joining method using MEGA 5.0 software.

259

260

261 Whole-cell FAME analysis

262 The fatty acid profile of isolate RZS6 demonstrated the presence of characteristic fatty acids

263 of M. barkeri (Aureobacterium, Corynebacterium; similarity index: 0.859) and M.

264 chocolatum (similarity index: 0.602) (16) (Sasser 2006).

265 BIOLOG identification

266 The pattern of carbon source utilization assays for the isolate RZS6 demonstrated a maximum

267 similarity index of 0.48 with M. paraoxydans. Based on the preliminary characteristics, 16S

268 rRNA gene sequencing, gas chromatographic analysis of FAMEs, and BIOLOG profiles, the

269 isolate RZS6 was identified as M. paraoxydans.

270 Production and assay of PHB depolymerase

271 Growth kinetics and PHB depolymerase activity

272 Isolate RZS6 grew well in MSS containing PHB. The isolate showed faster growth, produced

273 PHB depolymerase during the log phase of growth, and exhibited an optimum enzyme

274 activity of 6.675 U/mg/mL, obtained at 48 h. The activity gradually decreased from the



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275 beginning of the stationary phase (72 h) and was completely absent during the decline phase

276 (96–120 h) of growth (Fig 2).

277 Fig 2. Growth kinetics and PHB depolymerase activity of M. paraoxydans RZS6.

278

279 Production of PHB depolymerase from the isolates

280 M. paraoxydans RZS6 produced copious amounts of PHB depolymerase in MSM. After 48 h

281 (log phase) of incubation, RZS6 produced 6.6.75 U of PHB depolymerase with 0.247 mg/mL

282 protein content in 2 days at 30°C. Gowda and Srividya (2015) (29) reported the production of

283 4U of extracellular PHB depolymerase with a protein content of 0.05 mg/mL from

284 Penicillium expansum. Thus, our currently reported yields of the PHB depolymerase were

285 higher than those of previously reported yields.

286 Purification of the PHB depolymerase

287 Ammonium sulfate precipitation

288 The maximum protein precipitation in the culture supernatant was obtained at an ammonium

289 sulfate concentration of 70%. The protein concentrations, specific activities, and enzyme

290 activities in the dialyzed precipitate of M. paraoxydans RZS6 were 0.219 mg/mL, 0.321

291 mg/mL, and 6.6.75 U, respectively. Zhou et al. (2008) (30) also reported the precipitation of

292 PHB depolymerase from Escherichia coli and Penicillium sp. DS9701-D2 using 70% and

293 75% ammonium sulfate. Shivakumar et al. (2011) (31) reported efficient precipitation of

294 PHB depolymerase from Penicillium citrinum S2 using 80% ammonium sulfate.

295 Solvent purification method

296 Solvents adversely affected PHB depolymerase activity, with only approximately 45.66% and

297 51.14% remaining from M. paraoxydans RZS6. This significant loss in enzyme activity may



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298 be related to the precipitation of proteins (enzymes) by acetone and ethanol. Thus, the solvent

299 purification method proved to be inefficient.

300 Column chromatography

301 Out of the five fractions, fraction 3 exhibited the maximum enzyme activity of 7.703 U, with

302 0.247 mg/mL protein content. Purification of PHB depolymerase from various organisms has

303 been carried out using Sephadex columns, e.g., PHB depolymerase of Bacillus sp. (1.79

304 U/mg) (Shah et al. 2007) (32) Streptoverticillium kashmirense AF1, and Streptomyces

305 ascomycinicus (Garcia et al. 2013) (33). Wang et al. (2012) (28) reported the purification of

306 an extracellular PHB depolymerase from Pseudomonas mendocina DSWY0601 using a

307 DEAE-Sepharose column. Papaneophytou et al. (2009) (6) and Hsu et al. (2012) (7) purified

308 extracellular PHB depolymerases from Thermus thermophilus and Streptomyces

309 bangladeshensis 77T-4 respectively using column chromatography. Thus, column

310 chromatography using an octyl-sepharose CL-4B column resulted in the most efficient

311 purification.

312 Determination of the molecular weight of the purified enzyme

313 The purified protein fraction from M. paraoxydans RZS6 yielded single protein bands

314 corresponding to molecular weights of approximately 40 kDa (Fig 3). Sadocco et al. (1997)

315 (34) also reported a PHB depolymerase of 42.7 kDa from Aureobacterium saperdae. Calabia

316 and Tokiwa (2006) (35) identified a PHB depolymerase of 41 kDa from Streptomyces sp.

317 MG 41.

318 Fig 3. SDS-PAGE analysis of purified PHB depolymerase from M. paraoxydans RZS6.

319

320 Enzyme kinetics

321 Effects of temperature and determination of the thermostability of the enzyme



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322 The PHB depolymerase of RZS6 showed an optimum enzyme activity of 6.657 U with 0.247

323 mg/mL protein content at 30°C, indicating the mesophilic nature of the enzyme. The enzyme

324 activity decreased as the temperature increased, and the enzyme was completely inactivated

325 at 70°C (Fig 4). The decrease in the enzyme activity with the increase in temperature

326 reflected the thermolabile nature of the enzyme. Wang et al. (2012) (28) and Gowda and

327 Shivakumar (2015) (29) reported thermostable PHB depolymerases in Pseudomonas

328 mendocina DSWY0601 and Penicillium expansum, respectively. Calabia and Tokiwa (2006)

329 (35) reported optimum PHB depolymerase activity in Streptomyces sp. MG at 50°C.

330 Fig 4. Effects of temperature on PHB depolymerase activity of M. paraoxydans RZS6.

331

332 Effects of pH on enzyme activity and determination of the pH stability of the enzyme

333 The optimum activity of the PHB depolymerase of M. paraoxydans RZS6 was obtained at pH

334 7 (Fig 5), indicating the neutrophilic nature of the enzyme. Sadocco et al. (1997) (34) and

335 Jeong (1996) (36) reported an optimum pH for PHB depolymerase from A. saperdae in the

336 range of 7.0 to 9.0. The pH sensitivity of the PHB depolymerases of Penicillium expansum

337 and Pseudomonas mendocina DSWY0601 has been reported to be between 6.0 and 9.0

338 (Wang et al. 2012; Gowda and Srividya, 2015) (28,29). The enzymes from both isolates

339 remained stable at an acidic pH (4.0–6.5). Soam et al. (2012) (5) reported pH 7.0 as the

340 optimum pH for enzyme production in Bacillus mycoides. Calabia and Tokiwa (2006) (35)

341 reported that the optimum PHB depolymerase activity of Streptomyces sp. MG was in the pH

342 range from 6.5 to 8.5.

343 Fig 5. Effects of pH on PHB depolymerase activity of M. paraoxydans RZS6.

344

345 Effects of metal ions on enzyme activity



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346 The presence of Mg2+ ions significantly enhanced PHB depolymerases. However, Fe2+ ions

347 negatively affected the enzyme activity, decreasing activity to only 22.26%. The other metal

348 ions had a negligible effect on the enzyme activities of both isolates (Table 1). The increase

349 in PHB depolymerase activity in presence of Mg2+ ions was attributed to the enzyme

350 activator nature of these metal ions. Wang et al. (2012) (28) also reported a positive effect of

351 Mg2+ and Ca2+ ions on the PHB depolymerase of Pseudomonas mendocina DSWY0601.

352

353 Table 1. Effects of metal ions and chemicals on the PHB depolymerase of M. paraoxydans

354 RZS6.

Metal ion

(1 mM)

Enzyme

activity (U/mL)

Protein content

(mg/mL)

Specific activity

(U/mg/mL)

%

activity

No metal ions 0.146 0.247 0.6210 59.10

Mg2+ 0.194 0.247 0.7854 78.54

Ca2+ 0.192 0.247 0.7773 77.73

Mn2+ 0.129 0.247 0.5627 56.27

Cu2+ 0.099 0.247 0.4008 40.08

Co2+ 0.101 0.247 0.4089 40.89

Hg2+ 0.097 0.247 0.3927 39.27

Zn2+ 0.094 0.247 0.3805 38.05

Fe2+ 0.055 0.247 0.2226 22.26

Chemicals

No chemical 0.134 0.219 0.6118 61.18

NaCl 0.154 0.219 0.7196 71.96

NaNO3 0.148 0.219 0.6757 67.57

Methanol 0.145 0.219 0.6621 66.21



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KCl 0.138 0.219 0.6301 63.01

Ethanol 0.112 0.219 0.5114 51.14

Tween-80 0.11 0.219 0.5022 50.22

Tween-20 0.103 0.219 0.4703 47.03

Acetone 0.091 0.219 0.4155 41.55

EDTA 0.046 0.219 0.2100 21.00

Mercaptoethanol 0.023 0.219 0.1050 10.50



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356 Effects of different chemicals on enzyme activity

357 Mercaptoethanol caused the maximum inhibition (85%) of enzyme activity. NaCl enhanced

358 the activity of the PHB depolymerase (Table 1). The loss of enzyme activity in the presence

359 of chemicals and solvents occurs through denaturation and proteolysis of the enzyme.

360 Papaneophytou et al. (2009) (6) also reported mercaptoethanol as a strong inhibitor of PHB

361 depolymerase in Thermus thermophilus HB8. Wang et al. (2012) (28) have also reported the

362 negative effects of ethanol, acetone, Tween, and other chemicals on the PHB depolymerase

363 of Pseudomonas mendocina DSWY0601.

364 Scale up of the optimized process to a laboratory-scale bioreactor

365 Scale-up of the parameters optimized at the shake-flask level to the laboratory-scale

366 bioreactor level enhanced PHB depolymerase yield from 7.703 to 8.512 U and protein

367 content from 0.247 to 0.297 mg/mL.

368 Conclusion

369 Plastic-contaminated sites may harbor bioplastic (PHB)-degrading bacteria. The occurrence

370 of PHB-degrading bacteria in plastic-contaminated sites and their growth in the presence of

371 PHB indicated their ability to degrade bioplastic. Higher yields and optimal purification were

372 obtained with column chromatography. The growth of the organism and the observation of

373 maximum enzyme activity at higher temperatures are essential for the survival of the

374 organism under extreme environmental conditions. Thus, PHB depolymerase-producing

375 bacteria obtained from plastic-contaminated environments are expected to be important for

376 applications in the biodegradation of bioplastics. However, further studies on the growth of

377 Microbacterium paraoxydans and performance of PHB depolymerase under natural

378 conditions shall help in this direction.

379

380



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381 Acknowledgments

382 The authors extend their appreciation to the Deanship of Scientific Research at King Saud

383 University for funding this work through research group No (RG-1440-053).

384

385 Author Contributions

386 Conceptualization: Christabel Ndahebwa Muhonja, Gabriel Magoma.

387 Data curation: Christabel Ndahebwa Muhonja, Huxley Makonde, Gabriel Magoma.

388 Formal analysis: Christabel Ndahebwa Muhonja, Huxley Makonde, Gabriel Magoma.

389 Funding acquisition: Gabriel Magoma.

390 Investigation: Christabel Ndahebwa Muhonja, Huxley Makonde, Gabriel Magoma, Mabel

391 Imbuga.

392 Methodology: Christabel Ndahebwa Muhonja.

393

394 Data Availability Statement: All relevant data are within the paper.

395 Funding: This research is supported by the African Union Commission

396 (ADF/BD/WP/2013/68 to CNM) and the Japanese International Co-operation Agency (JICA)

397 (00025 to CNM). The funders had no role in study design, data collection, and analysis,

398 decision to publish, or preparation of the manuscript.

399 Competing interests: The authors have declared that no competing interests exist.

400



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