Accepted Manuscript

Pineapple peel bromelain extraction using gemini surfactant-based reverse mi-

celle Role of spacer of gemini surfactant

Jingjing Guo, Zhitong Miao, Jing Wan, Xia Guo

PII: S1383-5866(17)31550-2
DOI: http://dx.doi.org/10.1016/j.seppur.2017.08.051
Reference: SEPPUR 13993

To appear in: Separation and Purification Technology

Received Date: 15 May 2017

Revised Date: 17 August 2017
Accepted Date: 20 August 2017

Please cite this article as: J. Guo, Z. Miao, J. Wan, X. Guo, Pineapple peel bromelain extraction using gemini
surfactant-based reverse micelle Role of spacer of gemini surfactant, Separation and Purification Technology
(2017), doi: http://dx.doi.org/10.1016/j.seppur.2017.08.051

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Pineapple peel bromelain extraction using gemini

surfactant-based reverse micelle Role of spacer of

gemini surfactant

Jingjing Guo, Zhitong Miao, Jing Wan, Xia Guo*

School of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou,

Jiangsu, 225002, P. R. China

CORRESPONDING AUTHOR: Prof. Dr. Xia Guo

EMAIL ADDRESS: guoxia@yzu.edu.cn

Tel: +86-0514-87975590-9513

Fax: +86-0514-87975244

1
1 Pineapple peel bromelain extraction using gemini

2 surfactant-based reverse micelle Role of spacer of

3 gemini surfactant
4

5 Jingjing Guo, Zhitong Miao, Jing Wan, Xia Guo*

6 School of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou,

7 Jiangsu, 225002, P. R. China
8
9 Abstract: In this study, the reverse micelles from gemini surfactants
10 pentamethylene-,-bis(dodecyldimethylammonium bromide), dodecamethylene-
11 ,-bis(dodecyldimethylammonium bromide), pentamethylene-,-bis(cetyl-
12 dimethylammonium bromide) and octamethylene-,-bis(cetyldimethylammonium
13 bromide) were used to extract bromelain from pineapple peel. The extraction
14 parameters including pH, ionic strength and surfactant content were studied. It was
15 found that the reverse micelle from dodecamethylene-,-bis
16 (dodecyldimethylammonium bromide) could extract more active bromelain in a much
17 wider range of pH, surfactant content and ionic strength than the reverse micelles
18 from the other gemini surfactants, and the optimum activity recovery, purification fold
19 and protein extraction efficiency with dodecamethylene-,-bis
20 (dodecyldimethylammonium bromide) were 160 %, 2.7 and 59 %, respectively.
21 Pineapple peel accounts for the largest waste proportion of pineapple. This study
22 investigated the effect of gemini surfactant structure on extraction efficiency of
23 bromelain from pineapple peel by reverse micelle extraction, and hence, should be
24 helpful for understanding the potency of gemini surfactant in protein extraction.
25

26 Keywords: surfactant; reverse micelle extraction; pineapple peel; bromelain; gemini

27 surfactant

2
28 1. Introduction

29

30 Bromelain is a chief sulfhydryl proteolytic enzyme and abundant in fruit and stem

31 of pineapple. Pineapple wastes such as peel, core, crown and leaves also contain

32 bromelain. Since bromelain has been exploited commercially in diverse industrial

33 applications [1, 2], the interest in its extraction has been motivated. Pineapple

34 bromelain is commonly concentrated by precipitation, ultrafiltration, adsorption and

35 chromatography, but some drawbacks are existed, such as low activity recovery,

36 complex operation process, high cost, and/or environmentally unfriendly [2, 3].

37 Reverse micelle extraction is a novel purification technology and some of the

38 advantages are no loss of native function/activity, high capacity, ease of scale up, and

39 potential for continuous operation [2, 3]. Recently, reverse micelles from conventional

40 surfactants, including di-2-ethylhexyl sodium sulfosuccinate (AOT), cetyl trimethyl

41 ammonium bromide (CTAB) and benzil dodecyl bis(hydroxylethyl) ammonium

42 chloride (BDBAC), were used to extract bromelain from pineapple pulp, core and

43 stem [3-9]. When AOT was used, the extraction efficiency was very low, while when

44 CTAB was used, about 97 % of activity could be recovered from pineapple pulp and

45 102-106 % from core and stem (with the protein extraction efficiency being ca. 20 %)

46 [6, 7]. The maximum purification factor of pulp bromelain with BDBAC could be ca.

47 3 (while the activity recovery and the content of recovered protein were not reported)

48 [5]. The combination of reverse micelle extraction with ultrafiltration could increase

49 the purification fold of core bromelain [8]. In spite of all these studies, the extraction

50 of bromelain from pineapple peel has been still a problem. It should be mentioned that

51 since the peel is the largest waste proportion of pineapple, the extraction of peel

52 bromelain seems to have the most promise for bromelain extraction.

3
53 Reverse micelles are aggregates of surfactant molecules, dispersed in a continuous

54 organic solvent medium and containing an inner core of water molecules [10].

55 Reverse micelle extraction is still in laboratory stage. It has been generally considered

56 that the target protein should be smaller than the water core of reverse micelle and

57 electrostatic interaction between protein and surfactant plays a key role in protein

58 transfer between aqueous and reverse micellar phases [3, 4, 11-13]. Many parameters,

59 such as aqueous phase pH, ionic strength, and structures and contents of cosurfactant

60 and oil, can affect protein extraction and have been studied very well [3-5, 7, 11,

61 14-17]. However, the surfactants used so far have been almost exclusively

62 conventional surfactants, and the reports about the modification of surfactant structure

63 have been limited yet.

64 Major limitation for reverse micelle extraction is the unfolding or denaturation of

65 protein and how to extract enzyme efficiently from biological system has been

66 challenging. Gemini surfactant is a kind of novel surfactant; it has two hydrophilic

67 head groups and two hydrophobic chains; the two head groups are linked by a spacer

68 via covalent bonds [18, 19]. The properties of gemini surfactant are closely related to

69 the spacer [18, 19]. Recently, nano-structured gemini surfactant-based supramolecular

70 solvent was prepared and used for microextraction of small organic compounds [20,

71 21]. In the present study, the reverse micelles from gemini surfactants 12-5-12,

72 12-12-12, 16-5-16 and 16-8-16 (Scheme 1) were used to extract bromelain from

73 pineapple peel, and the effect of surfactant structure on the extraction efficiency was

74 investigated.

75

76 2. Materials and Methods

77

4
 78 2.1 Chemicals and Reagents

79

80 n-Hexane ( 97.0 %), 1-hexanol ( 98.0 %), disodium hydrogen phosphate (

81 99.0 %), citric acid ( 99.5 %), sodium hydroxide ( 96.0 %), acetic acid ( 99.5 %),

82 sodium acetate ( 99.0 %), sodium dihydrogen phosphate ( 99.0 %), sodium chloride

83 ( 99.5 %), potassium bromide ( 99.0 %), sodium bromide ( 99.0 %) and glycine (

84 99.0 %) were bought from Chinese Chemicals (Sinopharm, Shanghai, China).

85 EDTA2Na ( 99.0 %) was bought from Amresco (USA). Casein was biotechnology

86 grade, bought from Chinese Chemicals (Solarbio, Beijing, P.R.China). Matured

87 pineapple was from the local market (Ananas comosus L. Merryl, produced in Hainan,

88 P.R.China). Deionized water, prepared through an UPDROEDIMB system (Ulupure

89 Co., Sichuan, P.R.China), was used in all the experiments. Gemini surfactants 12-5-12,

90 12-12-12, 16-5-16 and 16-8-16 (Scheme 1) were prepared according to the literatures

91 [22, 23]. N,N-dimethylhexadecylamine ( 95 %), N,N-dimethyldodecylamine (97 %),

92 1,5-Dibromopentane (97 %), 1,8-dibromooctane (98 %) and 1,12-dibromododecane

93 (96 %) were purchased from Aldrich (USA) and used to synthesize the gemini

94 surfactants.

95

96 2.2 Apparatus

97

98 A NMR spectrometer (AVANCE 600, Bruker, Germany) was used to identify the

99 gemini surfactant structure, same as in the literature [22, 23] and our previous study

100 [24, 25]. A centrifuger (Allegra 64R centrifuge, Beckman Coulter, USA) was used to

101 separate the aqueous and reverse micellar phases. The absorbance was determined on

102 a Uv-vis spectrophotometer (UV-1600, Shimadzu, Japan). The water content in

5
103 reverse micelle was measured by using a volumetric Karl-Fischer titrator (Y20,

104 Mettler Toledo, Switzerland).

105

106 2.3 Methods

107

108 2.3.1 Reverse micelle preparation

109
110 The reverse micelles were prepared by mixing known quantities of n-hexane,

111 1-hexanol, surfactant and water. The surfactant content was varied from 0.003-0.05

112 g/ml, and the molar ratio of water to surfactant was 15. The volume ratio of n-hexane

113 to n-hexanol was 9 since under this condition, the extraction performance was most

114 efficient (exemplified by Fig. S1).

115

116 2.3.2 Crude enzyme extract preparation

117
118 The extraction buffer, containing 5 mM of EDTA, was prepared from 0.04 M of

119 sodium phosphate (pH 6.0). The peel was separated manually from pineapple and

120 crushed in an equal weight of the extraction buffer for 5 min. After filtration, the

121 filtrate was centrifuged at 9168 g for 25 min and the supernatant separated was the

122 crude enzyme extract. The activity and the specific activity of bromelain in crude

123 extract were around 406 CDU/ml and 3404 CDU/mg, respectively. Here, CDU meant

124 casein digestion unit (see Section 2.3.4 for more detail).

125
126 2.3.3 Bromelain extraction
127
128 The crude enzyme extract was diluted 2-40 times with disodium hydrogen

129 phosphate/citric acid (pH 8.0) or glycine/sodium hydroxide (pH > 8.0) buffer,

6
130 which was used as the forward aqueous phase. The pH of the forward aqueous phase

131 was between 8.0 and 12.0, and the content of salt was 0-0.50 M. The forward aqueous

132 phase was mixed with an equal volume of reverse micelle and vortexed for 25 min at

133 room temperature. The mixture was centrifuged at 17968 g for 30 - 50 min, and then,

134 the organic phase could be separated.

135 In backward extraction, acetic acid/sodium acetate (10 mM, pH: 3.8 - 5.7) and

136 disodium hydrogen phosphate/sodium dihydrogen phosphate (10 mM, pH: 5.8 - 8.0)

137 solutions were used as stripping solution. The organic phase (obtained from forward

138 extraction) was mixed with an equal volume of the stripping solution; the salt content

139 in the stripping solution was from 0.25 to 2.0 M. After the mixture was centrifuged at

140 17968 g for 30 - 50 min, the aqueous phase could be separated, which was used to

141 determine the extraction performance.

142

143 2.3.4 Bromelain activity measurement

144

145 Casein digestion unit (CDU) method was based on proteolytic hydrolysis of casein

146 (0.6 %) and applied in the present study to determine bromelain activity [26, 27]. The

147 reaction was carried out under standard condition (i.e. at 37 oC and pH 7.0 for 10 min).

148 One unit of bromelain activity suggested 1 g of tyrosine was released in 1 min per

149 ml of sample. The amount of solubilized casein was determined by measuring the

150 absorbance of the clear filtrate at 275 nm.

151

152 2.3.5 Total protein content measurement

153

154 Bradford method was used to determine the total protein content in aqueous phase.

7
155 BSA was used as the standard and the sample analyses were performed against

156 respective blank solutions [28]. The extraction performance was evaluated by activity

157 recovery and purification fold of bromelain and protein extraction efficiency, which

158 were calculated by using Eqs. (1) - (3).

159

160 Protein extraction efficiency (%) =

161 Recovered protein content (mg/ml)/Protein content in feed (mg/ml) 100 (1)

162 Activity recovery (%) =

163 Recovered bromelain activity (CDU/ml)/Bromelain activity in feed (CDU/ml) 100 (2)

164 Purification fold =

165 Specific activity of recovered bromelain (CDU/mg)/Specific activity of bromelain in feed (CDU/mg) (3)

166

167 3. Results

168

169 Reverse micellar extraction of proteins includes two steps: forward extraction and

170 backward extraction [12, 13]. In forward extraction, target protein is solubilized into

171 the organic phase (also called reverse micelle phase) and in backward extraction, the

172 target protein in reverse micelle is back-extracted into water. In the present paper, the

173 forward and backward extraction parameters, including aqueous phase pH, ionic

174 strength and surfactant concentration, were studied first with the 25-times diluted

175 crude enzyme extract used as the forward aqueous phase. Then, the effect of dilution

176 multiple of crude enzyme extract on reverse micellar extraction performance was

177 investigated.

178

179 3.1 Effect of pH

180

181 Fig. 1A-D represented the effects of forward aqueous phase pH on the activity

8
182 recovery (AR) and purification fold (PF) of the recovered bromelain as well as protein

183 extraction efficiency (OEF); the backward aqueous phase pH remained 4.2.

184 From Symbols in Fig. 1A-D, it could be seen that AR was the highest at the pH

185 of the forward aqueous phase being 10.0-10.5, and the maximum values of AR were

186 ca. 113 % with 12-5-12 (Fig. 1A), 160 % with 12-12-12 (Fig. 1B), 99 % with 16-5-16

187 (Fig. 1C), and 108 % with 16-8-16 (Fig. 1D). Symbols in Fig. 1A-D indicated that

188 the maximum values of OEF were 68 % with 12-5-12 (at pH 11.0), 75 % with

189 12-12-12 (at pH 10.5), 57 % with 16-5-16 (at pH 10.0-10.5), and 60 % with 16-8-16

190 (at pH 10.5) reverse micelles. Moreover, the maximum values of PF were ca. 1.8 with

191 12-5-12 and 16-5-16 (at pH being 9.5-10.5, Symbols in Fig. 1A and C), ca. 2.7 with

192 12-12-12 (at pH being 10.0, Symbols in Fig. 1B), and 2.0 with 16-8-16 (at pH

193 being 9.5, Symbols in Fig. 1D). Since activity should be the primary index to

194 enzyme quality, the optimum values of pH in forward extraction were 10.5 with

195 12-5-12, 16-5-16 and 16-8-16, and 10.0 with 12-12-12, and the AR and OEF at the

196 optimum pH were 113 % and 64 % with 12-5-12, 160 % and 59 % with 12-12-12, 99 %

197 and 57 % with 16-5-16, and 108 % and 60 % with 16-8-16, respectively.

198 Fig. 1E-H showed the variation of AR, OEF and PF with the backward aqueous

199 phase pH. The backward aqueous phase pH was varied from 3.8 to 8.0 and the

200 forward extraction was done at the optimum pH. From Fig. 1E-H, the values of the

201 maximum OEF were ca. 57-64 % with these four gemini surfactants, observed at the

202 pH of the stripping solution (i.e. backward aqueous phase) being ca. 4.2-5.0 (Symbols

203 in Fig. 1E-H). AR became the highest at the pH of the stripping solution being ca.

204 4.2, which were 113 % with 12-5-12 (Symbols in Fig. 1E), 160 % with 12-12-12

205 (Symbols in Fig. 1F), 99 % with 16-5-16 (Symbols in Fig. 1G), and 108 % with

206 16-8-16 (Symbols in Fig. 1H). Therefore, the optimum pH in backward extraction
9
207 should be 4.2. Interestingly, at the backward aqueous phase pH being 3.8-8.0,

208 satisfying values of AR (110-160 %) could be observed with 12-12-12 (Symbols in

209 Fig. 1F), while the values of AR with the other gemini surfactants were decreased

210 obviously at pH lower or higher than 4.2 (for example, at pH 8.0, they were dropped

211 to 74 % with 12-5-12 (Symbols in Fig. 1E) and 39 % with 16-5-16 and 16-8-16

212 (Symbols in Fig. 1G and H)).

213

214 3.2 Effect of ionic strength

215

216 NaCl should favor protein transfer into reverse micelle phase while KBr should be

217 good for protein recovery (i.e. back into water phase) [7, 29, 30]. In the present study,

218 when 12-5-12, 16-5-16 and 16-8-16 were used, 0.05 M or higher content of NaCl

219 was necessary to make sure a clear interface between the two phases in the forward

220 extraction, while when 12-12-12 was used, NaCl was not necessary. In backward

221 extraction, KBr must be added to ensure the clear interface despite surfactant

222 structure. In the case of clear stratification, the dependence of extraction

223 performance on ionic strength was evaluated. It was found that 0.10 M of NaCl in

224 forward aqueous phase could ensure the maximum AR with 12-5-12, 12-12-12,

225 16-5-16 and 16-8-16 (Symbols , Fig. 2A-D). The maximum values for OEF were

226 obtained at 0.10 M of NaCl when 12-5-12 (Symbols , Fig. 2A) and 16-8-16

227 (Symbols , Fig. 2D) were used, and OEF with 12-12-12 (Symbols , Fig. 2B)

228 remained almost unchanged at NaCl content not higher than 0.10 M and then, it was

229 increased with the increase of NaCl content. When 16-5-16 was used (Symbols ,
10
230 Fig. 2C), the highest OEF was observed at 0.05-0.10 M of NaCl. Fig. 2E-H showed

231 the effect of KBr concentration (in backward aqueous phase) on bromelain

232 extraction. The highest OEF (around 60 %) was observed at 0.50 M of KBr when

233 12-5-12 and 16-5-16 were used (Fig. 2E and G, Symbols ) or at 0.25 M when

234 16-8-16 was used (Fig. 2H, Symbols ), while OEF with 12-12-12 remained around

235 60 % at KBr content not higher than 1.0 M (Fig. 2F, Symbols ). Furthermore, to get

236 the highest AR, the concentrations for KBr were 0.50-0.75 M when 12-5-12 was

237 used (Fig. 2E, Symbols ) and 0.50 M when 16-5-16 and 16-8-16 were used (Fig.

238 2G and H, Symbols ). When 12-12-12 was used, AR was changed very little at

239 KBr content being 0.50-1.0 M (Fig. 2F, Symbols ). Based on these data, 0.10 M of

240 NaCl was included in the forward aqueous phase in Figs. 1, 2 (Panels E-H), 3 and 4,

241 and 0.50 M of KBr was included in the backward aqueous phase in Figs. 1, 2 (Panels

242 A-D), 3 and 4.

243

244 3.3 Dependence on surfactant content

245

246 Symbols in Fig. 3A, C and D showed that the values of AR with 12-5-12,

247 16-5-16 and 16-8-16 were increased with increasing surfactant content until 0.01

248 g/ml, and then, decreased gradually; the highest values of AR were 113 % with

249 12-5-12, 99 % with 16-5-16, and 108 % with 16-8-16. When 12-12-12 was used (Fig.

250 3B, Symbols ), AR was high (140 - 160 %) at 0.005-0.04 g/ml of surfactant, and

251 the highest value (160 %) was found at 0.03 g/ml. OEF was also related to surfactant

11
252 content. When 12-5-12, 16-5-16 and 16-8-16 were used, the variation tendency for

253 OEF with surfactant content was similar to that for AR; at the surfactant content

254 being 0.01 g/ml, the highest OEF was observed, 64 % with 12-5-12 (Fig. 3A,

255 Symbols ), 57 % with 16-5-16 (Fig. 3C, Symbols ), or 60 % with 16-8-16 (Fig.

256 3D, Symbols ). The maximum OEF with 12-12-12 (ca. 59 %) could be obtained at

257 12-12-12 content being 0.01-0.03 g/ml; at 12-12-12 content lower than 0.01 g/ml or

258 higher than 0.03 g/ml, OEF was found lower (Fig. 3B, Symbols ). Therefore, the

259 optimum surfactant content should be 0.01 g/ml for 12-5-12, 16-5-16 and 16-8-16,

260 under which condition, the values of AR, OEF and PF were 113 %, 64 % and 1.8,

261 respectively, with 12-5-12, 99 %, 57 % and 1.7, respectively, with 16-5-16, and

262 108 %, 60 % and 1.9, respectively, with 16-8-16. When 12-12-12 was used, the

263 optimum surfactant content should be 0.03 g/ml for the highest AR (160 %), and the

264 values of OEF and PF under this condition were 59 % and 2.7, respectively. Here, it

265 should be noted that at 12-12-12 content being 0.005-0.04 g/ml, the values of AR

266 kept satisfying, 140 - 160 %, and the values of PF were around 2.2 - 3.3, much

267 higher than the optimum AR and PF with 12-5-12, 16-5-16 and 16-8-16.

268

269 3.4 Effect of initial crude enzyme content

270

271 When 12-5-12, 16-5-16 and 16-8-16 reverse micelles were used, the forward

272 extraction had to be carried out with crude enzyme solution diluted at least two times,

273 otherwise, emulsification occurred. However, dilution was not necessary for a clear

12
274 interface when 12-12-12 reverse micelle was used. Therefore, 12-12-12 could make

275 the reverse micelle accommodate more protein than 12-5-12, 16-5-16 and 16-8-16.

276 Furthermore, from Fig. 4A, the dilution of crude enzyme could make AR and OEF

277 increased with 12-5-12. From Fig. 4B, OEF and AR with 12-12-12 reached the

278 highest at the dilution multiple being 15, then AR was changed very little while OEF

279 was decreased a little. Fig. 4C and D showed that when 16-5-16 and 16-8-16 reverse

280 micelles were used, the optimum AR was found at the dilution multiple being 25 or

281 higher, while OEF was increased fast until the dilution multiple reached 10 and then,

282 changed very little. Therefore, 12-12-12 reverse micelle should be most efficient,

283 same as the result from Figs. 1-3.

284

285 3.5 Water content of reverse micelle during extraction

286

287 To understand protein transfer between the two phases, we determined the water

288 contents in reverse micelles after forward and backward extractions. The reverse

289 micelle separated after forward extraction was called filled reverse micelle after

290 forward extraction, and that separated after backward extraction was called filled

291 reverse micelle after backward extraction. For comparison, we also mixed the

292 reverse micelle with forward buffer (without crude enzyme) and separated the

293 organic phase, which was called unfilled reverse micelle after forward extraction.

294 This unfilled reverse micelle after forward extraction was then mixed with

295 stripping solution and the organic phase was separated, too, which was called

13
296 unfilled reverse micelle after backward extraction. Fig. 5A-D showed the water

297 percentage and Wo in filled reverse micelle with surfactant content. Here, Wo was

298 defined as the molar ratio of water to initial surfactant in the organic phase. It could

299 be seen that (1) the water percentage in organic phase (Fig. 5A and C) was increased

300 while Wo (Fig. 5B and D) was decreased continuously with the increase of surfactant

301 content, and (2) the water content in reverse micelle after forward extraction was

302 higher than that after backward extraction (Fig. 5A vs. C or B vs. D). Fig. 5E-F

303 compared the water contents in filled and unfilled reverse micelles, from which, the

304 water content in unfilled reverse micelle was only a little lower than that in filled

305 reverse micelle. Interestingly, Fig. 5A-B also showed the water content in 12-12-12

306 reverse micelle after forward extraction was much lower than those in 12-5-12,

307 16-5-16 and 16-8-16 reverse micelles.

308

309 4. Discussion

310

311 The surfactant concentration (i.e. from 0.003 to 0.05 g/ml) in the present study

312 was much higher than the critical micelle concentration (cmc, which were, for

313 example, 0.19 mM (i.e. 0.12 mg/ml) for 12-12-12 and 0.12 mM (i.e. 0.09 mg/ml) for

314 12-5-12, respectively, in organic solvent [31]). Figs. 1 - 4 showed that the reverse

315 micelles from the gemini surfactants could be used to extract pineapple peel

316 bromelain. SDS-PAGE electrophoresis experiment (Fig. S2) showed the quality of

317 the recovered bromelain was quite satisfactory and the molecular weight (around 23

14
318 kDa) was within the range reported (i.e. 23-36 kDa) [3]. Pineapple peel bromelain

319 had an isoelectric point of 9.5 [7], and it could have more negative charges with the

320 increase of pH. The optimum forward aqueous phase pH (i.e. ca. 10.0-10.5, Fig.

321 1A-D) and backward aqueous phase pH (i.e. ca. 4.2, Fig. 1E-H) indicated that the

322 electrostatic interaction between bromelain and the positively charged gemini

323 surfactant played a key role in bromelain extraction; the electrostatic attraction

324 between bromelain and surfactant was advantageous to the transfer of bromelain into

325 reverse micelle phase while the repulsion was beneficial for the backward extraction.

326 Here, it should be noticed that when pH was higher than 11 (in the forward

327 extraction) or lower than 4.0 (in the backward extraction), bromelain might become

328 denatured or unstable and as a result, the extraction efficiency could be decreased.

329 Salt could affect the strength of electrostatic interaction; the increase of ionic

330 strength could decrease repulsive force between surfactant head groups and shield

331 the attractive interaction of protein with surfactant. The combination of these two

332 effects made low content of salt (Fig. 3A-D and Fig. S3A-B) helpful for protein

333 transfer into reverse micelles while high content of salt (Fig. 3E-H and Fig. S3C-D )

334 beneficial for protein back into water (when salt content was too high in backward

335 extraction, salting out effect might become obvious, which might make protein

336 retained in reverse micelle due to its hydrophobic patches). The surfactant content

337 could affect bromelain transfer, too. With the increase of surfactant content, on the

338 one hand, the reverse micelle number could increase, but on the other hand, reverse

339 micelles could collapse. The former should be advantageous to the transfer of protein

15
340 into organic phase, while the latter should be not. As a result, the extraction

341 efficiency was increased first with increasing surfactant content, and then, at high

342 surfactant content, it was decreased (Fig. 3). It should be mentioned that the effects

343 of pH, salt and surfactant content discussed above were similar to those in the

344 literatures and also well-known [3-9, 32]. A notable result here should be that

345 although the difference in cmc for the surfactants in organic solvent was quite small,

346 the structure of gemini surfactant could modulate the extraction performance; among

347 12-5-12, 12-12-12, 16-5-16 and 16-8-16, 12-12-12 made (1) the reverse micelle load

348 the highest amount of crude enzyme, (2) the recovered bromelain have the highest

349 activity and purification fold, and (3) the extraction carried out in the widest range of

350 pH, surfactant content and ionic strength.

351 The water core size of reverse micelle was directly related to Wo; higher the Wo,

352 bigger the water core size. It was generally thought that a higher water content in

353 reverse micelle could be helpful for protein extraction. However, from Fig. 5,

354 12-12-12 reverse micelle had the lowest water content. This might suggest the effect

355 of spacer on the transfer of protein between the two phases. To further support this

356 assumption, we added water or crude enzyme solution dropwise into reverse micelle

357 until saturation. Then, we determined the water content in reverse micelle and found

358 that the solubility of water was the lowest in 12-12-12 reverse micelle and highest in

359 12-5-12 reverse micelle (Fig. S4). Fig. S4B also suggested that the solubility of

360 crude enzyme in 12-12-12 reverse micelle was the lowest. We further chose BSA as

361 the model protein and determined its solubility in these reverse micelles and found

16
362 the solubility of BSA in 12-12-12 reverse micelle was the lowest, too.

363 To transfer protein into the organic phase, the two-phase surfactant-laden interface

364 should deform around the protein to form a reverse micelle [33]. Therefore, the

365 surfactant-laden interfacial resistance was not good for the transfer of protein. As a

366 result, 16-5-16 reverse micelle extraction system was more sensitive to pH; when

367 16-5-16 was used, the interface between oil and water phases was not clear at

368 forward aqueous phase pH lower than 9.5 (different from the case when 12-5-12 was

369 used). A longer spacer could decrease the interfacial resistance and helpful for

370 protein transfer, which made (1) a clear interface observed at forward aqueous phase

371 pH lower than 9.5 when 16-8-16 was used (Fig. 1D), and (2) a satisfying OEF (ca.

372 56 %) observed with 12-12-12 once the forward aqueous phase pH reached 8.5,

373 much higher than that (30 %) with 12-5-12 at the same pH (Symbols in Fig. 1B vs.

374 1A).

375 Fig. 5A vs. C or B vs. D showed that after backward extraction, the water content

376 was decreased, indicating that the reverse micelle core should become smaller. The

377 decrease of core size was considered good for squeezing protein out. However, by

378 comparing the water content of the organic phase after forward extraction with that

379 after backward extraction (Fig. 5A vs. C or B vs. D), it could be found that the

380 change in the water content was the smallest when 12-12-12 was used, which could

381 be seen more easily by Fig. 5E and F. Then, how to understand the highest efficiency

382 of 12-12-12? It was mentioned above that the recovery of bromelain was driven by

383 electrostatic repulsion (Fig. 1E-H and Fig. 2E-H). Besides, the coalescence of the

17
384 protein-containing micelle with the bulk interface contributed to the backward

385 extraction [34]. Compared with -(CH2)5- and -(CH2)8-, -(CH2)12- should be more

386 flexible and hence, better for the coalescence; actually, bromelain could be recovered

387 efficiently at backward aqueous phase pH being 3.8-8.0 when 12-12-12 was used

388 (Fig. 1F), while when the spacer was changed to -(CH2)5- or -(CH2)8-, the backward

389 extraction had to be carried out at pH 4.2, otherwise, the extraction performance was

390 decreased markedly (Fig. 1G and H).

391 During extraction, the interaction between protein and surfactant might change

392 protein conformation and affect protein activity. From Fig. 3, for 12-5-12, 16-5-16

393 and 16-8-16 extraction systems, AR was dropped to ca. 60-75 % when surfactant

394 content reached 0.03 g/ml (Symbols in Fig. 3A, C and D), however, when

395 12-12-12 was used, the recovered bromelain remained 140 % activity even when

396 12-12-12 content reached 0.04 g/ml (Symbols in Fig. 3B). A couple of reports had

397 suggested that attributed to the strong hydrophobic interaction between gemini

398 surfactant and protein, the gemini surfactant might be used in renaturation of

399 proteins [35, 36], and a longer spacer was more effective in stabilizing protein

400 structure [19]. In addition, the tolerance of the extraction system to salt was related

401 to surfactant structure (Fig. 2); 12-12-12 extraction system could exhibit satisfying

402 performance in a wider range of salt content than 12-5-12, 16-5-16 and 16-8-16

403 extraction systems. This might be related to (1) the higher ionization degree for

404 12-12-12 micelle than 12-5-12, 16-5-16 and 16-8-16 micelles [22], and (2) the

405 stronger ability for spacer (CH2)12 (compared with (CH2)5 and (CH2)8) to shield the

18
406 repulsive force between the head groups due to the higher flexibility.

407

408 5. Conclusions

409

410 In summary, gemini surfactant structure could affect the transfer of pineapple peel

411 bromelain between water and oil phases in reverse micelle extraction. Compared

412 with 12-5-12, 16-5-16 and 16-8-16, 12-12-12 exhibited the much better extraction

413 performance and the extraction could be carried out in a wider range of surfactant

414 content, pH and ionic strength. Therefore, a longer spacer seemed more beneficial

415 for bromelain transfer. Since so far there have been very limited reports about

416 application of gemini surfactant in protein purification, this study should indicate

417 that devoting more attention to gemini surfactant in reverse micelle extraction

418 technology seems worthwhile.

419

420 Acknowledgements

421

422 This study was supported by national natural scientific foundation of China (No.

423 21373179) and a project funded by the priority academic program development of

424 Jiangsu higher education institutions (PAPD). And the authors declare that they have

425 no competing interests.

426

427 Supporting information available: Effects of contents of n-hexanol and salt on

428 extraction efficiency, SDS-PAGE electrophoresis of purified bromelain by reverse

19
429 micelle, and the solubility of water and crude enzyme in reverse micelles.

20
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512 surfactants C12-S-C12 2Br in n-heptane/n-hexanol solution: Effect of the spacer chains on the
513 critical reverse micelle concentrations, J. Colloid Interface Sci. 300 (2006) 310-313.
514 [32] Wan, J., Guo, J., Miao, Z., and Guo, X. Reverse micellar extraction of bromelain from pineapple
515 peel Effect of surfactant structure, Food Chem. 197 (2016) 450-456.
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517 proteins, Anal. Sci. 24 (2008) 901-906.

22
518 [34] Dungan, S. R., Bausch, T., Hatton, T. A., Plucinski, P., and Nitsch, W. Interfacial transport
519 processes in the reversed micellar extraction of proteins, J. Colloid Interface Sci. 145 (1991)
520 33-50.
521 [35] Li, Y., Wang, X., and Wang, Y. Comparative studies on interactions of bovine serum albumin with
522 cationic gemini and single-chain surfactants, J. Phys. Chem. B 110 (2006) 8499-8505.
523 [36] Gull, N., Sen, P., Khan, R. H., and Kabir-ud-Din. Interaction of bovine (BSA), rabbit (RSA), and
524 porcine (PSA) serum albumins with cationic single-chain/gemini surfactants: a comparative
525 study, Langmuir 25 (2009) 11686-11691.
526

23
527 Figure Captions
528
529 Scheme 1 Structures of gemini surfactants
530
531 Fig. 1 Dependences of protein extraction efficiency (OEF), activity recovery (AR)
532 and purification fold (PF) of bromelain on forward aqueous phase pH (A-D) and
533 backward aqueous phase pH (E-H). 0.10 M of NaCl was present in forward aqueous
534 phase and 0.50 M of KBr in backward aqueous phase. Backward aqueous phase pH
535 (Panels A-D): 4.2. In Panels E-H, optimum pH from Panels A-D was used in forward
536 extraction. Surfactant: 12-5-12 (Panels A and E), 12-12-12 (Panels B and F), 16-5-16
537 (Panels C and G) and 16-8-16 (Panels D and H). Optimum surfactant content from Fig.
538 3 was used.
539
540 Fig. 2 Effects of NaCl content in forward aqueous phase (Panels A-D) and KBr
541 content in backward aqueous phase (Panels E-H) on OEF, AR and PF of bromelain
542 extracted with 12-5-12 (Panels A and E), 12-12-12 (Panels B and F), 16-5-16 (Panels
543 C and G) and 16-8-16 (Panels D and H) reverse micelles. In Panels A-D, 0.50 M of
544 KBr was present in backward aqueous phase. In Panels E-H, 0.10 M of NaCl was
545 present in forward aqueous phase. Optimum pH from Fig. 1A-D was used in forward
546 extraction experiment and that from Fig. 1 E-H used in backward extraction
547 experiment. Optimum surfactant content from Fig. 3 was used.
548
549 Fig. 3 Effects of surfactant content on OEF, AR and PF of bromelain extracted with
550 12-5-12 (Panel A), 12-12-12 (Panel B), 16-5-16 (Panel C) and 16-8-16 (Panel D)
551 reverse micelles. 0.10 M of NaCl was present in forward aqueous phase and 0.50 M
552 of KBr in backward aqueous phase. Optimum pH from Fig. 1A-D was used in
553 forward extraction experiment and that from Fig. 1E-H was used in backward
554 extraction experiment.
555
556 Fig. 4 Effects of crude enzyme content in feed on OEF, AR and PF of bromelain
557 extracted with 12-5-12 (Panel A), 12-12-12 (Panel B) , 16-5-16 (Panel C) and 16-8-16
558 (Panel D) reverse micelles. 0.10 M of NaCl was present in forward aqueous phase and
559 0.50 M of KBr in backward aqueous phase. Optimum pH from Fig. 1A-D was used in
560 forward extraction experiment and that from Fig. 1E-H was used in backward
24
561 extraction experiment.
562
563 Fig. 5 Panels A-D showed the water percentage and Wo in filled reverse micelle with
564 surfactant content. Panels E-F compared the water contents in filled and unfilled
565 reverse micelles when surfactant content was 10 mg/ml.

25
566
567

568
569 12-5-12 12-12-12

570

571
572 16-5-16 16-8-16

573
574
575

576 Scheme 1

577

26
 120 AR 3.0 160 AR 3.5
OEF 140 OEF
PF PF 3.0
100 2.5
120
2.5

OEF (%)
AR (%)
OEF (%) 100
AR (%)
80 2.0 2.0

PF

PF
80
60 60 1.5
1.5
40 40 1.0
1.0 20 0.5
20 0
0.5 0.0
8 9 10 11 12 8 9 10 11 12
pH pH
578
579 A B

110 3.5 3.0

AR 120 AR
OEF OEF
100 PF 3.0 100 2.5
PF
90 2.5 80 2.0
OEF (%)
AR (%)

OEF(% )
AR(% )
80 PF

PF
2.0 60 1.5
70
1.5 40 1.0
60
1.0 20 0.5
50
0.5 0 0.0
9 10 11 12 8 9 10 11 12
pH pH
580
581 C D

120 3.0 160 AR 4.0

AR
110 OEF OEF
2.5 140 PF 3.5
100 PF
120
OEF (%)

90
AR (%)

3.0
OEF (%)
AR (%)

2.0

PF
80 100
PF

2.5
70 80
1.5
60 2.0
60
50 1.0
40 1.5
40
30 0.5 20 1.0
4 5 6 7 8 4 5 6 7 8
pH pH
582
583 E F

100 AR 3.0 120 AR 3.0

90 OEF OEF
PF 2.5 PF 2.5
80 100
OEF (%)
AR (%)

2.0 2.0
OEF(%)

70
AR(%)

80
PF

PF

60 1.5 1.5
50 60
1.0 1.0
40
0.5 40 0.5
30
20 0.0 20 0.0
4 5 6 7 8 4 5 6 7 8
pH pH
584
585 G H
586
587 Fig. 1
588
27
 120 AR 4.0 160 3.5
AR
110 OEF 3.5 OEF 3.0
PF 140 PF
100 3.0
120 2.5

OEF (%)
90
OEF (%)

AR (%)
AR (%) 2.5
2.0

PF
80

PF
2.0 100
70 1.5
1.5 80
60
1.0 1.0
50 60
40 0.5 0.5
40
30 0.0 0.0
0.05 0.10 0.15 0.20 0.25 0.30 0.00 0.10 0.20 0.30 0.40 0.50
[NaCl] (M) [NaCl] (M)
589
590 A B

100 3.0 3.0

AR 120 AR
OEF OEF
90 PF
2.5 PF 2.5
100
2.0
OEF (%)

80 2.0
AR (%)

80

OEF(%)
AR(% )
PF

PF
70 1.5 1.5
60
60 1.0 1.0
40
50 0.5 0.5
20
40 0.0 0.0
0.05 0.10 0.15 0.20 0.25 0.30 0.05 0.10 0.15 0.20 0.25 0.30
[NaCl] (M) [NaCl] (M)
591
592 C D

120 AR 3.0 210 AR 6.0

OEF OEF
PF 180 PF 5.0
100 2.5
OEF (%)

OEF (%)

150 4.0
AR (%)
AR (%)

PF

2.0

PF
80
120 3.0
60 1.5
90 2.0
40 1.0 60 1.0

20 0.5 30 0.0
0.4 0.8 1.2 1.6 2.0 0.2 0.4 0.6 0.8 1.0
[KBr] (M) [KBr] (M)
593
594 E F

120 3.5 OEF 3.5

AR 120
105 OEF AR
PF
3.0 PF 3.0
100
90 2.5 2.5
OEF (%)
AR (%)

OEF(%)
AR(%)
PF

75 2.0 80 2.0
PF

60 1.5 1.5
60
45 1.0 1.0
40
30 0.5 0.5
15 0.0 20 0.0
0.4 0.8 1.2 1.6 2.0 0.2 0.4 0.6 0.8 1.0
[KBr] (M) [KBr] (M)
595
596 G H
597
598 Fig. 2
599

28
 120 180 AR 6.0
AR 5.0
OEF 160 OEF
105 PF PF 5.0
4.0 140
4.0
OEF (%)
90

OEF (%)
120
AR (%)

AR (%)
3.0

PF
PF
75 100 3.0
2.0 80
60 2.0
60
45 1.0 1.0
40
30 0.0 20 0.0
0.00 0.01 0.02 0.03 0.04 0.05 0.00 0.01 0.02 0.03 0.04
[12-5-12] (g/ml) [12-12-12] (g/ml)
600
601 A B

105 AR 3.0 120 3.5

AR
OEF
90 PF 2.5 OEF 3.0
100 PF
2.5
OEF (%)

75 2.0
AR (%)

OEF(%)
80 2.0
PF

AR(% )

PF
60 1.5
60 1.5
45 1.0
1.0
30 0.5 40
0.5
15 0.0 20 0.0
0.00 0.01 0.02 0.03 0.04 0.05 0.00 0.01 0.02 0.03 0.04
[16-5-16] (g/ml) [16-8-16] (g/ml)
602
603 C D
604
605
606 Fig. 3
607

29
 160 4.0 180 6.0
AR AR
140 OEF 3.5 160 OEF
PF PF 5.0
120 3.0 140
OEF (%)

OEF (%)
AR (%) 4.0

AR (%)
2.5 120
100

PF
PF
2.0 100 3.0
80
1.5 80
2.0
60
1.0 60
40 40 1.0
0.5
20 0.0 20 0.0
0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35
Dilution multiple Dilution multiple
608
609 A B

120 AR 3.0 120 OEF 3.0

OEF AR
100 PF 2.5 PF 2.5
100
OEF (%)

80 2.0 2.0
AR (%)

OEF(%)
AR(%)
80
PF

PF
60 1.5 1.5
60
40 1.0 1.0
20 0.5 40 0.5
0 0.0 20 0.0
0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40 45
Dilution multiple Dilution multiple
610
611 C D
612
613
614 Fig. 4
615

30
 Filled reverse micelle

Percentage of water in organic phase

Filled reverse micelle
8 after forward extraction 70 after forward extraction
12-5-12
12-5-12
7 12-12-12 12-12-12
16-5-16 60 16-5-16
6 16-8-16 16-8-16

5 50

Wo
4
40
3
2 30
1
20
0
0.00 0.01 0.02 0.03 0.04 0.05 0.00 0.01 0.02 0.03 0.04 0.05
[Surfactant] (g/ml) [Surfactant] (g/ml)
616
617 A B
Filled reverse micelle
Filled reverse micelle
Percentage of water in organic phase

after backward extraction

after backward extraction
4
12-5-12 60 12-5-12
12-12-12
16-5-16 12-12-12
16-5-16
16-8-16 50 16-8-16
3
40

Wo
2
30
1
20

0 10
0.00 0.01 0.02 0.03 0.04 0.05 0.00 0.01 0.02 0.03 0.04 0.05
[Surfactant] (g/ml) [Surfactant] (g/ml)
618
619 C D
After forward extraction
Unfilled reverse micelle After backward extraction
Filled reverse micelle Unfilled reverse micelle
50 30 Filled reverse micelle

25
40
20
30
Wo
Wo

15
20
10
10 5
0 0
12-5-12 12-12-12 16-5-16 16-8-16 12-5-12 12-12-12 16-5-16 16-8-16
Surfactant Surfactant
620
621 E F

622

623
624 Fig. 5
625

31
626 Supporting Information

627

628 Pineapple peel bromelain extraction using gemini

629 surfactant-based reverse micelle Role of spacer of

630 gemini surfactant

631

632 Jingjing Guo, Zhitong Miao, Jing Wan, Xia Guo*

633 School of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou,

634 Jiangsu, 225002, P. R. China

635

636 CORRESPONDING AUTHOR: Prof. Dr. Xia Guo

637 EMAIL ADDRESS: guoxia@yzu.edu.cn

638 Tel: +86-0514-87975590-9513

639 Fax: +86-0514-87975244

640

32
641 Supporting Information

642
643 Fig. S1 Effect of n-hexanol content on OEF, AR and PF of bromelain extracted with
644 12-5-12 reverse micelle. 0.10 M of NaCl was present in forward aqueous phas and
645 0.50 M of KBr was present in backward aqueous phase. Surfactant content: 10 mg/ml.
646 Forward aqueous phase pH: 10.5. Backward aqueous phase pH: 4.2.
647
648 Fig. S2 SDS-PAGE electrophoresis (12.5 % polyacrylamide gel). Lane 1: Marker,
649 Lane 2: Purified bromelain by reverse micelle (before loaded onto the gel, the
650 aqueous phase of backward extraction was dialyzed and lyophilized).
651
652 Fig. S3 Effects of salt content in forward (Panels A and B) and backward (Panels C
653 and D) aqueous phases on OEF, AR and PF of bromelain extracted with 12-5-12
654 reverse micelle. In Panels A and B, 0.50 M of KBr was present in backward aqueous
655 phase and in Panels C and D, 0.10 M of NaCl was present in forward aqueous phase.
656 Surfactant content: 10 mg/ml. Forward aqueous phase pH: 10.5. Backward aqueous
657 phase pH: 4.2.
658
659 Fig. S4 The solubilities of water in reverse micelles by adding water (left) and crude
660 enzyme (right) into reverse micelles until saturation (surfactant content: 10 mg/ml).
661
662

33
663 Supporting Information

664
665
666

120 AR 3.0
OEF
110 PF 2.5
100
2.0

OEF(%)
AR(%) 90

PF
1.5
80
1.0
70
60 0.5
50 0.0
8 10 12 14 16 18 20 22 24 26
Vhexanol/Vhexane (%)
667
668
669
670
671
672 Fig. S1
673

34
674 Supporting Information

675
676
677
678
679

1 2

680
681
682
683
684
685 Fig. S2
686
687

35
688 Supporting Information

689
690

100 AR 3.0 100 AR 3.0

90 OEF 90 OEF
PF 2.5 PF 2.5
80 80
OEF(%)

OEF(%)
2.0 2.0
AR(%)

AR(%)
70 70

PF

PF
60 1.5 60 1.5
50 50
1.0 1.0
40 40
30 0.5 30 0.5
20 0.0 20 0.0
0.05 0.10 0.15 0.20 0.25 0.30 0.05 0.10 0.15 0.20 0.25 0.30
[KBr] (M) [NaBr] (M)
691
692 A B

120 AR 5.0 5.0

120 AR
OEF OEF
100 PF 4.0 PF 4.0
100
OEF(%)
AR(%)

80
OEF(%)

3.0 3.0
AR(%)

80
PF

PF
60
2.0 60 2.0
40
20 1.0 40 1.0

0 0.0 20 0.0
0.2 0.4 0.6 0.8 1.0 0.2 0.4 0.6 0.8 1.0
[NaCl] (M) [NaBr] (M)
693
694 C D
695
696
697
698
699 Fig. S3
700

36
701 Supporting Information

702
703
704

35 30
30
25
25
20
Wo

Wo
20
15
15
10 10

5 5
0 0
12-5-12 12-12-12 16-5-16 16-8-16 12-5-12 12-12-12 16-5-16 16-8-16
surfactant Surfactant
705
706 A B
707
708
709
710
711 Fig. S4
712
713 Highlights
714
715 Extraction efficiency was closely related to spacer of gemini surfactant.
716 Longer spacer could increase the extraction efficiency obviously.
717 Longer spacer could make extraction performed under more extensive conditions.
718

37