Various Types of Lipases Immobilized on Dendrimer-Functionalized

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Various Types of Lipases Immobilized on Dendrimer-Functionalized Magnetic Nanocomposite and Application in Biodiesel Preparation Yanli Fan, Caixia Ke, Feng Su, Kai Li, and Yunjun Yan Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/acs.energyfuels.7b00036 • Publication Date (Web): 22 Feb 2017 Downloaded from http://pubs.acs.org on February 24, 2017

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Energy & Fuels

1

Various Types of Lipases Immobilized on Dendrimer-Functionalized

2

Magnetic Nanocomposite and Application in Biodiesel Preparation

3 4

Yanli Fan, Caixia Ke, Feng Su, Kai Li, Yunjun Yan*

5 6

Key Laboratory of Molecular Biophysics of the Ministry of Education, College of

7

Life Science and Technology, Huazhong University of Science and Technology,

8

Wuhan 430074, P. R.China

9 10

*

11

Phone/Fax: +86-27-87792213;

12

E-mail: [email protected]

Corresponding author: Yunjun Yan

13 14 15 16 17 18 19 20 21 22

1

ACS Paragon Plus Environment

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ABSTRACT

24

Three sub-group lipases of Burkholderia cepacia lipase (BCL), Rhizomucor miehei

25

lipase (RML), and Candida rugosa lipase (CRL) were covalently immobilized on

26

dendrimer functionalized magnetic carbon nanotube and used as catalysts to catalyze

27

biodiesel production. The effects of imprinting molecule, organic solvent, water,

28

methanol, temperature and time interval of methanol addition on the yield of biodiesel

29

were optimized. The results showed that bioimprinting could greatly enhance catalytic

30

performances of the three immobilized lipases. The obtained lipases were then

31

employed to catalyze biodiesel production, and the achieved optimum conditions were:

32

for BCL, water content 5 wt.%, reaction temperature 35°C and with t-butanol as

33

reaction medium, methanol : oil molar ratio 4 : 1, its highest biodiesel yield attained

34

96.4%; for RML, water content 10 wt.%, reaction temperature 50°C, n-octane as the

35

reaction medium, methanol : oil molar ratio of 5 : 1, the utmost biodiesel conversion

36

rate was up to 96.2%; and for CRL, water content 7.5 wt.%, reaction temperature

37

40°C, isooctane as the reaction medium, methanol : oil molar ratio of 4 : 1, the best

38

yield reached 85.1%. It was borne out that the effect of time interval of methanol

39

addition on the biodiesel conversion was more obvious for the immobilized RML and

40

CRL than BCL. Furthermore, waste vegetable oil was also explored for biodiesel

41

preparation vs soybean oil. It reveals that the immobilized RML exhibited best

42

catalysis toward both feedstock in its corresponding solvent systems.

43

Keywords: Biodiesel; Bioimprinting; Lipase; Magnetic nanocomposite; Waste

44

vegetable oil

2


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1. INTRODUCTION

46

To substitute fossil fuels, biodiesel, i.e. fatty acid alkyl esters (FAAEs), has drawn

47

increasing attention as a biodegradable and renewable fuel not only because of lower

48

exhaust emissions, such as CO, SOx and HC 1, but also its being satisfactory and

49

practical for both direct-injection and indirect-type diesel engines 2. It is usually

50

prepared by transesterification of oils/fats or esterification of fatty acids with short

51

chain alcohols. Enzymatic approaches (lipase catalysis) occupy the advantage relative

52

to chemical methods due to the lower energy consumption, easy recovery of product,

53

more environmentally-benign process, and compatibility with a wide variety of

54

feedstock, especially with high free fatty acid content 3.

55

Lipases (triacylglycerol hydrolases, EC 3.1.1.3) widely distribute in animals,

56

plants and microbes, among which, those from microbes are the main resources 4. So

57

far, over 65 microbial species are known to produce lipases 5, especially in the genera

58

of Mucor, Rhizopus, Yarrowia, Candida, Bacillus, Pseudomonas, Burkholderia, etc.

59

Based on substrate specificity, Pleiss et al. 6 and Naik et al. 7 subdivided lipases into

60

three sub-groups: (a) lipases with a crevice-like binding site located near the protein

61

surface (such as lipases from Rhizomucor and Rhizopus); (b) lipases with a funnel-like

62

binding site (for example, lipases from Candida antarctica, Pseudomonas, and

63

Burkholderia); and (c) lipases with a tunnel-like binding site (for instance, lipase from

64

Candida rugosa). Typical commercial lipases of the above three sub-groups, such as

65

Rhizomucor miehei lipase, Burkholderia cepacia lipase, Candida rugosa lipase, were

66

abundantly reported to be employed for biodiesel production 3


8-10

. In particular,

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67

immobilized forms are more favored, mainly because high operational stability and

68

reusability of the immobilized enzymes make it possible to employ them in a batch

69

reaction (easy recovery), or in a continuous procedure for a long time. This technique

70

finally defrays biofuel production cost.

71

Till now, various kinds of carriers are widely used for enzyme immobilization.

72

Among them, several types of magnetic nanostructured materials have been proved

73

to have great potential for immobilization of lipases like magnetic nanoparticles,

74

nanotubes and grapheme etc.

75

properties that enhance the efficiency of biocatalysts, including effective enzyme

76

loading, higher surface area, and reducing mass transfer resistance

77

the conjugates endowed with magnetism can be easily separated from the reaction

78

medium by using a magnetic field.

79

11-13

. Nanoscale materials possess many unique

14

. Furthermore,

The methods of enzymatic immobilization mainly include adsorption, entrapping 15

80

cross-linking and covalent attachment

. Thereinto, covalent attachment has drawn

81

increasing attention because it has the advantage of strong interactions between the

82

support and the enzyme which makes enzyme leakage uncommon

83

covalent bonds formation usually adversely affect the conformation of the enzyme,

84

leading to decreased catalytic activity. But adopted proper approach such as

85

oriented-immobilization might eliminate this adverse effect 17.

16

. However,

86

In addition, researches demonstrate that molecular bioimprinting and interfacial

87

activation are the effective methods to improve enzymatic activity and stability in

88

non-aqueous media

18-19

. Meanwhile, proper organic solvents can also boost the

4


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89

activity of lipase in biodiesel synthesis

90

solvent in transesterification reactions are to shape a homogeneous reaction system,

91

which can decrease the viscosity of the reaction mixture, increase the diffusion rate

92

and reduce mass transfer problems around the enzyme

93

enzymatic biodiesel preparation with different lipases has been elaborated by Babaki

94

et al. 22 where Candida antarctica lipase B (CALB), Thermomyces lanuginosus lipase

95

(TLL) and Rhizomucor miehei lipase (RML) were immobilized onto SBA-epoxy. In

96

our

97

dendrimer-functionalized magnetic carbon nanotube (mMWCNTs-PAMAM) was

98

successfully developed

99

stability and excellent operational reusability for biodiesel production. Therefore, in

100

this work, to generalize this new immobilization method, based on the substrate

101

specificity of lipases and the merits of mMWCNTs-PAMAM matrix possessing

102

magnetic properties and massive active sites to increase the loading of enzyme, the

103

typical representatives of three sub-group lipases with different substrates specificity,

104

B. cepacia lipase (BCL), R. miehei lipase (RML) and C. rugosa lipases (CRL), were

105

selected as target proteins to be immobilized on mMWCNTs-PAMAM. To further

106

achieve

107

bioimprinting–immobilization was also adopted to modify lipases. The effects of

108

organic solvents, water, methanol content, reaction temperature and time interval of

109

methanol addition into the reaction mixture on the methanolysis catalyzed by the three

110

immobilized lipases were investigated. Additionally, a comparison of the catalytic

previous

more

work,

a

process

. The main reasons for the use of organic

of

employing

21

. A detailed comparison on

immobilized

lipase

on

23

. The immobilized lipase exhibited easy recovery, high

satisfactory

catalytic

performance,

the

5


strategy

based

on

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111

efficiencies in transesterification for biodiesel synthesis from soybean oil and waste

112

vegetable oil by the three immobilized lipases was carried out.

113 114

2. MATERIALS AND METHODS

115

2.1. Materials. Burkholderia cepacia lipase (BCL, initial specific activity 4.51×103

116

U/g-protein) powder with protein content 0.8% was purchased from Amano Enzyme

117

Inc. (Nagoya, Japan). Candida rugosa lipase (CRL, 7.94×103 U/g-protein) powder of

118

4.2% (mixtures of isozymes) and Rhizomucor miehei lipase (RML, 4.61×103

119

U/g-protein) of 3.7 mg/mL was brought from Sigma Aldrich (St. Louis, MO, USA).

120

Multi-walled carbon nanotubes (MWCNTs, purity > 95%) were commercially got

121

from Nanotech Port Co. Ltd. (Shenzhen, China). Waste vegetable oil was obtained

122

from ZTE Agri-valley Co. Ltd (Hubei, China). The acid value was at 11.6 mg KOH/g.

123

Soybean oil with 99% purity was purchased from local market. Other reagents such as

124

methyl acrylate, ethylenediamine (EDA), 3-aminopropyltriethoxysilane (APTES) and

125

glutaraldehyde (GA, a commonly used non-toxic cross-linker) produced by

126

Sinopharm Chemical Reagent Co., Ltd (Shanghai, China) were of analytical grade

127

and used as received without any further purification.

128

2.2. Analysis of the 3D-Structure of BCL, CRL and RML. 3D structural models

129

of BCL, RML and CRL (pdb identifiers: BCL, 3LIP; RML, 4TGL; CRL, 1CRL, all

130

open configuration) obtained from the NCBI (http://www.ncbi.nlm.nih.gov/) were

131

employed to analyze surface-exposed amino acid groups using PyMOL (2.7.6) 24.

132

2.3 Synthesis and Functionalization of mMWCNTs-PAMAM. mMWCNTs-

6


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133

PAMAM was synthesized and characterized according to the reported article

134

Briefly, the MWCNTs were oxidized by the nitro/sulfuric acid for 4 h to obtain the

135

carboxyl grafted MWCNTs. Then, magnetic iron oxide nanoparticles were loaded

136

onto the surfaces of MWCNTs (mMWCNTs) by the impregnation method, which was

137

detailed in the Supporting

138

(mMWCNTs-NH2) were prepared with APTES via postsynthetic grafting.

139

Subsequently, PAMAM dendrimer was grafted on the surface of mMWCNTs-NH2

140

(Scheme S1). The detailed preparation procedures are provided in the Supporting

141

Information.

142

2.4. Lipase Immobilization. mMWCNTs-PAMAM composites (100 mg) were

143

dispersed in absolute ethanol and followed by adding an amount of glutaraldehyde

144

(five different concentration at 2.5, 5, 7.5, 10, 12.5 wt.%) and shaken in a

145

thermostatic shaker at a stirring speed of 200 rpm at 30°C for 10 h. The product was

146

taken out by magnetic separation, washed several times with de-ionized water to

147

remove

148

mMWCNTs-PAMAM-GA. Then, BCL (150, 200, 250, 300, 350 mg), CRL (10, 20,

149

30, 40, 50 mg) powder respectively were dissolved in 5 mL phosphate buffer

150

solution (0.05 M) and RML (0.4, 0.5, 0.6, 0.7, 0.8 mL) sample were added to 4.6,

151

4.5, 4.4, 4.3 and 4.2 mL phosphate buffer solution, respectively. The three enzymes

152

solution were mixed with the carriers (mMWCNTs-PAMAM-GA, 100 mg) in a

153

rotary shaker at a stirring speed of 200 rpm at various temperature (25, 30, 35, 40,

154

45, 50oC) and time (1, 2, 2.5, 3, 3.5, 4, 4.5 h). The mixture was separated

excess

Information.

glutaraldehyde.

The

.

Amine-funtionalized mMWCNTs

obtained

sample

7


was

defined

as

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magnetically thoroughly and rinsed with fresh buffer to remove unbound and

156

nonspecific absorption lipases.

157

2.5. Bioimprinting Procedure. Bioimprinting procedure as per the method of Lv et

158

al.

159

improve the activity of the immobilized lipases. BCL (250 mg), CRL (30 mg)

160

powder were respectively dissolved in 4.9 mL 0.05 M phosphate buffer solution and

161

RML (0.5 mL) sample in 4.4 mL. Bioimprinting molecules (lauric acid, oleic acid,

162

linoleic acid, triolein, olive oil) were resolved in the mixture solvent of isopropanol

163

(0.1 mL) and Tween 60 (100 mg) as surfactant. The mixture was added to the

164

enzyme solution (the final concentration of the bioimprinting molecule was 0.075

165

mmol/mL) and incubated under the conditions of rotating speed 200 rpm at room

166

temperature for 20 min. After incubation, the bioimprinted lipases were immobilized

167

according to the procedures described in “Section 2.4”. Then, the bioimprinting

168

molecules

169

immobilized–bioimprinted lipases were collected using a magnet and dried in a

170

vacuum desiccator at room temperature.

171

2.6. Lipase Activity Assay. The activities of the immobilized and free lipases were

172

analyzed using the method described previously

173

immobilized and free lipases were added to 10 mL mixture containing 1-dodecanol

174

(0.2 M) and lauric acid (0.2 M) in isooctane with addition 0.01 mL water, and the

175

reactions were implemented at a certain temperature for 30 min with continuous

176

shaking at 200 rpm. 1 mL sample was sampled and mixed with 15 mL of

25

with slight modification was carried out prior to immobilization procedure to

were

removed

with

5

mL

isooctane,

and

then

the

26

. A certain amount of the

8


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ethanol-acetone (1:1, v/v) to stop the reaction. The remaining acid in the sample was

178

detected by titration with NaOH solution (0.05 M). Phenolphthalein solution (1%,

179

w/v) was used as pH indicator. One unit of enzyme activity (U) was defined as the

180

amount of lipase that consumed 1µmol of lauric acid per minute under the assay

181

conditions. The amount of immobilized enzyme was detected as per the method from

182

Bradford with bovine serum albumin (BSA) as the standard 27. The immobilization

183

efficiency (%), activity recovery (%) and specific activities (U/g-protein) were

184

calculated via Eqs. (1) - (3) 28. immobilized protein × 100% total loading protein

185

Immobilization efficiency (%) =

186

Activity recovery (%) =

187

Specific activity (U/g-protein) =

(1)

specific activity of immobilized lipase × 100% (2) specific activity of adding free lipase

initial activity (3) protein content of immobilized lipase

188

2.7. General Procedures of Transesterification Reactions for Biodiesel Synthesis

189

in Organic Solvents and GC Analysis. The reactions were conducted in a

190

stoppered 50 mL shake flask in organic solvent system under a stirring rate of 200

191

rpm. The reaction mixture includes soybean oil (2.19 g), methanol, immobilized

192

lipases (10 wt.%, the specific activities of BCL is 5.62×104 U/g-protein, RML

193

1.58×105 U/g-protein and CRL 5.71×103 U/g-protein), organic solvent (20 wt.%)

194

and some water. All dosage percentages were based on the oil mass, unless

195

otherwise stated. To avoid the inhibitory effect of methanol on the immobilized

196

lipases, methanol were respectively added in three steps at the same interval.

9


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GC analysis: some samples were collected from the reaction mixture at

198

specified time and centrifuged at 13800×g for 5 min to obtain the supernatant. 10 µL

199

of supernatant, 290 µL hexane and 300 µL of 1.0 mg/mL heptadecanoic acid methyl

200

ester (as internal standard, hexane as solvent) were mixed thoroughly for gas

201

chromatographic analysis. The methyl ester content was analyzed using a GC-9790

202

gas chromatograph (Agilent HP-INNOWAX capillary column 30 m × 0.25 mm ×

203

0.25 µm, J&W Scientific, Folsom, CA). The operating conditions: the mixed sample

204

(1.0 µL) above was injected into the GC, and the column initial temperature was

205

180°C and increased to 230°C at a rate of 3°C min-1 and then maintained at 230°C

206

for 3 min. The injector and detector temperature were set at 230°C and 280°C,

207

respectively. The biodiesel yield (%) was defined as the total FAAE content in the

208

conversion oil sample. The biodiesel yield was calculated with Eqs. (4) and (5) 28,

209

Biodiesel yield (%) =

210

f0 =

Asamplef0 AinternalWinternal

WsampleAinternl WinternalAsample

(4) (5)

211

where, Asample: the peak area of the free fatty acids in sample; f0: the response factor;

212

Ainternal: the peak area of the internal standard; Winternal: the mass (g) of the internal

213

standard; Wsample: the mass (g) of the sample.

214

2.8. Statistics Analysis. All trials were conducted in three parallel replicates, and the

215

data were analyzed by the software Origin 8.0 (OriginLab Co., Northampton, MA,

216

USA).

217 218

3. RESULTS AND DISCUSSION 10


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3.1 Synthesis of Immobilized Lipases. Three subgroup lipases, B. cepacia lipase

220

(BCL), R. miehei lipase (RML) and C. rugosa lipases (CRL) were immobilized on

221

mMWCNTs-PAMAM. During the immobilization process, the immobilization

222

conditions will change due to the difference in lipase sources. In our previous work,

223

the effects of glutaraldehyde concentration, lipase loading, pH value, immobilization

224

temperature and coupling time on the immobilization efficiency and activity

225

recovery of RML were investigated

226

parameters on BCL and CRL were further examined. As shown in Tables 1and 2, all

227

the lipases immobilized on the dendrimer-functionalized magnetic carbon nanotube

228

showed excellent activity recovery expect CRL. When the glutaraldehyde

229

concentration was at 7.5 wt.%, the activity recovery of the three immobilized lipases

230

all arrived at their highest values (BCL: 185%, RML: 2,769%, CRL: 42.2%). The

231

probable reason is because the same amount of glutaraldehyde was required to

232

completely activate the amino group of the same carrier. At this concentration (7.5

233

wt.%) of glutaraldehyde, the immobilization efficiencies of the three lipases were

234

86.4% (BCL), 89.4% (RML) and 90.7% (CRL), and the corresponding specific

235

activities were 8.38×103 U/g (BCL), 1.276×105 U/g (RML) and 3.34×103 U/g

236

(CRL), respectively. However, the effects of other parameters on the immobilization

237

of the three enzymes varied to some extent. The optimum dosage of enzymes used

238

differed owning to the different protein contents (BCL: 0.8%, RML: 3.7 mg/mL,

239

CRL: 4.2%) and amino groups contents on the surface of the enzyme molecules

240

(BCL: 2.2%, RML: 2.6%, CRL: 3.7%). In fact, the higher amount of the enzyme

23

. Herein, the effects of immobilization

11


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241

loading, the easier the aggregation of enzyme would occur, which could decline the

242

activity recovery. Actually, the highest activity recovery was not obtained at the

243

highest loading of the enzymes. While the dosage of enzymes were respectively 250

244

mg (BCL), 0.5mL (RML) and 30mg (CRL), the activity recovery of the three

245

immobilized lipases reached the highest points (Table 2). Meanwhile, pH value is a

246

critical factor in enzyme immobilization. Panzavolta et al.

247

value had little effect on immobilization efficiency while great influence on the

248

esterification activity. It is because enzymes are differently charged in various pH

249

values, which is conducive to the active conformation. As per the theory of “pH

250

memory”, enzymes maintain the ionization as they turn towards organic phase from

251

aqueous. So, various conformation was preserved

252

recovery were obtained with the pH value severally in 7.5 (BCL), 7 (RML) and 6.5

253

(CRL).

30

29

have reported that pH

and the highest activities

254

For the immobilization temperature, enzyme activity generally increases with

255

the elevation of temperature to a certain level, and thereafter too much high

256

temperature will lead to protein denaturation and thus decline the activity recovery.

257

The denaturation and inactivation temperatures of the three lipases are different, so

258

the most appropriate immobilization temperatures are also different. The optimum

259

temperatures were 30oC for BCL, 45oC for RML and 35oC for CRL. In addition, the

260

activity of enzyme in water environment will decrease with prolonging of coupling

261

time. In general, the activity recovery increases with the extension of the coupling

262

time and then decreases as the coupling time extension. Nevertheless, the rate of

12


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enzyme activity decreased and the time required to maintain enzyme activity for

264

different enzymes are also different. As the results shown, the optimum coupling

265

times for BCL, RML and CRL were 2.5 h, 4 h and 2.5 h, respectively. Under the

266

optimal conditions, the activity recovery of the immobilized BCL and RML were

267

dramatically improved compared with the immobilized CRL. The reason is not only

268

related to the unique characteristics of the lipase structure, but also to the

269

immobilization method. As is well known, the mechanism for improving the activity

270

and stability of the immobilized lipase is extremely complicated. It is mainly

271

attributable to a combination of the following factors. First, the active centers of most

272

lipases are covered by a so-called “lid” structure, which controls access of the

273

substrate(s) to the active site. The secondary structure of the lipase would probably

274

change during immobilization, and the “lid” might be opened to some extent for the

275

substrate(s), which would provide an easier access, leading to an increase in lipase

276

activity 31. The second factor is related to the immobilization method. Here, the three

277

lipases, BCL, RML and CRL, were respectively immobilized on a uniform matrix by

278

covalent bond according to the distribution of amino groups on enzyme molecules,

279

because most amino groups are located far from the catalytic active center in

280

comparison to other functional groups (carboxyl, hydroxyl and sulfhydryl groups). As

281

shown in the Figure S1, the distributions of ε-NH2 residues of BCL and RML are both

282

non-uniform, mainly far from the catalytic center (Fig. S1b and d). However, its

283

distribution in CRL (Fig. S1e and f) is nearly uniform with some amino residues

284

distributed near the catalytic center, which may be the main cause resulting in loss of

13


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17

285

activity after immobilization

286

hydroxyl and sulfhydryl

287

immobilization methods such as adsorption, entrapment, and encapsulation might be a

288

better choice for the immobilization of CRL.

289

. Other active groups of CRL, such as carboxyl,

groups, are also

unsuitable. Therefore, physical

There are some reports about the influences of the support on the loading and the 11, 32-33

290

enzymatic activity of the immobilized lipases on magnetic nanocomposites

291

Table 3 presents a comparison between mMWCNTs-PAMAM in this investigation

292

and previously reported immobilization on other magnetic micro/nanocomposites

293

support. As mentioned above, the transesterification performance of the three

294

proposed

295

mMWCNTs-PAMAM were comparable or better than that obtained from other

296

immobilized lipases. Moreover, the three immobilized lipase have higher water

297

tolerance and reusability than the other immobilized lipases (Table 3). Thus, the

298

immobilized lipases developed in this study seem to be more effective and have the

299

potential for practical applications in enzymatic biodiesel synthesis processes,

300

especially for non-edible oil with high content of water as substrate.

lipases

with

different

substrates

specificity

immobilized

.

on

301

----------------------------- Fig. S1, Table 1, Table 2, Table 3-----------------------------

302

3.2. Bioimprinting. The strategy of molecular bioimprinting is to cause a

303

ligand-induced beneficial conformational change in the enzyme in aqueous solution,

304

and later employ it in non-aqueous media where the enzyme is supposed to maintain

305

the imprinted conformation and keeps high catalysis activity. Generally, fatty acid 14


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Energy & Fuels

306

substrate analogues are utilized as templates for the bioimprinting of lipases 34. Herein,

307

the effects of five imprinting molecules on the biocatalytic activity of the three lipases

308

were examined (Fig. 1). Lauric acid was used as template, the activity recovery of the

309

immobilized BCL was enhanced from 192.0% to 1,244%. Oleic acid chosen as

310

template, the maximal activity recovery of CRL was 72.6%, and that of RML was

311

3,437%, which were 1.47-fold and 1.25-fold enhancement over the non-bioimprinted

312

immobilized enzymes, respectively. Moreover, the immobilization efficiency of the

313

three lipases had no obvious change after imprinting with different imprinting

314

molecules. The reasonable explanation for this dramatic enhancement of activity

315

imprinted with lauric acid to BCL and oleic acid to CRL and RML was probably that

316

the resemblance of these molecules to the natural substrates of the enzymes

317

contributes to forming an enzyme-support complex with a very suitable open

318

conformation favorable for the access of substrates

319

immobilizing Candida rugosa lipase on polypropylene by physical adsorption. The

320

activity

321

bioimprinting-immobilization technique, with specific activity enhancement of near

322

70% with respect to non-bioimprinted CRL. Similarly, the activity recovery of

323

Yarrowia lipolytica lipase LIP2 were also significantly improved after imprinting with

324

imprinting molecule compared with non- treated YlLIP2 37. These results manifest that

325

different lipases all can be enhanced to some degree via imprinting technology.

326

of

the

immobilized

lipase

was

35

. Foresti et al.

greatly

------------------------------Fig. 1----------------------------

15


36

reported

enhanced

via

Energy & Fuels

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

327

3.3 The Effects of Organic Solvents and Water Content on the Biodiesel

328

Production. It is widely recognized that organic solvent features not only affect the

329

mass transfer in the reaction system, but also greatly influence on the enzyme

330

structure and activity. Enzyme shows different catalytic activity, substrate selectivity,

331

operating stability and kinetic characteristics in different solvents. Lu et al. 38 studied

332

the performances of Candida sp. 99-125 catalyzing methanolysis of glycerol

333

trioleate in twelve different solvents and found that the fundamental influence of

334

organic solvents on enzymatic catalysis was hydrophobicity (log P, logarithm of the

335

partition coefficient (P) of the solvent in 1-octanol and water two-phase system). An

336

optimal organic solvent could improve the mutual solubility of hydrophilic alcohols

337

and hydrophobic triglycerides and thus protect the enzymes from denaturation at

338

high alcohol concentrations. Table 4 enucleates the effect of different solvents on the

339

three lipases with methanol added only once, and Table 5 lists biodiesel yield of

340

methanolysis reaction in varied solvents systems and water amounts. It can be

341

clearly seen that the yield of the reaction is remarkably dependent on the type of

342

solvents.

343

The lipase in various solvents also required different optimal amount of water

344

to retain its maximum activity. As shown in Table 4, biodiesel yield catalyzed by the

345

immobilized BCL did not exceed 70% except t-butanol, which showed a yield of

346

80.6%. Similarly, when the immobilized RML was used as a catalyst, the highest

347

biodiesel yield was obtained in n-octane, which was close to 50%. While isooctane

348

was the optimal organic solvent for the immobilized CRL (35.3%). In the more

16


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Energy & Fuels

349

hydrophilic solvents systems, such as phenol, dihydroxybenzene and N,

350

N-dimethylformamide, the yields were all not very high. Some former studies have

351

elucidated that enzymes showed higher activity in relatively hydrophobic organic

352

solvents (log P > 2) which have been tried as reaction medium for biodiesel

353

production 39. However, some research showed that there was not a linear correlation

354

between the solvents and the biodiesel yield, but an approximate S-shaped curve 38.

355

In this study, we also found that there was not a linear correlation between solvents

356

and the biodiesel yields for the three lipases in transesterification reaction. The

357

explanation might due to the fact that different lipase sources with specific

358

immobilized method would have unique characteristics in organic solvents. In

359

addition, it can be found that t-butanol was a relatively excellent medium for these

360

three immobilized lipases, which was perhaps because that methanol and glycerol

361

have good solubility in t-butanol solvent. So, the negative effects on lipases activity

362

and stability caused by methanol and glycerol could be weaken, and lipases still

363

exhibited fairly stability in such reaction medium. Meanwhile, the yields catalyzed

364

by the immobilized RML and CRL compared with BCL were very low in most cases,

365

revealing that the activities of RML and CRL might be more severely inhibited by

366

the excess methanol. When three-step methanol addition strategy was employed, all

367

biodiesel yields were markedly enhanced in their corresponding optimal organic

368

solvents (Table 5). Furthermore, RML and CRL are known to exhibit much higher

369

tolerance to water

370

n-octane were beneficial to RML and CRL due to the fact that they do not partially

40-41

, so several hydrophobic solvents such as isooctane and

17


Energy & Fuels

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371

replace the residual protein-surface bound water. Accordingly, t-butanol, n-octane

372

and isooctane were employed as the reaction media for further transesterification

373

catalyzed by the immobilized BCL, RML and CRL, and three-step methanol

374

addition procedure was selected in the following experiments.

375

Water content of the reaction mixture has significant influence on the catalytic

376

activity and stability of lipases, especially in different solvent systems, which is not

377

only related to the unique characteristics of lipases acting at oil-water interface, but

378

also correlated with the reaction equilibrium 42. Moreover, the optimum water content

379

generally depends on the type of lipases. So, water amount varied from 0 to 12.5 wt.%

380

was determined for each immobilized lipases. From Table 5, it was found that a

381

certain amount of water was required in the reaction mixtures for the three lipases.

382

The biodiesel yields catalyzed by the three immobilized lipases increased to different

383

degree with the increase of water amount in isooctane, n-octane and t-butanol system.

384

And the highest conversion rate of 84.5% was obtained with 5 wt.% water for the

385

immobilized BCL in t-butanol system. Meanwhile, the biodiesel yield slightly

386

increased to a high yield at 10 wt.% of water content in n-octane system for the

387

immobilized RML, and 7.5 wt.% for CRL in isooctane system.

388

----------------------------- Table 4, Table 5-----------------------------

389

3.4 The Effects of Methanol Concentration on Biodiesel Production. Methanol

390

serves as a reaction substrate of transesterification and excessive methanol tends to

391

push the reaction process to the synthesis direction, but is harmful to the lipases.

18


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Energy & Fuels

392

Addition of appropriate amounts of alcohols to the reaction mixture can increase

393

reaction rate and degree of transesterification reaction. So, the optimal amount of

394

methanol added to the reaction for each lipase was tested. As shown in Figure 2, the

395

biodiesel yields grew with the increase of methanol amount, while higher methanol

396

content would significantly lower the enzyme activities of the three immobilized

397

lipases. Consequently, the minimal stoichiometric methanol to oil ratio of 5 : 1 for

398

the immobilized RML, 4 : 1 for BCL and CRL were chosen in further experiments.

399

-----------------------------Fig. 2-------------------------------

400

3.5 The Effects of Reaction Temperature on Biodiesel Production. In order to

401

prevent the lipases from thermal inactivation, the enzymatic transesterification is

402

generally conducted at lower temperature compared with chemical reactions.

403

Meanwhile, increasing the reaction temperature tends to push the reaction process to

404

endothermic direction, which is propitious to the biodiesel production 43. Therefore,

405

temperature effects on methanolysis catalyzed by the immobilized lipases (BCL,

406

RML and CRL) in organic solvents were examined. The optimum operational

407

temperatures for the three immobilized lipases varied greatly. The biodiesel yield of

408

the immobilized BCL, RML and CRL showed their maximum values at 35, 50 and

409

40°C, respectively, further increase of temperature will result in decrease of biodiesel

410

yield (Fig. 3).

411

-------------------------------Fig. 3------------------------------

19


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Page 20 of 41

412

3.6 The Effects of Time Interval of Methanol Addition on Biodiesel Production.

413

It is known that over 1/2 M equivalent of alcohols added to the reaction mixture at

414

the beginning will inactivate the activity of enzyme. Stepwise addition of alcohols to

415

the system is a widely adapted strategy to prevent enzyme from inactivation caused

416

by alcohols

417

great influence on the enzyme activity. Fan et al.

418

obtained 93.1% yield with the time interval of 10 h , which was higher than that of 6

419

h time interval. Thus, the effects of time interval of methanol addition on biodiesel

420

production for the three lipases were investigated. As can be seen from Figure 4a,

421

there was no significant increase in conversion rates with extension of time interval

422

of methanol addition for the immobilized BCL. However, the biodiesel yields

423

catalyzed by the immobilized RML and CRL were remarkably improved by

424

lengthening the time interval (Fig. 4b, c). The results indicated that bacterial lipase

425

(BCL is from B. cepacia) was more resistant to organic solvents than fungal lipases

426

(RML and CRL are from R. miehei and C. rugose, respectively) 45.

44

. Moreover, the length of time interval of alcohol addition also has 23

used the three-step method and

427

-------------------------------Fig. 4-------------------------------

428

3.7 A Comparison of Biodiesel Production from Soybean Oil and Waste

429

Vegetable Oil. The above researches have enunciated that the soybean oil can be

430

effectively converted into biodiesel with the three immobilized lipases as catalysts

431

under their corresponding optimal conditions. However, the cost of feedstock

432

accounts for a large portion in biodiesel production cost. So, a kind of cheaper

20


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Energy & Fuels

433

feedstock, waste vegetable oil was further investigated. Some specifications of waste

434

vegetable oil have been tested and listed in Table S1, and can be seen that there are

435

obvious distinctions between soybean oil and waste vegetable oil, especially in water

436

contents, free fatty acids (FFAs), stearic acid, oleinic acid and linoleic acid.

437

The temporal kinetics of methanolysis for soybean oil and waste vegetable oil

438

by the three immobilized lipases were presented in Figure 5a-c. For the immobilized

439

BCL, the initial reaction rate of waste vegetable oil was higher than that of soybean

440

oil owing to the higher FFAs content; nevertheless, the final biodiesel yield of waste

441

vegetable oil was much lower than that of the soybean oil, which might result from

442

the negative effect of water existing in the waste vegetable oil (2.01%) and

443

continuously produced by esterification of FFAs (Fig. 5a)

444

yield of waste vegetable oil was similar with that of soybean oil for the immobilized

445

RML, probably due to its higher tolerance to water up to 20% (w/w) 40. While for the

446

immobilized CRL, the final yield of waste vegetable oil was much higher than that

447

of soybean oil, which is perhaps because CRL is a versatile lipolytic enzyme with

448

five individual isoforms

449

their catalytic efficiencies 47. Actually, Kuo et al.

450

activities of CRL isozyme (CRL1–CRL4) for three non-edible oils and found that

451

CRL2 and CRL4 exhibited superior catalytic efficiencies for producing biodiesel

452

from Jatropha curcas seed oil.

453

46

20

. In comparison, the

and different isoforms exhibit remarkable variation in 47

compared the transesterification

---------------------------------Fig. 5, Table S1---------------------------------

21


Energy & Fuels

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

454

A comparison of the transesterification catalyzed by the immobilized BCL, CRL

455

and RML showed that biodiesel yields of BCL and RML were both up to 96% when

456

soybean oil was used as raw material. However, the reaction time when the highest

457

yield was achieved by the immobilized BCL was 10 h much shorter than that of the

458

immobilized RML. As waste vegetable oil was used as raw material, the final

459

biodiesel yield was 80.8%, much lower than that of the soybean oil for the

460

immobilized BCL; while for the immobilized RML, it was up to 92.1%, much close

461

to that of the soybean oil; for the immobilized CRL, less than 90%, though higher

462

than that of soybean oil. The results are probably due to the following reasons: (i) the

463

characteristics (such as trans-/esterification ability and substrate specificity) of the

464

enzymes are much different; (ii) the compositions of the two feedstock differ from

465

each other; (iii) it is closely related to the nature of the carrier and the immobilization

466

method. Reasons (i) and (ii) have been respectively elaborated in “Section 3.2 and

467

3.7”. Moreover, previous report also suggested that the immobilized CRL could play a

468

better role in the preparation of biodiesel by replacing organic solvents with ionic

469

liquids

470

production from raw oils having lower water content and FFAs, meaning it has higher

471

ability to catalyze transesterification than esterification and less tolerance to the

472

negative effect of moisture. In contrast, higher water content and FFAs such as waste

473

vegetable oil would be suitable for RML, which showed best performance among the

474

three lipase for biodiesel synthesis, indicating its high catalysis abilities for

475

transesterification and esterification.

48

. Additionally, the immobilized BCL could work better for biodiesel

22


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Energy & Fuels

476

3.8 Operational Stability of Biocatalysts. One of the objectives of the immobilized

477

enzyme is to design more efficient biocatalysts which can easily be recovered and

478

reused. To investigate the reusability of the three biocatalysts for biodiesel production,

479

the immobilized enzymes was recovered by magnetic separation after each batch and

480

washed with their corresponding solvents for the subsequent batch and the next batch

481

was carried out with fresh substrates under the same reaction conditions as described

482

previously. The reusability of the immobilized enzymes were presented in Table 6. As

483

can be seen, the biodiesel yields after 10 cycles for the immobilized BCL, RML and

484

CRL were 89.4%, 80.5% and 58.3%, respectively. The results indicate that the

485

immobilized lipases show good capability to be repeatedly used. The yield of

486

biodiesel, catalyzed by immobilized Candida antarctica lipase on SBA-15 via

487

physical adsorption, decreased by 8.9% after eight recycles 49. The gradual reduction

488

in biodiesel yield was ascribed to both the leaching of the enzyme and loss of activity

489

of the immobilized lipase. Compared to physical adsorption, covalently linkage could

490

strongly diminish the leaching of the enzyme. In addition, the stability and reusability

491

of BCL was better than that of RML and CRL, which attributed to the tolerance of the

492

enzyme and the system solvent. Isooctane and n-octane (log P > 4) are high

493

hydrophobic solvent, which beneficial for CRL and RML in single batch reaction.

494

However, with the increase of the recycling number, methanol and by-product

495

glycerol will adsorb onto the surface of the immobilized lipase due to their poor

496

solubility in isooctane and n-octane solvents, enlarging mass transfer resistance and

497

causing gradual inactivation of the enzyme, resulting in a reduction in the biodiesel

23


Energy & Fuels

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

498 499

yield. ---------------------------------Table 6---------------------------------

500 501

4. CONCLUSION

502

Typical representatives, BCL, RML and CRL, of three sub-group lipases with

503

different substrates specificity, were respectively immobilized on a uniform magnetic

504

nanocomposites material by covalent binding, and their characteristics for

505

transesterification reaction were further investigated. Bioimprinting could improve

506

catalytic performance of the three lipases. In the optimized conditions, all

507

immobilized lipases could achieved high biodiesel yields, showing advantage of using

508

CNTs as carriers for catalytic aim over other less durable nanomaterials. The

509

immobilized BCL, RML and CRL exhibited well operational stability and reusability.

510

However, the biodiesel yields achieved by the immobilized BCL and RML were

511

higher than that of CRL. It was also demonstrated that waste vegetable oil could be

512

effectively converted into biodiesel by the immobilized RML. This study implies that

513

the immobilization strategy employed in this study can significantly enhance the

514

catalytic activity and stability of lipases, but the enhancement is different to some

515

extent for different enzymes. Specific conditions are still required for each lipase to

516

catalyze transesterification and the immobilized RML may offer a promising solution

517

to industrial scale biodiesel production for low-cost inedible oil. Furthermore, this

518

work provides a new choice for immobilization of other enzymes with similar surface

519

amino distribution and substrate specificity.

24


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Energy & Fuels

520 521

AUTHOR INFORMATION

522

Corresponding Author

523

Phone/Fax: +86-27-87792213; E-mail: [email protected]

524

Notes

525

The authors declare no competing financial interest.

526 527

The Supporting Information includes:

528

Experimental Procedures

529

1.1 Preparation of mMWCNTs

530

1.2 Synthesis of mMWCNTs-NH2 by Aminosilane

531

1.3 Surface Modification with PAMAM Dendrimer

532

Table S1

533

Scheme S1 and Figure S1

534 535

ACKNOWLEDGEMENTS

536

This study was financially supported by the National Natural Science Foundation

537

of China (No. 31170078), the National High Technology Research and Development

538

Program of China (Nos. 2011AA02A204 and 2013AA065805), the Natural Science

539

Foundation of Hubei Province (No. 2015CFA085), and the Fundamental Research

540

Funds for HUST (Nos. 2014NY007 and 2014QN119). The authors thank Ms. Chen

541

Hong, from the Centre of Analysis and Test, Huazhong University of Science and

542

Technology for biodiesel analysis and Pro. Xiaotao Han from the National High 25


Energy & Fuels

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

543

Magnetic Field Center, HUST.

544 545 546 547

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355-363. 27


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

602

(40) Alzuhair, S. Biofuel. Bioprod. Bior. 2007, 1, 57-66.

603

(41) Kaieda, M.; Samukawa, T.; Kondo, A.; Fukuda, H. British J. Learning Disabilities 2001,

604

41, 13-21.

605

(42) Su, F.; Li, G. L.; Fan, Y. L.; Yan, Y. J. Fuel Process. Technol. 2015, 137, 298-304.

606

(43) Gumel, A. M.; Annuar, M. S. M.; Heidelberg, T.; Chisti, Y. Bioresour. Technol. 2011,

607 608 609 610 611 612 613

102, 8727-8732. (44) Shimada, Y.; Watanabe, Y.; Sugihara, A.; Tominaga, Y. J. Mol. Catal. B Enzym. 2002, 17, 133-142. (45) Yang, W. J.; He, Y. J.; Xu, L.; Zhang, H. J.; Yan, Y. J. J. Mol. Catal. B Enzym. 2016, 126, 76-89. (46) Chang, S. W.; Li C. F.; Lee G. C.; Yeh T.; Shaw J. F. J. Agr. Food Chem. 2011, 59, 6710-6719.

614

(47) Kuo, T. C.; Shaw, J. F.; Lee, G. C. Bioresour. Technol. 2015, 192, 54-59.

615

(48) Su, F.; Peng, C.; Li, G. L.; Xu, L.; Yan, Y. J. Renew. Energ. 2016, 90, 329-335.

616

(49) Arumugam, A.; Ponnusami, V. J. Sol. Gel. Sci. Techn. 2013, 67, 244-250.

617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 28


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Page 29 of 41

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Energy & Fuels

632

Table 1 The optimized single factor conditions for the three lipases in the

633

immobilization process† Optimum value

Parameters BCL

RML

CRL

Glutaraldehyde concentration

7.5 wt.%

7.5 wt.%

7.5 wt.%

Amounts of lipase

250 mg

0.5 mL

30 mg

pH value

7.5

7.0

6.5

Reaction temperature

30°C

45°C

35°C

Coupling time

2.5 h

4h

2.5 h

634

†

635

lipase. The optimal conditions of five parameters of RML have been presented in the published

636

paper 23. Note: ± indicates the standard deviation (SD).

BCL: Burkholderia cepacia lipase; RML: Rhizomucor miehei lipase; CRL: Candida rugosa

637 638 639 640 641 642 643 644 645 646 647 648

29


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Page 30 of 41

649 650

Table 2 The corresponding activity recovery, immobilization efficiency and specific activity of the three immobilized lipases under their

651

optimized single factor conditions for the five key parameters† Activity recovery (%)

Parameters BCL Glutaraldehyde concentration Amounts of lipase

RML

Immobilization efficiency (%)

Specific activity (U/g)

CRL

BCL

RML

CRL

BCL

RML

CRL

185 ± 5.2

2,769 ± 25.3

42.2 ± 1.7

86.4 ± 2.2

89.4 ± 2.1

90.7 ± 3.0

(8.38 ± 0.25)×103

(1.276 ± 0.012)×105

(3.34 ± 0.12)×103

187 ± 4.7

2,775 ± 27.8

43.1 ± 2.1

85.1 ± 2.5

88.6 ± 2.0

89.6 ± 2.5

(8.44 ± 0.23)×103

(1.280 ± 0.013)×105

(3.42 ± 0.17)×103

pH value

190 ± 3.9

2,781 ± 26.4

45.3 ± 1.9

87.2 ± 2.1

89.1 ± 1.9

91.1 ± 2.1

(8.59 ± 0.17)×103

(1.283 ± 0.012)×105

(3.59 ± 0.15)×103

Reaction temperature

191 ± 5.0

2,789 ± 30.8

46.8 ± 1.9

87.2 ± 1.8

90.4 ± 2.4

90.6 ± 2.6

(8.64 ± 0.23)×103

(1.286 ± 0.014)×105

(3.71 ± 0.15)×103

Coupling time

193 ± 4.6

2,799 ± 32.5

49.4 ± 2.4

86.7 ± 1.9

89.3 ± 2.2

89.8 ± 2.4

(8.73 ± 0.20)×103

(1.291 ± 0.015)×105

(3.92 ± 0.20)×103

652

†

653

RML have been presented in the published paper 23. Note: ± indicates the standard deviation (SD).

BCL: Burkholderia cepacia lipase; RML: Rhizomucor miehei lipase; CRL: Candida rugosa lipase. The activity recovery and immobilization efficiency of

654 655

30


Page 31 of 41

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Energy & Fuels

656 657

Table 3 Comparison the reaction components/conditions and biodiesel yields using the immobilized lipases in present work and previous works Support

Immobilization method

Magnetic

Covalent

nanoparticles

bonding

Magnetic

Covalent

microsphere Magnetic silica nanocomposite

Dendrimer-coated magnetic carbon nanotubes

Enzyme binding efficiency (%) 84%

83%

bonding

Methanolysis of soybean oil (12 h); oil: alcohol molar ratio 1:3; 50 oC; No water; solvent-free system; 40% immobilized lipase Thermomyces lanuginosa (w/w oil) Methanolysis of soybean oil (24 h) ; oil: alcohol molar ratio 1:5; 35 oC; 0.1% water; solvent-free system; 50% immobilized lipase Candida rugosa (w/w oil)

Biodiesel yield (%) 87%

Reuse (reaction cycle) 5 (50%

Ref. [11]

conversion) 86%

5 (47%

[33]

conversion)

Methanolysis of olive oil (30 h); oil: alcohol molar ratio 1:4; 40 oC; Physical adsorption

97%

87% (BCL) Covalent bonding

10% water; solvent-free system; 11% immobilized lipase Burkholderia

92%

10 (60% conversion)

96% (BCL) 96% (RML) 85% (CRL)

10 (89% conversion) 10 (81% conversion) 10 (58% conversion)

sp. C20 (w/w oil)

89% (RML) 90% (CRL)

658

Reaction condition

Methanolysis of soybean oil (24 h); oil: alcohol molar ratio 1:4; 35oC; 5% water; 20% t-butanol; 10% immobilized BCL (w/w oil). Methanolysis of soybean oil (36 h); oil: alcohol molar ratio 1:5; 50oC; 10% water; 20% n-octane; 10% immobilized RML (w/w oil). Methanolysis of soybean oil (40 h); oil: alcohol molar ratio 1:4; 40oC; 7.5% water; 20% isooctane; 10% immobilized CRL (w/w oil).

Note: BCL: Burkholderia cepacia lipase; RML: Rhizomucor miehei lipase; CRL: Candida rugosa lipase.

31


[32]

This study

Energy & Fuels

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Page 32 of 41

659

Table 4 Effect of solvents with different log P values on the enzymatic biodiesel

660

production

661

Organic solvents

†

Yield (%)

log P BCL

RML

CRL

Solvent free

--

42.5 ± 3.6

30.5 ± 2.8

21.4 ± 1.7

n-Nonane

5.1

40.4 ± 4.1

32.1 ± 4.6

23.8 ± 0.9

Isooctane

4.7

69.4 ± 2.9

44.6 ± 3.4

35.3 ± 0.7

n-Octane

4.5

52.9 ± 4.8

49.6 ± 2.6

29.5 ± 3.2

n-Heptane

4.0

62.1 ± 3.1

36.7 ± 1.9

26.3 ± 4.3

n-Hexane

3.5

45.8 ± 2.9

31.4 ± 2.5

25.8 ± 2.5

n-Pentane

3.0

39.2 ± 2.5

29.9 ± 3.1

26.8 ± 0.8

Toluene

2.5

43.5 ± 2.8

38.5 ± 1.7

27.9 ± 0.8

Benzene

2.0

40.3 ± 4.0

30.6 ± 0.9

29.6 ± 1.5

Phenol

1.5

38.6 ± 2.3

28.4 ± 1.8

20.7 ± 3.8

Dihydroxybenzene

1.0

40.8 ± 2.6

26.6 ± 2.3

25.4 ± 2.0

t-Butanol

0.8

80.6 ± 4.7

42.2 ± 4.1

30.9 ± 3.4

N,N-dimethylforma

-1.0

37.6 ± 1.7

20.5 ± 2.6

26.7 ± 0.7

mide

662

†


663

lipase. The reactions were performed at 40 °C, 200 rpm for 12 h. 10 wt.% immobilized lipases

664

were added to 2.19 g soybean oil containing 20 wt.% solvent (from log P = 5.1 to log P = -1.0)

665

and 5 wt.% water (all dosage percentages were based on the oil mass), methanol were added only

666

once by the molar ratio of methanol : oil molar ratio 4:1. The values are average and ± indicates

667

the standard deviation (SD).

668 669 670

32


Page 33 of 41

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Energy & Fuels

Table 5 Biodiesel yield of methanolysis catalyzed by three immobilized lipases in different solvents systems and water amounts†

671

Yield (%) Solvents Isooctane n-Octane t-Butanol

Control

2.5 wt.% water

BCL

RML

CRL

36.6 ± 2.6 29.6 ± 3.4 25.6 ± 2.9

18.8 ± 3.1 20.4 ± 4.3 15.4 ± 3.5

19.5 ± 2.8 14.6 ± 2.1 6.1 ± 1.9

BCL 65.4 ± 2.8 41.7 ± 2.9 68.9 ± 0.7

5 wt.% water

RML

CRL

BCL

RML

CRL

26.4 ± 3.0 30.3 ± 3.2 28.8 ± 4.5

28.6 ± 3.6 20.9 ± 2.2 7.8 ± 3.7

47.2 ± 2.6 38.2 ± 3.7 50.3 ± 2.6

39.8 ± 3.4 44.9 ± 1.8 41.4 ± 2.0

40.4 ± 1.5 32.6 ± 2.3 25.5 ± 2.7

(continued)

672

Yield (%) Solvents Isooctane n-Octane t-Butanol

7.5 wt.% water

10 wt.% water

12.5 wt.% water

BCL

RML

CRL

BCL

RML

CRL

BCL

RML

CRL

47.2 ± 2.6 38.2 ± 3.7 50.3 ± 2.6

39.8 ± 3.4 44.9 ± 1.8 41.4 ± 2.0

40.4 ± 1.5 32.6 ± 2.3 25.5 ± 2.7

38.6 ± 3.7 24.4 ± 1.5 29.1 ± 1.6

46.9 ± 2.6 55.7 ± 2.1 45.7 ± 0.9

30.7 ± 1.4 26.1 ± 2.7 31.7 ± 1.8

30.6 ± 1.6 21.7 ± 2.4 28.7 ± 2.4

43.7 ± 2.9 48.4 ± 2.3 42.5 ± 1.7

23.4 ± 1.6 23.6 ± 1.6 20.8 ± 2.8

673

†

674

wt.% immobilized lipases were added to 2.19 g soybean oil containing 20 wt.% solvent (all dosage percentages were based on the oil mass), methanol were

675

respectively added to the system in three steps at 0 h, 4 h and 8 h by the molar ratio of methanol : oil molar ratio 4:1. The values are average and ± indicates the

676

standard deviation (SD).

BCL: Burkholderia cepacia lipase; RML: Rhizomucor miehei lipase; CRL: Candida rugosa lipase. The reactions were performed at 40 °C, 200 rpm for 12 h. 10

677 678

33


Energy & Fuels

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Table 6 The reusability of the three immobilized lipases

679

BCL (24 h)

†

†

Biodiesel yield (%)

Cycles

680

Page 34 of 41

a

RML (36 h)b

CRL (40 h)c

1

95.4 ± 1.9

96.3 ± 1.3

84.6 ± 1.5

2

93.8 ± 2.6

92.9 ± 2.0

80.2 ± 2.1

4

94.3 ± 1.5

89.2 ± 1.7

76.5 ± 2.4

6

93.1 ± 2.1

86.9 ± 1.4

69.9 ± 1.2

8

90.7 ± 1.8

84.1 ± 2.5

65.7 ± 1.6

10

89.4 ± 2.3

80.5 ± 1.6

58.3 ± 2.0


681

lipase. The reaction conditions of three immobilized lipases were the same as described

682

previously. a, b, c refer to the reaction time of the immobilized BCL, RML and CRL for each

683

batch is 24 h, 36 h and 40 h, respectively. Note: ± indicates the standard deviation (SD).

684 685 686

34


Page 35 of 41

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Energy & Fuels

687

Figure captions

688

Fig. 1 Illustration of bioimprinting affecting on the biocatalytic capability of three

689

lipases. The reaction conditions: immobilized lipases loading 0.1 g, stirring

690

speed 200 rpm, room temperature, incubated time 1 h, bioimprinting

691

molecular amount 0.075 mmol/mL).

692

Fig. 2 Effects of methanol concentration on biodiesel production. The reactions were

693

performed at 40°C, 200 rpm for 12 h, 10 wt.% immobilized lipases were

694

added to 2.19 g soybean oil containing 20 wt.% solvent, methanol were

695

respectively added in three steps at 0 h, 4 h and 8 h by the molar ratio of

696

methanol : oil between 2:1 and 6:1, the water content 5 wt.% for immobilized

697

BCL (Burkholderia cepacia lipase) in t-butanol system, 7.5 wt.% for CRL

698

(Candida rugosa lipase) in isooctane system and 10 wt.% for RML

699

(Rhizomucor miehei lipase) in n-octane system.

700

Fig. 3 Effects of temperature on biodiesel production. The reactions were performed

701

at the temperature from 30 to 55°C, 200 rpm for 12 h, 10 wt.% immobilized

702

lipases were added to 2.19 g soybean oil containing 20 wt.% solvent,

703

methanol were respectively added in three steps at 0 h, 4 h and 8 h by

704

methanol : oil molar ratio of 5 : 1 for immobilized RML (Rhizomucor miehei

705

lipase), 4 : 1 for BCL (Burkholderia cepacia lipase) and CRL (Candida

706

rugosa lipase), the water content 5 wt.% for immobilized BCL in t-butanol

707

system, 7.5 wt.% for CRL in isooctane system and 10 wt.% for RML in

708

n-octane system.

35


Energy & Fuels

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

709

Fig. 4 Effects of time interval of methanol addition on biodiesel production. The

710

reactions condition: 10 wt.% immobilized lipases, 2.19 g soybean oil

711

containing 20 wt.% solvent, 200 rpm, (a) methanol : oil molar ratio of 4 : 1,

712

the water content 5 wt.%, 35°C for immobilized BCL (Burkholderia cepacia

713

lipase) in t-butanol system; (b) methanol : oil molar ratio of 5 : 1, the water

714

content 10 wt.%, 50°C, for immobilized RML (Rhizomucor miehei lipase) in

715

n-octane system; (c) methanol : oil molar ratio of 4 : 1, the water content 7.5

716

wt.%, 40°C, for immobilized CRL (Candida rugosa lipase) in isooctane

717

system.

718

Fig. 5 Biodiesel production from soybean oil and waste vegetable oil. The reactions

719

condition: 10 wt.% immobilized lipases, 2.19 g soybean oil (molecular

720

weight: 877)，1.98 g waste vegetable oil (molecular weight: 792) containing

721

20 wt.% solvent, 200 rpm, (a) methanol : oil molar ratio of 4 : 1, the water

722

content 5 wt.%, 35°C for immobilized BCL (Burkholderia cepacia lipase) in

723

t-butanol system; (b) methanol : oil molar ratio of 5 : 1, the water content 10

724

wt.%, 50°C, for immobilized RML (Rhizomucor miehei lipase) in n-octane

725

system; (c) methanol : oil molar ratio of 4 : 1, the water content 7.5 wt.%,

726

40°C, for immobilized CRL (Candida rugosa lipase) in isooctane system.

727 728 729 730

36


Page 36 of 41

Page 37 of 41

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Energy & Fuels

731 732

Fig. 1

733 734

* Significant at P < 0.05

735 736 737 738 739 740 741 742 743 37


Energy & Fuels

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744 745

Fig. 2

746

747 748 749 750 751 752 753 754 755

38


Page 38 of 41

Page 39 of 41

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Energy & Fuels

756 757

Fig. 3

758

759 760 761 762 763 764 765 766

39


Energy & Fuels

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767 768

Fig. 4

769

770

40


Page 40 of 41

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Energy & Fuels

771 772

Fig. 5

773

774 775 776 777

41


Various Types of Lipases Immobilized on Dendrimer-Functionalized

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