Professional Documents
Culture Documents
Enzymatic Hydrolysis 06
Enzymatic Hydrolysis 06
Enzymatic Hydrolysis 06
PII: S0959-6526(17)30365-7
DOI: 10.1016/j.jclepro.2017.02.139
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
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form.
Please note that during the production process errors may be discovered which could affect the
content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
13
14
15
1
ACCEPTED MANUSCRIPT
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
36
37 Key words:
38 Dehairing protease; Bacillus cereus; Collagen hydrolysate; Chrome shavings; Aqueous two
39 phase system
2
ACCEPTED MANUSCRIPT
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).
3
ACCEPTED MANUSCRIPT
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
4
ACCEPTED MANUSCRIPT
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
94 economical downstream processing method, aqueous two phase system (ATPS) have also been
95 studied.
5
ACCEPTED MANUSCRIPT
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.
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
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.
6
ACCEPTED MANUSCRIPT
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
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 ±
135 The method of Bradford (1976) was followed for protein content estimation using bovine
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.
7
ACCEPTED MANUSCRIPT
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
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
152 nm.
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
8
ACCEPTED MANUSCRIPT
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-
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.
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
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
9
ACCEPTED MANUSCRIPT
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.
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
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
10
ACCEPTED MANUSCRIPT
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
215 Thus, the medium containing 15 g/L of molasses, 12 g/L of CHCS and trace elements was
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.
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
11
ACCEPTED MANUSCRIPT
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
12
ACCEPTED MANUSCRIPT
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
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
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
13
ACCEPTED MANUSCRIPT
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
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).
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
14
ACCEPTED MANUSCRIPT
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
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
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
15
ACCEPTED MANUSCRIPT
327 in protease production was found to be beneficial in enhancing the stability of the enzyme as
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
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.
341 References
342 Amid, M., Manap, Y., Azmira, F., Hussin, M., Sarker, Z.I., 2015. A novel liquid/liquid
345 Amid, M., Shuhaimi, M., Sarker, M.Z.I., Manap, M.Y.A., 2012. Purification of serine protease
346 from mango (Mangifera Indica Cv. Chokanan) peel using an alcohol/salt aqueous two phase
16
ACCEPTED MANUSCRIPT
348 Arte, E., Rizzello, C.G., Verni, M., Nordlund, E., Katina, K., Coda, R., 2015. Impact of
349 enzymatic and microbial bioprocessing on protein modification and nutritional properties of
351 Arulnathan, N., Murugan, M., Balakrishnan, V., 2013. Proximate principles, fibre fraction and
352 Mineral content of Black gram husk (Vigna mungo). Int. J. Livest. Res. 3, 24-30.
353 Bolen, D.W., 2001. Protein stabilization by naturally occurring osmolytes. Protein structure,
355 Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities
356 of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.
357 Cabeza, L.F., McAloon, A.J., Yee, W.C., Taylor, M.M., Brown, E.M., Marmer, W.N., 1998a.
358 Process simulation and cost estimation of treatment of chromium-containing leather waste. J.
360 Cabeza, L.F., Taylor, M.M., Di Maio, G.L., Brown, E.M., Marmer, W.N., Carrio, R., Celma,
361 P.J., Cot, J., 1998b. Processing of leather waste: pilot scale studies on chrome shavings.
362 Isolation of potentially valuable protein products and chromium. Waste Manage. 18, 211-
363 218.
364 Cantera, C.S., 2003. Adding value to chrome shavings: hydrolysates as retanning materials.
366 Choudhary, R.B., Jana, A.K., Jha, M.K., 2004. Enzyme technology applications in leather
368 Davis, B.J., 1964. Disc electrophoresis–II method and application to human serum proteins. Ann.
17
ACCEPTED MANUSCRIPT
370 Gupta, V.K., Tuohy, M.G., O' Donovan, A., Lohani, M., 2015. Bioactive compounds in cereals-
371 technological and nutritional properties, in: Biotechnology of bioactive compounds: sources
372 and applications. John Wiley & Sons, West Sussex, UK, p.112.
373 Han, S.-W., Chee, K.-M., Cho, S.-J., 2015. Nutritional quality of rice bran protein in comparison
375 Horikoshi, K., 1990. Enzymes of alkalophiles, in: Microbial enzymes and biotechnology.
377 Kaur, S., Vohra, R.M., Kapoor, M., Beg, Q.K., Hoondal, G.S., 2001. Enhanced production and
378 characterization of a highly thermostable alkaline protease from Bacillus sp. P-2. World J.
380 Kijima, T., Yamamoto, S., Kise, H., 1996. Study on tryptophan fluorescence and catalytic
381 activity of α-chymotrypsin in aqueous-organic media. Enzyme Microb. Technol. 18, 2-6.
382 Kirk, O., Borchert, T.V., Fuglsang, C.C., 2002. Industrial enzyme applications. Curr. Opin.
384 Kresalkova, M., Hnanickova, L., Kupec, J., Kolomaznik, K., Alexy, P., 2002. Application of
385 protein hydrolysate from chrome shavings for polyvinyl alcohol-based biodegradable
387 Kumar, A.G., Swarnalatha, S., Sairam, B., Sekaran, G., 2008. Production of alkaline protease by
388 Pseudomonas aeruginosa using proteinaceous solid waste generated from leather
390 Kumar, C.G., Takagi, H., 1999. Microbial alkaline proteases: from a bioindustrial viewpoint.
18
ACCEPTED MANUSCRIPT
392 Kumaraguru, S., Sastry, T., Rose, C., 1998. Hydrolysis of tannery fleshings using pancreatic
393 enzymes: a biotechnological tool for solid waste management. J. Am. Leather Chem. Assoc.
395 Manta, C., Ferraz, N., Betancor, L., Antunes, G., Batista-Viera, F., Carlsson, J., Caldwell, K.,
396 2003. Polyethylene glycol as a spacer for solid-phase enzyme immobilization. Enzyme
398 Mukherjee, A.K., Adhikari, H., Rai, S.K., 2008. Production of alkaline protease by a
399 thermophilic Bacillus subtilis under solid-state fermentation (SSF) condition using Imperata
400 cylindrica grass and potato peel as low-cost medium: characterization and application of
402 Nilegaonkar, S.S., Zambare, V.P., Kanekar, P.P., Dhakephalkar, P.K., Sarnaik, S.S., 2007.
403 Production and partial characterization of dehairing protease from Bacillus cereus MCM B-
405 Ooi, C.W., Tey, B.T., Hii, S.L., Kamal, S.M.M., Lan, J.C.W., Ariff, A., Ling, T.C., 2009.
408 Otto, A., Knud, A., Helle, O., 1974. Dehairing of leather. U.S. Patent 3,840,433.
409 Pasupuleti, V.K., Braun, S., 2008. State of the art manufacturing of protein hydrolysates.
411 Pillai, P., Archana, G., 2012. A novel process for biodegradation and effective utilization of
412 chrome shavings, a solid waste generated in tanneries, using chromium resistant Bacillus
19
ACCEPTED MANUSCRIPT
414 Purohit, C., Rajyalakshmi, P., 2011. Quality of products containing defatted groundnut cake
416 Raju, A.A., Chandrababu, N.K., Samivelu, N., Rose, C., Rao, N.M., 1996. Eco-friendly
417 enzymatic dehairing using extracellular proteases from a Bacillus species isolate. J. Am.
419 Rao, J.R., Thanikaivelan, P., Sreeram, K.J., Nair, B.U., 2002. Green route for the utilization of
420 chrome shavings (chromium-containing solid waste) in tanning industry. Environ. Sci.
422 Ravindran, B., Kumar, A.G., Bhavani, P.A., Sekaran, G., 2011. Solid-state fermentation for the
423 production of alkaline protease by Bacillus cereus 1173900 using proteinaceous tannery solid
425 Reis, I.A., Campos, A.F., Santos, P.H., Santos, S.B., Soares, C.M., Lima, Á.S., 2015. Potassium
426 phosphate salts-based aqueous two-phase systems applied in the extraction of gallic acid
428 Sastry, T., Sehgal, R., Ramasami, T., 2005. Value added eco-friendly products from tannery
430 Shanthi, C., Banerjee, P., Babu, N.C., Rajakumar, G., 2013. Recovery and characterization of
431 protein hydrolysate from chrome shaving by microbial degradation. J. Am. Leather Chem.
433 Shanthi, C., Shelly, D.C., Stennett, B., 2003. Immobilization of degradative enzyme onto
434 collagen hydrolysate films. J. Am. Leather Chem. Assoc. 98, 6-12.
435 Shanthi, C., Suseela Rajakumar, G., 2012. A process for the preparation of protein hydrolysate
20
ACCEPTED MANUSCRIPT
437 Singleton, V., Rossi, J.A., 1965. Colorimetry of total phenolics with phosphomolybdic-
439 Sivasubramanian, S., Manohar, B.M., Rajaram, A., Puvanakrishnan, R., 2008. Ecofriendly lime
440 and sulfide free enzymatic dehairing of skins and hides using a bacterial alkaline protease.
442 Sumathi, C., Mohanapriya, D., Mandal, A.B., Sekaran, G., 2012. Production of different
443 proteases from fish gut microflora utilizing tannery fleshing. Eng. Life Sci. 12, 223-237.
444 Sundar, V.J., Gnanamani, A., Muralidharan, C., Chandrababu, N.K., Mandal, A.B., 2011.
445 Recovery and utilization of proteinous wastes of leather making: a review. Rev. Environ. Sci.
447 Sundararajan, S., Kannan, C.N., Chittibabu, S., 2011. Alkaline protease from Bacillus cereus
448 VITSN04: Potential application as a dehairing agent. J. Biosci. Bioeng. 111, 128-133.
449 Taneja, S., Ahmad, F., 1994. Increased thermal stability of proteins in the presence of amino
451 Tianwei, T., Qing, H., Qiang, L., 2002. Purification of glycyrrhizin from Glycyrrhiza uralensis
452 Fisch with ethanol/phosphate aqueous two phase system. Biotechnol. Lett. 24, 1417-1420.
453 Tomarelli, R., Charney, J., Harding, M.L., 1949. The use of azoalbumin as a substrate in the
454 colorimetric determination or peptic and tryptic activity. J. Lab.Clin. Med. 34, 428.
455 Zaslavsky, B.Y., 1994. Partitioning of solutes in aqueous two phase systems, in: Aqueous two-
456 phase partitioning: physical chemistry and bioanalytical applications. CRC Press. pp. 153-
457 217.
21
ACCEPTED MANUSCRIPT
458 Zia-Ul-Haq, M., Iqbal, S., Ahmad, S., Imran, M., Niaz, A., Bhanger, M., 2007. Nutritional and
459 compositional study of desi chickpea (Cicer arietinum L.) cultivars grown in Punjab,
461
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
22
ACCEPTED MANUSCRIPT
Fig. 1
ACCEPTED MANUSCRIPT
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
ACCEPTED MANUSCRIPT
Fig.3
ACCEPTED MANUSCRIPT
Fig. 4
Fig.6
ACCEPTED MANUSCRIPT
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
ACCEPTED MANUSCRIPT
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
ACCEPTED MANUSCRIPT
Table 3
Fluorescence spectral analysis of enzyme in the presence of osmolytes and denaturant
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
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