Accepted Manuscript

Exploration of chrome shaving hydrolysate as substrate for production of dehairing

protease by Bacillus cereus VITSN04 for use in cleaner leather production

Sundararajan Shakilanishi, Narasimhan Kannan Chandra Babu, Chittibabu Shanthi

PII: S0959-6526(17)30365-7

DOI: 10.1016/j.jclepro.2017.02.139

Reference: JCLP 9065

To appear in: Journal of Cleaner Production

Received Date: 03 September 2016

Revised Date: 16 February 2017

Accepted Date: 19 February 2017

Please cite this article as: Sundararajan Shakilanishi, Narasimhan Kannan Chandra Babu,
Chittibabu Shanthi, Exploration of chrome shaving hydrolysate as substrate for production of
dehairing protease by Bacillus cereus VITSN04 for use in cleaner leather production, Journal of
Cleaner Production (2017), doi: 10.1016/j.jclepro.2017.02.139

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1 Total word count:6064

2 Exploration of chrome shaving hydrolysate as

3 substrate for production of dehairing protease by
4 Bacillus cereus VITSN04 for use in cleaner leather
5 production
6
7 Sundararajan Shakilanishia, Narasimhan Kannan Chandra Babub,
8 Chittibabu Shanthia, *
9 a School of Bio Science and Technology, VIT University, Vellore 632014, India
10 b Chief Scientist, Tannery Division, CSIR-CLRI, Adyar, Chennai 600020, India
11 *Corresponding author. Tel.:+91 416 2202549; fax: +91 416 2243092.

12 E-mail address: cshanthi@vit.ac.in (C. Shanthi).

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17

18 ABSTRACT

19 Chrome shavings (chromium complexed collagenous waste scrapings), one of the major

20 proteinous solid wastes of leather industry are posing a pollution threat. Recovery and reuse of

21 protein component of the waste can prevent their disposal as landfills. In the present work, the

22 collagen hydrolysate derived from chrome shavings was screened as an inexpensive protein

23 source in comparison with agro based protein wastes for the cost-effective production of

24 dehairing protease. The chrome shavings used in the study were obtained during processing of

25 goat skins. Maximum enzyme production of 203±0.07 U/mL by Bacillus cereus VITSN04 was

26 observed with the formulated medium (pH 8.0) containing, 12 g/L of collagen hydrolysate from

27 chrome shavings, 15 g/L of molasses, 3 g/L of K2HPO4, 2 g/L of NaCl and 0.04 g/L of CaCl2.

28 Fluorescence spectral analysis confirmed that collagen hydrolysate stabilized the protease and

29 prolonged its activity. The protease was purified with a yield of 88.1% using 22% (w/v) of 2-

30 propanol and 14% (w/v) of K2HPO4 aqueous two phase system. The purification and activity of

31 the enzyme was confirmed by native-polyacrylamide gel electrophoresis. The purification yield

32 of protease with aqueous two phase system was much higher than that with ultrafiltration system.

33 Thus, approach made in the present study provides an attractive option for recycling chrome

34 shavings waste as a cheaper protein source in the production of dehairing enzyme for use in

35 cleaner leather processing.

36

37 Key words:
38 Dehairing protease; Bacillus cereus; Collagen hydrolysate; Chrome shavings; Aqueous two
39 phase system

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41

42 1. Introduction

43 Leather industry, a well-established industrial sector has been contributing to the economic

44 development in many developing and underdeveloped countries through industrial growth and

45 employment generation. However, the industry has been facing waste disposal problems for the

46 past few decades (Kumaraguru et al., 1998; Cabeza et al., 1998a). In processing one metric ton

47 of raw hides, about 0.6 t of solid waste is generated out of which 0.1 t is accounted for by the

48 chrome shavings (Sundar et al., 2011). Chrome shavings are waste scrapings of leathers obtained

49 during thickness adjustment of the chrome tanned leather prior to post tanning operations.

50 Collagenous protein (on dry weight) is the major (75-85%) component of the shavings and other

51 components include chromium, salts, fats and oils (Shanthi et al., 2013). Nearly, 0.8×106 t of

52 chrome shavings (Rao et al., 2002) are produced from global leather industries annually, of

53 which major part is dumped as landfills (Sastry et al., 2005). There have been many research

54 efforts for processing the waste to obtain protein and chromium fractions for various end

55 applications (Cabeza et al., 1998a; Shanthi and Suseela, 2012; Shanthi et al., 2013). Attempts

56 have also been made to use protein fractions of chrome shavings for producing biodegradable

57 plastic (Kresalkova et al., 2002), support for immobilizing degradative enzymes (Shanthi et al.,

58 2003), technical grade gelatin (Cabeza et al., 1998b) and in retanning formulations (Cantera,

59 2003).

60 A better management option would be the utilization of the recycled chrome shavings for a

61 value added application in enzyme production for use in cleaner leather processing. Earlier, the

62 protein hydrolysates obtained from animal and plant sources have been screened as a source of

63 nitrogen in the bacteriological media for induction of proteases (Pasupuleti and Braun, 2008).

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64 The present work has investigated the use of collagen hydrolysate obtained from chrome

65 shavings (CHCS) by microbial degradation (Shanthi et al., 2013) as a protein source for

66 production of alkaline protease with the dehairing property for use in leather processing. In

67 leather industry, enzymatic dehairing has been receiving serious attention as a greener alternative

68 for the conventional chemical based dehairing process (Sivasubramanian et al., 2008;

69 Sundararajan et al., 2011). Enzymatic dehairing, if adopted by the leather industry, would require

70 proteases in large amounts (Choudhary et al., 2004). Though enzymes can be obtained from

71 plant, animal and microbial sources, the latter is preferred for enzyme production for commercial

72 uses. It is due to the fact that the process is inexpensive and microbes, especially bacteria are

73 efficient enzyme producers. Species belonging to genus Bacillus have been extensively studied

74 for the production of dehairing proteases (Otto et al., 1974; Raju et al., 1996; Nilegaonkar et al.,

75 2007). However, the use of microbial protease for dehairing application is limited due to the

76 higher cost of enzyme production (Mukherjee et al., 2008). About 30 to 40% of the production

77 cost of industrial enzymes is due to the cost of growth medium components (Kirk et al., 2002).

78 The cost can be considerably reduced, by using cheaper media components for growth of

79 organism and adoption of efficient protease purification methods (Amid et al., 2012). In this

80 aspect, a well known cheaper source, agro industrial wastes have been used in protease

81 production for decades (Kaur et al., 2001). Several attempts have been made to screen various

82 tannery solid wastes in the production of proteases by Bacillus cereus 1173900, Pseudomonas

83 aeruginosa and fish gut microflora (Ravindran et al., 2011; Kumar et al., 2008; Sumathi et al.,

84 2012).

85 Chrome shavings, being a protein-rich waste can also be used for the purpose. There has been

86 an attempt to utilize the waste as such without any pretreatment for the production of

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87 keratinolytic enzyme (Pillai and Archana, 2012). However, the presence of heavy metal ion,

88 chromium can pose a serious problem and there might be problem with the easy bioavailability

89 of nitrogen due to high molecular weight of collagen. Hence, the present study has investigated

90 the recycling of CHCS as an inexpensive protein source for production of dehairing enzyme in

91 higher yield and in a cost-effective and sustainable manner. The suitability of the CHCS as

92 protein source was studied in comparison with agro wastes. Standardization of media for

93 enhanced production of protease and further reduction in production cost by means of an

94 economical downstream processing method, aqueous two phase system (ATPS) have also been

95 studied.

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97 2. Materials and methods

98 2.1. Materials

99 Chrome shavings were obtained from tannery, CSIR-CLRI, Chennai, India during processing

100 of goat skins and hydrolysed to obtain collagen hydrolysate free from chromium as per

101 procedure reported earlier (Shanthi et al., 2013). The chromium in the hydrolysate was estimated

102 as 3.14±2.0 µg/g (on dry weight basis) as against 32 mg/g in the chrome shavings. Protein based

103 agro wastes such as Bengal gram husk, black gram husk, groundnut cake, wheat bran and rice

104 bran were purchased from the local market, Vellore, India and used in the study. All other

105 chemicals used were of analytical grade. Bacillus cereus VITSN04 isolated from protein-rich

106 dumping sites (Sundararajan et al., 2011) was used in this study.

107 2.2. Enzyme production using CHCS and agro wastes

108 Protease production with CHCS (2 to 14 g/L) as protein source and molasses (0.5 to 50 g/L)

109 as carbon source was standardized. The production media (pH 7.0) contained 3 g/L of K2HPO4, 2

110 g/L of NaCl, and 0.04 g/L of CaCl2 as mineral components. Sterile media of 50 mL were

111 inoculated with 3.8×107 cells and incubated at 35 °C in an orbital shaker. Samples were

112 withdrawn at regular intervals to measure the proteolytic activity. Peptone and molasses

113 containing medium was used as control.

114 Five gram each of different agro wastes were taken in 250 mL Erlenmeyer flasks and 50 mL

115 of 0.05 M phosphate buffer (pH 7.0) were added and sterilized. Nutrient broth of pH 7.0 was

116 used as control. The inoculum and culture conditions were the same as that was used for CHCS.

117 Parallel experiments were also carried out using each of the agro wastes after the removal of

118 polyphenols. Polyphenols were extracted by treating agro wastes (appropriate amount) with 1%

119 acidified methanol and water in the ratio 4:1 in shaking water bath at 65 °C for an hour.

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120 Extraction was repeated thrice to remove polyphenols and the samples were dried in hot air oven

121 at 60 °C. The extracted polyphenol was estimated using Folin-Ciocalteu reagent, as described by

122 Singleton and Rossi (1965). The concentration of total phenols was expressed as gram gallic acid

123 equivalent per 100 g of agro wastes.

124 2.2. Protease assay and protein estimation

125 Proteolytic activity was determined using azocasein as substrate according to the method of

126 Tomeralli et al. (1949). Azocasein (7.5 mg/mL) was dissolved in 50 mM Tris-HCl (pH 8.0).

127 Reaction mixture containing 180 µL of substrate and 120 µL of the cell-free supernatants were

128 incubated at 40 °C for 1 h. One milliliter of 0.6 M trichloroacetic acid was added to the test

129 sample to precipitate unhydrolysed azocasein and to the control samples prior to enzyme

130 addition. The test and controls samples were centrifuged at 12,000 rpm for 5 min. One milliliter

131 of 0.1 M NaOH was added to the supernatants and the absorbance was measured at 440 nm using

132 UV-Visible spectrophotometer (Shimadzu, Japan). One unit of protease activity is equivalent to

133 change in the optical density of 0.01 per minute. The results are expressed as mean value ±

134 standard deviation.

135 The method of Bradford (1976) was followed for protein content estimation using bovine

136 serum albumin as standard.

137 2.3. Influence of pH and incubation time on protease production

138 To determine the effect of pH on protease production, the pH of the standardized media used

139 in the study were adjusted ranging from 7 to 9 and the proteolytic activity was determined after

140 24 h.

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141 To study the effect of incubation time on growth and protease production, culture samples

142 were withdrawn at regular time intervals up to 48 h for determining the cell number and

143 proteolytic activity.

144 2.5. Effect of CHCS on the stability of protease

145 Fluorescence spectral analysis was carried out to check the stability of purified protease

146 (Sundararajan et al., 2011) in the presence of 12 g/L of CHCS and compared with 0.5 M of

147 osmolytes (hydroxyproline, glycine and glycerol) and 0.5 M of denaturant (guanidine

148 hydrochloride). Trypsin was used as a control enzyme in the study. The enzymes were mixed

149 with CHCS, osmolytes and denaturant in separate tubes in the ratio of 2:1 and pre-incubated at

150 40 °C for 3 h. Emission spectra of intrinsic tryptophan were recorded on Cary Eclipse

151 fluorescence spectrophotometer (Agilent Technologies, USA) at an excitation wavelength of 295

152 nm.

153 2.6. Purification of CHCS induced protease

154 2.6.1. Partitioning of enzyme using polymer/salt and alcohol/salt biphasic systems

155 ATPS based extraction was carried out by adding predetermined amounts of polymers/salts

156 and alcohols/salts (Table 4) to 20% of cell free supernatant obtained from the culture grown in

157 collagen hydrolysate molasses medium. Deionized water was added to make a final volume of 1

158 mL, mixed thoroughly by gentle agitation and allowed to stand at 4 °C for 15 min. After

159 equilibration, phase separation was induced by centrifugation at 4000 rpm for 10 min.

160 Parameters such as partition co-efficient, selectivity, purification factor and yield of enzyme

161 were calculated as described in the literature (Ooi et al., 2009). Partitioning efficiency of

162 protease was optimized using ATPS (2-propano1/K2HPO4) with different phase compositions as

163 presented in Table 5.

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164 2.6.2. Native-polyacrylamide gel electrophoresis of the protease purified by ATPS

165
166 Native-polyacrylamide gel electrophoresis (native-PAGE) was performed as described by

167 Davis (1964). The protein samples were prepared as follows (i) 2-propanol (top phase) was

168 evaporated and proteins were dissolved in 0.05 M Tris-HCl buffer (pH 8.0) and (ii) K2HPO4

169 (bottom phase) was subjected to dialysis. The protein samples were then mixed with non-

170 denaturing sample buffer and loaded on to the gel.

171 Gelatin (10 mg/mL) was used as substrate and copolymerized in the resolving gel of the

172 native-PAGE for zymography. After the run, the gel was washed with 0.05 M Tris-HCl buffer

173 (pH 8.0), incubated at 37 °C for an hour, washed and stained with Coomassie Brilliant Blue R-

174 250.

175 2.7. Enzyme concentration and dehairing studies

176 The enzyme from 500 mL of cell free supernatant was partitioned in the solvent phase of

177 ATPS (22% w/v of 2-propanol and 14% w/v of K2HPO4) and concentrated on rotary evaporator

178 at 40 °C. The resultant protease was assayed for proteolytic activity and used in dehairing

179 studies.

180 The cell-free supernatant (500 mL) was also concentrated separately by ultrafiltration (UF)

181 using stirred-cells (Amicon, model 8400, USA) with 10 kDa membrane (Ultracel®). The feed

182 side of filtration system was pressurized using nitrogen gas. The resultant concentrate was

183 assayed for proteolytic activity and used in dehairing studies.

184 Two samples of dimension 10×10 cm were cut from the butt portions (identical locations on

185 left and right side on either side of backbone) of wet salted goat skin for enzyme application

186 trials. The pieces were labeled and soaked with two changes of 300% water each time on the wet

187 salted weight. Then they were drained free of excess water and the soaked weight for each piece

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188 was noted. The enzymes from ATPS and UF purification systems were applied on the flesh side

189 of each piece (left side piece with UF enzyme and right one with ATPS enzyme) at 10% on the

190 soaked weight. Both the pieces were folded with flesh side inside and left overnight (16 h). Next

191 day, the hair was manually removed to test for dehairing efficacy of the enzymes.

192 3. Results and Discussion

193
194 3.1. Influence of different proteinous wastes on protease production

195 Proteins, apart from being the substrates for proteases, act also as inducers for the production

196 of proteases (Kumar and Takagi, 1999). In the present study, CHCS was used as protein source

197 and compared with the other selected protein containing agro wastes such as wheat bran (Arte et

198 al., 2015), rice bran (Han et al., 2015), Bengal gram husk (Zia-Ul-Haq et al., 2007), black gram

199 husk (Arulnathan et al., 2013) and groundnut cake (Purohit and Rajyalakshmi, 2011). The results

200 from the study on the effect of different agro wastes on protease production are presented in

201 Table 1. It is evident that the enzyme activity has improved by more than 70% with all the agro

202 wastes on removal of the polyphenols confirming their role in the reduction of enzyme induction

203 probably due to the complexation of proteins with polyphenols (Gupta, 2015). The highest

204 proteolytic activity of 86.0±0.05 U/mL was recorded for wheat bran but the highest percent

205 increase after the removal of polyphenols was observed in the case of black gram husk (~120%).

206 The highest protease production with wheat bran may be due to the fact that solubilization is

207 higher in sub-aleurone layer than the aleurone layer rich in polyphenols, as reported earlier (Arte

208 et al., 2015).

209 The effect of varying concentrations of CHCS ranging from 2 to 14 g/L on protease

210 production was studied. Protease production increased (64.9±0.05 U/mL) up to a concentration

211 of 12 g/L and hence this concentration was used in formulating the media for optimization of

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212 other parameters (Table 2). The medium was formulated using CHCS as protein source and

213 molasses as carbon source. The influence of varying concentrations of molasses (Fig.1) on

214 proteolytic activity showed an increase (139.6±0.02 U/mL) up to a concentration of 15 g/L.

215 Thus, the medium containing 15 g/L of molasses, 12 g/L of CHCS and trace elements was

216 chosen for standardization of pH and time.

217 3.2. Effect of physical parameters on media containing wastes

218 The pH of the growth medium influences the availability of metabolic ions and membrane

219 permeability thereby effecting growth of the organism and enzyme production. The proteolytic

220 activity was maximal at pH of 8.0 for all agro wastes and CHCS (Fig. 2). The highest proteolytic

221 activity of 165.7±0.05 U/mL was observed with CHCS. Bacillus cereus has been reported earlier

222 to have optimal activity in the slightly alkaline condition (Horikoshi, 1990).

223 Fig. 3 depicts the growth and protease production profile using protein wastes by Bacillus

224 cereus VITSN04. Results showed that there is a substantial change in the secretion of enzyme

225 with the growth and time. CHCS containing medium showed highest protease production

226 (203±0.07 U/mL) at 42 h indicating that hydrolysate is a better inducer compared to other protein

227 wastes. The longer duration for protease production could be due to initial difficulty in digesting

228 the collagen hydrolysate. The maximal proteolytic activity of 116.6±0.04, 103.5±0.04, 98.4±0.2,

229 85±0.08 and 81.6±0.05 U/mL was observed for wheat bran, rice bran, black gram husk, Bengal

230 gram husk and groundnut cake at 24, 18, 24, 24 and 30 h respectively. Overall, the results clearly

231 indicate that there is less protease production with agro wastes when compared to CHCS.

232 3.3. Effect of CHCS on stability of protease

233 The stability of the enzyme is a crucial limiting factor for commercial production and its

234 potential industrial use. The nature of the protein (native/denatured) in their microenvironment is

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235 better understood from fluorescence signal of intrinsic tryptophan residue in the protein excited

236 at 295 nm. Stoke shifts of tryptophan residue depend on the immediate environment being

237 hydrophobic or hydrophilic (Kijima et al., 1996). The fluorescence spectral parameters such as

238 maximum intensity (Imax) and maximum emission wavelength (λmax) were measured to check the

239 stability of the protease in the presence of CHCS, Osmolytes and denaturant. The normalized

240 fluorescence spectra of protease in the presence of CHCS are depicted in Fig. 4. The result shows

241 variation in emission spectrum of intrinsic tryptophan when pre-incubated in the presence or

242 absence of CHCS. The purified protease dissolved in 50 mM Tris-HCl buffer (pH 8.0) at 25 °C

243 showed a λmax of 340 nm indicating probable exposure of tryptophan to solvent even in their

244 native condition. The enzymes in the presence of CHCS showed λmax of 339 nm indicating that

245 there is no change in the native condition of the enzymes. But, when CHCS and enzyme were

246 pre-incubated for 3 h at 40 °C, a red shift (337 to 347 nm) was observed indicating maximum

247 exposure of tryptophan to solvent with simultaneous decrease in Imax due to unfolding of protein.

248 Besides being a protein source, CHCS is also rich in natural osmolytes like glycine, proline and

249 hydroxyproline. These osmolytes enhance protein stability (Bolen, 2001) and protect enzyme

250 from heat inactivation (Taneja and Ahmad, 1994). Table 3, describes glycerol, a polyol osmolyte

251 stabilizing the enzyme by shifting λmax to lower wavelength (blue shift), whereas the guanidine

252 hydrochloride caused unfolding of protein and destabilization of the enzyme with decreased Imax.

253 Overall, the micro-environment created by CHCS containing osmolytic amino acids makes the

254 enzyme more stable with enhanced proteolytic activity as evidenced from the results presented in

255 the Table 3.

256 3.4. Purification of protease

257 3.4.1. ATPS

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258 Purification of enzyme is crucial to standardize a cost-effective dehairing application method.

259 Hence, in search of cheaper and efficient methods to purify the enzyme, ATPS method was

260 investigated (Zaslavsky, 1994). Partitioning studies were carried out using polymers/salts and

261 alcohols/salts (Table 4) to determine the suitable ATPS for the maximum separation of the

262 enzyme with high purity. The enzyme partitioned well in alcohol phase of 22% (w/v) of 2-

263 propanol and 18% (w/v) of K2HPO4 ATPS with the yield of 80.6%, purification factor of 3.3 and

264 selectivity of 4.5. A similar observation was made by Amid et al. (2012) with protease from

265 mango (Mangifera indica cv. Chokanam) in 2-propanol/potassium phosphate ATPS.

266 Among the polymer/salt ATPSs, 22% (w/v) of PEG 6000 and 16% (w/v) of K2HPO4 ATPS

267 had enhanced maximum partitioning of the enzyme in polymer phase with a yield of 76.9%

268 which was close to that obtained with 2-propanol/potassium phosphate ATPS. Partitioning of

269 enzyme using polymer/salt ATPS has drawbacks like slow separation and difficulty in re-

270 extraction of enzymes. Though PEG has wide industrial applications in enzyme immobilization

271 (Manta et al., 2003), its use in dehairing was limited as the penetration of PEG bound enzyme

272 into hair-roots of skin may be difficult. The recovery of enzyme from the propanol system was

273 relatively easier (Tianwei et al., 2002) and hence 2-propanol/ K2HPO4 ATPS was chosen for

274 further partitioning studies.

275 3.4.2. Optimization of 2-propanol/ K2HPO4 ATPS

276 Six ATPSs were prepared to optimize the partitioning efficiency of the enzyme. The results

277 presented in Table 5 show that the highest partitioning of the enzyme was achieved with 22%

278 (w/v) of 2-propanol and 14% (w/v) of K2HPO4 with the maximum yield of 88.1% and

279 purification factor of 4.9. The increase in selectivity of 4.1 was found with 14% (w/v) K2HPO4.

280 As the salt concentration increased, the selectivity and protease yield decreased probably due to

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281 salting out of proteins in the lower salt phase. Higher salt concentration might affect the phase

282 pH influencing the deprotonation of biomolecules and hence its partition (Reis et al., 2015).

283 Study on the effect of varying concentrations (10, 20, 30 and 40% v/v) of cell free supernatant in

284 ATPS experiments showed that increase in cell free supernatant above 20% (v/v) led to poor

285 partitioning of the enzyme. This may be due to the increased amount of the biomolecules present

286 in the cell-free supernatant, which may affect the volume ratio and partitioning efficiency of

287 enzyme in the system. Loading higher amount of cell free supernatant may also lead to the

288 accumulation of the target protein and other biomolecules at the interphase as a precipitate

289 (Amid et al., 2015). 20% (v/v) of cell free supernatant was found to be the optimum

290 concentration for ATPS (22% w/v of 2-propanol and 14% w/v of K2HPO4) and also favored the

291 maximum recovery of protease.

292 3.4.3. Native-PAGE analysis

293 Electrophoretic pattern (Fig. 5A) of crude enzyme (E) and partitioned proteins in top (T) and

294 bottom phase (B) obtained from ATPS of 22% (w/v) of 2-propanol and increasing concentrations

295 of K2HPO4 viz., 14, 16, 18 and 20% (w/v) respectively, were run on native-PAGE. It is evident

296 from the gel that there is a variation in band pattern between two phases. The enzyme was highly

297 concentrated in alcohol phase (lane aT) of 2-propanol/K2HPO4 ATPS with a composition of

298 22/14% (w/v). The activity of the enzyme was confirmed by zymography on gelatin gels.

299 Proteolysis of gelatin by the enzyme was seen as clear zones in the zymogram (Fig. 5B).

300 3.5. Enzymatic dehairing of goat skins

301 The protease produced using CHCS has been partially purified by ATPS and compared with

302 that purified by ultrafiltration system and tested for the dehairing efficacy in order to authenticate

303 the proposed process suitable for the purpose. Fig. 6 depicts goat skins after dehairing

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304 confirming that enzyme is still active without much loss of activity throughout the purification

305 process to dehair the skins. It was observed that the concentrated enzyme with specific activity of

306 8.2×103 U/mg of protein from ATPS showed complete removal of hairs (Fig. 6A), whereas

307 enzyme (7.8×102 U/mg of protein) from UF in diluted form caused only partial removal (Fig.

308 6B). The results confirm that ATPS proves to be an efficient purification system to obtain

309 concentrated enzyme without the loss of activity.

310 3.6. Sustainable management of waste

311 From the present study, it could be estimated that about 0.07 t of protein content of collagen

312 hydrolysate recovered from 0.1 t chrome shavings could induce the bacterium to produce

313 11.8×108 units of protease of which 9.4×108 of units are extractable through ATPS. With

314 reference to the previous report (Sundararajan et al., 2011), this partitioned enzyme would dehair

315 about 1.5 t of raw skins, about the same mass of the material from which the chrome shaving is

316 generated. Thus, the CHCS could be considered as the potential candidate for the enhanced

317 production of an enzyme which could be used in the same industry generating the chrome

318 shavings wastes. The stabilizing effect of CHCS on enzyme could also be profitably exploited to

319 improve storage stability of enzyme for industrial application.

320 4. Conclusions

321 Among various protein sources, a newly formulated medium (pH 8.0) containing: 12 g/L of

322 CHCS, 15 g/L of molasses, 3 g/L of K2HPO4, 2 g/L of NaCl, and 0.04 g/L of CaCl2 showed

323 maximum protease production (203±0.07 U/mL) by Bacillus cereus VITSN04 at 42 h. Thus, the

324 present study confirms that the hydrolysate from chrome containing collagenous solid waste

325 obtained during processing of goat skins can be recycled as an inexpensive protein source for

326 production of dehairing protease for use in cleaner leather processing. CHCS apart from helping

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327 in protease production was found to be beneficial in enhancing the stability of the enzyme as

328 evidenced from fluorescence spectral studies. Isopropanol/K2HPO4 with a composition of

329 22/14% (w/v) ATPS was found to be efficient in the purification process with highest yield of

330 protease (88.1%) without loss of activity as evidenced by good dehairing efficacy.

331 From the results, it can be concluded that the recovered collagen hydrolysate can be put to

332 value added application in the production of enzyme paving way for the better waste

333 management option for the leather industry. The recovered collagen hydrolysate can be

334 completely recycled for cleaner production of more than the quantum of skins from which the

335 equivalent amount of chrome shaving has been originally generated.

336 Acknowledgements

337 Authors, Dr. C. Shanthi and S. Shakilanishi thank the management of VIT University for

338 providing the facilities to carry out this work. Two authors are also grateful to Dr. Anand Prem

339 Rajan, SBST, VIT University for providing fluorescence spectrophotometer facility and Dr.

340 Rambabu , SMBS, VIT University for providing ultrafiltration facility.

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347 system. Food Chem. 132, 1382-1386.

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348 Arte, E., Rizzello, C.G., Verni, M., Nordlund, E., Katina, K., Coda, R., 2015. Impact of

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461

462 Figure legends

463 Fig.1. Effect of CHCS at varying concentrations of molasses on growth of the organism ( )
464 and protease production ( )
465
466 Fig.2. Effect of pH of media containing wastes on protease production

467 Fig.3. Growth ( ) and protease ( ) production using protein based wastes, wheat bran
468 (WB), groundnut cake (GC), Bengal gram husk (BNGH), rice bran (RB), black gram
469 husk (BLGH), Molasses-collagen hydrolysate (MOL-CHCS) and nutrient broth (CON)
470
471 Fig.4. Fluorescence spectra of protease: E0 (enzyme), C0 (CHCS), T0 (trypsin), E+C0
472 (enzyme + CHCS) and T+C0 (trypsin+CHCS) are the samples measured without pre-
473 incubation at 25 °C. E3 (enzyme), C3 (CHCS), T3 (trypsin), E+C3 (enzyme+CHCS)
474 and T+C3 (trypsin+CHCS) are the sample measured after pre-incubation for 3 h at
475 40 °C.
476
477 Fig.5. Non-denaturing PAGE (10%) analysis on purity of partitioned protease
478 (A) Lane M: molecular weight markers; Lane E: crude enzyme obtained from culture
479 grown in collagen hydrolysate-molasses medium; Lane aT, aB, bT, bB, cT, cB, dT and
480 dB: protein samples procured from top and bottom phase of 2-propanol/K2HPO4 ATPSs
481 with a composition of 22/14, 22/16, 22/18 and 22/20% (w/v), respectively.
482 (B) Zymogram: lane M showing trypsin as enzyme marker.
483
484 Fig.6. Enzymatic dehairing of goat skins (A) Aqueous two phase partitioned enzyme and (B)
485 Ultra filtered enzyme

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Fig. 1
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Fig.2
200
pH 7.5 pH 8.0 pH 8.5 pH 9.0
180

160

140
Enzyme activity (U/mL)

120

100

80

60

40

20

0
Wheat bran Rice bran Black gram Molasses- Groundnut Bengal gram Nutrient
husk CHCS cake husk broth
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Fig.3
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Fig. 4

Note: Color image for web version

Note: Black and white image for print version

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Fig.6
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Table 1
Effect of protein based agro wastes on protease production
Protease activity before Protease activity after
Phenolic content
Agro wastes removal of polyphenols removing polyphenols
(% w/w)
(U/mL) (U/mL)
Wheat bran 0.004 45.7±0.2 86.0±0.05
Rice bran 0.005 39.5±0.09 72.9±0.08
Black gram
0.052 28.8±0.06 63.9±0.09
husk
Groundnut cake 0.039 29.7±0.1 51.1±0.1
Bengal gram
0.015 35.5±0.2 67.8±0.02
husk
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Table 2
Effect of protein concentration in CHCS on protease production

Protein Proteolytic
concentration in activity
CHCS (g/L) (U/mL)

2 3.7±0.05
4 13.5±0.03
6 20.3±0.1
8 38.7±0.1
10 54.4±0.1
12 64.9±0.05
14 50.1±0.06
Controla 67.5±0.06
a10 g of peptone
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Table 3
Fluorescence spectral analysis of enzyme in the presence of osmolytes and denaturant

Osmolytes and denaturant λmax (nm) after Protease

3 h incubation activity
at 40 °C (U/mL)
CHCS 331 -
Trypsin 336 130.4±0.1
Trypsin+hydroxyproline 329 133.2±0.08
Trypsin+glycine 328 134.0±0.07
Trypsin+hydroxyproline-glycine 325 137.3±0.16
Trypsin+glycerol 323 139.3±0.09
Trypsin+GnHCl 341 78.6±0.09
Trypsin+CHCS 329 141.1±0.05
Enzyme 337 121.5±0.09
Enzyme+hydroxyproline 330 123.0±0.1
Enzyme+glycine 329 123.6±0.07
Enzyme+hydroxyproline-glycine 324 127.8±0.06
Enzyme+glycerol 321 124.0±0.05
Enzyme+GnHCl 345 55.9±0.1
Enzyme+CHCS 327 135.7±0.06
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Table 4
Partitioning of protease using different ATPSs
Phase components Phase Selectivity Purification fold Protease yield
composition in top phase in top phase
(% w/v) (%)
PEG 8000/K2HPO4 20/14 2.3 1.6 62.5±0.06
PEG 8000/(NH4)2SO4 17/13 0.8 0.9 29.4±0.05
PEG 8000/Na3C6H5O7 15/10 1.9 1.4 61.3±0.03

PEG 6000/ K2HPO4 22/16 2.1 2.5 76.9±0.03

PEG 6000/(NH4)2SO4 18/13 1.0 2.0 40.4±0.02
PEG 6000/ Na3C6H5O7 17/13 1.5 2.3 57.4±0.1

PEG 4000/ K2HPO4 26/18 2.0 2.4 73.3±0.06

PEG 4000/(NH4)2SO4 20/12 1.1 2.12 44.8±0.07
PEG 4000/ Na3C6H5O7 22/16 0.8 1.75 45.4±0.06

1 propanol/ K2HPO4 26/10 1.9 1.7 61.7±0.2

1 propanol/(NH4)2SO4 24/10 0.4 0.4 11.1±0.05
1 propanol/ Na3C6H5O7 23/12 1.1 1.1 34.4±0.06

2 propanol/ K2HPO4 22/18 4.5 3.3 80.6±0.02

2 propanol/(NH4)2SO4 24/16 0.5 0.55 16.1±0.1
2 propanol/ Na3C6H5O7 28/14 1.2 1.22 47.6±0.1
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Table 5
Optimization of protease partitioning in 2 propanol/ K2HPO4 ATPS
Phase components Phase composition Selectivity Purification fold Protease yield
(% w/v) in top phase in top phase
(%)
2 propanol/ K2HPO4 22/12 2.8 1.6 55.4±0.06
2 propanol/ K2HPO4 22/14 4.9 4.1 88.1±0.2
2 propanol/ K2HPO4 22/16 4.6 3.1 81.0±0.05
2 propanol/ K2HPO4 22/18 4.5 3.3 80.6±0.06
2 propanol/ K2HPO4 22/20 4.2 3.0 76.9±0.1
2 propanol/ K2HPO4 22/22 2.2 1.3 50.5±0.07