HYDROLYSIS OF FISH OIL BY HYPERACTIVATED Rhizomucor 1

miehei IMMOBILIZED BY MULTIPOINT ANION EXCHANGE 2

3

4

Marco Filice2, Marzia Marciello2, Lorena Betancor3, Alfonso V. Carrascosa1, Jose M. 5

Guisan2* and Gloria Fernandez-Lorente 1* 6

7

1: Departament of Microbiology. Instituto de Fermentaciones Industriales. CSIC. 8

C/Juan de la Cierva 3, 2006 CSIC. Madrid. Spain. 9

2: Departament of Biocatalysis. Instituto de Catálisis. CSIC. Campus UAM. Cantoblanco. 10

28049 Madrid. Spain. 11

3: Madrid Institute for Advanced Studies. Campus UAM. Cantoblanco.. Pabellón C 12

Corresponding authors: 13

Prof José M. Guisán / Dr. Gloria Fernández Lorente 14

Instituto de Catálisis. CSIC. Campus UAM-Cantoblanco. 15

28049 Madrid. SPAIN 16

Tel: +34 91 585 48 09 17

E-mail: jmguisan@icp.csic.es gflorente@ifi.csic.es 18

ABSTRACT 19

Rhizomucor miehei lipase (RML) is greatly hyperactivated (around 20-25 fold towards 20

small substrates) in the presence of sucrose laurate. Hyperactivation seems to be an 21

intramolecular process because it is very similar for soluble enzymes and covalently 22

immobilized derivatives. The hyperactivated enzyme was immobilized (in the presence 23

of sucrose laurate) on CNBr activated agarose (very mild covalent immobilization 24

through the amino terminal residue), on glyoxyl agarose (intense multipoint covalent 25

immobilization through the region with the highest amount of Lys residues) and on 26

different anion exchangers (by multipoint anionic exchange through the region with 27

the highest density of negative charges). Covalent immobilization does not promote 28

the fixation of the hyperactivated enzyme, but immobilization on Sepharose Q retains 29

the hyper-activated enzyme, even in the absence of detergent. Hydrolysis of fish oils 30

by these hyperactivated enzyme derivatives was 7 fold faster than by covalently 31

immobilized derivatives and 3.5 fold faster than by enzyme hyperactivated on octyl-32

agarose. The open structure of hyperactivated lipase is fairly exposed to the medium 33

and no steric hindrance should interfere with the hydrolysis of large substrates. These 34

new hyperactivated derivatives seem to be more suitable for hydrolysis of oils by RML 35

immobilized inside porous supports. In addition, the hyperactivated derivatives are 36

fairly stable against heat and organic cosolvents. 37

38

Keywords: lipase hyperactivation, fixation of hyperactivated lipases, multipoint 39

interaction with activated supports 40

41

INTRODUCTION 42

Recently, the hydrolysis of fish oils by lipases immobilized on porous supports 43

has been reported (1). Immobilized lipases cannot undergo interfacial activation by 44

interaction with oil or solvent interfaces because oil drops are not able to penetrate 45

inside the porous structure of the catalyst. This lack of interfacial activation can be 46

compensated for by promoting the activation of lipase during its immobilization. 47

Hyperactivation of lipases by the adsorption of lipases on the surface of porous 48

hydrophobic supports already been reported (19 y 21). In this case, the 49

hyperactivation towards small substrates is usually higher than hyperactivation 50

towards large substrates (e.g., oils). The access of large substrates to the enzyme 51

active center can be hindered by the close contact between the open active center and 52

the support surface. 53

On the other hand, several lipases are also hyperactivated with the presence of low 54

concentrations of detergents, even when the active center is fairly well exposed to the 55

reaction medium. In homogeneous aqueous medium, lipases exist in two structural 56

forms, the closed one where a polypeptide chain (lid or flat) isolates the active center 57

from the medium, and the open form where this lid moves and the hydrophobic active 58

center is exposed to the medium (2, 3, 4). This equilibrium may be shifted towards 59

the open form in the presence of detergents that partially cover the hydrophobic 60

active center and make it much more hydrophilic (5). The presence of detergents 61

during the catalytic reaction could interfere with the adsorption of oils on the active 62

center of the immobilized lipase. If the open and active form of lipase promoted by 63

detergents could be fixed after detergent removal, the hyperactivated form of 64

immobilized lipase could be used for the hydrolysis of fish oils. Fixation of these open 65

forms of lipases can be developed on the basis of the distribution of the surface groups 66

of lipases and differences between the open and closed states. In this case, the open 67

structure of the lipase hyperactivated in the presence of an optimal concentration of 68

an optimal detergent could have a very different distribution of negative charges, 69

lysine groups, etc. than the distribution corresponding to the closed structure. This 70

open structure generated in the presence of detergents could be fixed via multipoint 71

physical or chemical immobilization. The immobilized fixed open structure of lipase 72

could then be used in the absence of detergents. 73

The release of omega 3 (e.g., eicosapentaenoic acid (EPA) and 74

docosahexaenoic acid (DHA)) from fish oil represents the first key step in the 75

preparation of highly enriched triglycerides (70 to 90% of content in one or both 76

polyunsaturated fatty acids, PUFAs). These triglycerides have been described as 77

excellent functional ingredients (6). Recently, there has been a dramatic surge in 78

interest among health professionals in the beneficial effects of omega-3 fatty acids 79

derived from fish oils, mainly consisting of EPA and DHA. DHA is required in high levels 80

in the brain and retina as a physiologically essential nutrient to provide for optimal 81

neuronal functioning (learning ability, mental development) and visual acuity in the 82

early stages of life (7). On the other hand, EPA is considered to have beneficial effects 83

in the prevention of cardiovascular diseases in adults (8,9). In this way, the 84

preparation of triglycerides enriched in both of the omega 3 acids (DHA and EPA) or in 85

only one of them could be very interesting. The first key step for the production of 86

triglycerides of omega 3 is the rapid and selective release of polyunsaturated fatty 87

acids from fish oils. 88

Rhizomucor miehei lipase exhibits interesting catalytic properties for the 89

hydrolysis of sardine oil. In addition to a good hydrolytic activity, RML exhibits a good 90

specificity towards EPA (eicosapentaenoic acid) compared with DHA (docosahexaenoic 91

acid), with an 80 % purity in EPA during the initial stages of release of both omega-3 92

acids. Improvement in the activity and/or selectivity could be quite interesting in order 93

to produce very pure omega-3 acids (1). 94

This work studies the hyperactivation of RML by different concentrations of 95

different detergents. Then, the fixation of the most active open form was attempted 96

via intense multipoint anion exchange or intense multipoint covalent attachment. The 97

activity and selectivity of the immobilized and hyperactivated RML were finally 98

evaluated for the hydrolysis of sardine oil. 99

100

101

102

103

104

105

106

107

MATERIALS AND METHODS 108

Materials 109

Sucrose laurate was a generous gift from the Mitsubishi-Kagaku Food 110

Corporation (cat. No. C-1216). Triton® X-100 (TX) was obtained from Sigma. 1,4-111

Dioxane and p-nitrophenyl butyrate (p-NPB) were purchased from Fluka. Octyl 112

sepharoseTM, Q-sepharose, CNBr activated Sepharose 4B (CNBr-agarose) and inert 113

agarose (Sepharose 4B) were purchased from GE Healthcare. The enzyme from 114

Rhizomucor miehei was kindly donated by Novozymes (Palatase 2000L). Sardine oil was 115

a kind gift from BTSA (Madrid, Spain). Docosahexaenoic acid (DHA), eicosapentaenoic 116

acid (EPA) and sodium borohydride (NaBH4) were obtained from Sigma Chemical Co. 117

(St. Louis, USA). Other reagents and solvents used were of analytical or HPLC grade. 118

MANAE y PEI. 119

120

Methods 121

Enzymatic activity assay 122

The activities of the soluble lipases and their immobilized preparations were 123

analyzed spectrophotometrically by measuring the increase in absorbance at 348 nm 124

( = 5150 M-1cm-1) produced by the release of p-nitrophenol (pNP) in the hydrolysis of 125

0.4 mM pNPB in 25 mM sodium phosphate buffer at pH 7 and 25°C. To initialize the 126

reaction, 0.05-0.2 ml of lipase solution (blank or supernatant) or suspension was added 127

to 2.5 ml of substrate solution. Enzymatic activity is given as μmol of pNP produced per 128

minute per mg of enzyme (IU) under the conditions described above. 129

130

131

132

Hyperactivation of RML performed by different detergents 133

To a solution of crude Palatase, (1 ml of crude extract - 1.6 mg of RML - diluted with 19 134

ml of 25 mM NaH2PO4 buffer solution at pH 7.0) various concentrations of different 135

detergents (Triton X-100, Sucrose laurate and CTAB) were added and the activity was 136

periodically checked by the enzymatic activity assay described above. 137

138

Immobilization of RML 139

Usually, the enzyme was incubated with the desired support, and a similar 140

suspension was prepared under identical conditions but using inert agarose. Samples 141

of the suspensions were withdrawn using cut pipette tips, and samples of the 142

supernatants were taken using a filter pipette tip. 143

To perform immobilization and stability studies, each preparation was loaded 144

with 2 mg/g of pure RML in order to prevent diffusion problems. In the enzymatic 145

hydrolysis, highly loaded derivatives (up to 10 mg/g) were prepared. 146

147

Immobilization of RML on octyl-Sepharose 148

A volume of 1.5 ml of Palatase commercial solution (6,5 mg total protein/ml, about 1,6 149

mg RML as determined by Bradford’s assay) (10) were mixed with 30 ml of 10 mM 150

sodium phosphate at pH 7.0 and 4ºC, and then 1 g of octyl-Sepharose previously 151

equilibrated with the immobilization buffer was added. The supernatant and 152

suspension activities were periodically checked by the method above described. After 153

immobilization, the enzyme derivative was recovered by filtration under vacuum. 154

These adsorbed lipases were used as biocatalysts or to purify RML. 155

156

Desorption of the RML from the octyl-Sepharose with sucrose laurate 157

To obtain a pure lipase, RML was desorbed from the RML-octyl-Sepharose; 1 g 158

of this immobilized preparation was suspended in 10 ml of 0.5% (v/v) sucrose laurate 159

in 25 mM NaH2PO4 buffer solution at pH 7.0 for 1 h. The progress of the desorption 160

was monitored by periodically checking the enzymatic activity of the supernatant and 161

suspension until they became equal. The final concentration of the purified lipase 162

solution was 0.2 mg lipase/ml. The enzymatic solution obtained was then used for 163

immobilization of the different supports, when a pure enzyme was required. 164

165

Immobilization of lipases on different ionic supports 166

Standard studies were performed as previously described. Then, 1 g of different 167

ionic exchanger supports (MANAE (11), Q-Sepharose or PEI(12)) equilibrated with the 168

immobilization buffer were added to the pure RML solution obtained as described 169

above (about 2 mg in 10 ml), and the pH was adjusted to 7. The mixture was then 170

stirred at 25ºC. To finalize the immobilization, the supernatant was removed by 171

filtration and the supported lipase was washed several times with distilled water. 172

173

174

Immobilization of lipase on a CNBr-activated support 175

These support and immobilization conditions were used as reference 176

conditions for lipase behavior. In general, immobilization of enzymes under these 177

conditions will occur by only a few bonds (very likely even just one bond), and these 178

immobilized preparations result in a good model of the enzyme properties in absence 179

of intermolecular phenomena (13-16). Lipases have been shown to exhibit a strong 180

tendency to form lipase-lipase aggregates, making it very complex to study isolated 181

lipase molecules in a soluble form (13-16). 182

Commercial CNBr activated Sepharose support was activated prior to use by its 183

suspension in an acidic aqueous solution (pH 2-3) for one hour. Then, the support was 184

dried by filtration under vacuum. Once activated, 1 g of CNBr-Sepharose was added to 185

15 ml of the purified lipase solution (0,2 mg/ml). The mixture was then stirred at 4ºC 186

and 250 rpm for 20 minutes. After that, the solution was removed by filtration, the 187

supported lipase was washed twice with 100 mM NaHCO3 at pH 8 and then re-188

suspended in 15 ml of 1 M ethanolamine at pH 8 for 90 minutes to block the unreacted 189

imido carbonate reactive groups (17). Subsequently, the reaction mixture was filtered 190

and washed with abundant distilled water. The immobilization yield was 60% with a 191

protein loading amount of about 2 mg/g support. Following immobilization, the 192

enzymatic activity assay described above was performed. 193

194

Immobilization of RML on glyoxyl-agarose 195

Glyoxyl agarose support (1 g) (18) was added to 13 ml of RML solution (0.2 mg of 196

enzyme/ml) purified as described above. Immobilization was carried out at pH 10.2 197

over 24 h at 25 ºC under very gentle stirring. To finalize the immobilization reaction, 198

solid NaBH4 was added to the suspension at 1 mg/ml and the suspension was gentle 199

stirred for 30 minutes. Borohydride reduces the Schiff’s bases formed between the 200

enzyme and the support and the unreacted aldehyde groups remaining in de support. 201

Subsequently, the suspension was filtered and the glyoxyl derivative was washed with 202

abundant distilled water. The percentage of immobilization was 60% with about 2 203

mg/g support. The immobilization was followed by the enzymatic assay described 204

above. 205

206

Thermal inactivation of different RML immobilized derivatives. 207

The different RML derivatives loaded at low levels (0.5 mg of enzyme/g of 208

support) were incubated in 25 mM sodium phosphate at pH 7.0 and 50ºC. Samples 209

were withdrawn periodically and their activities were measured using the pNPB assay. 210

The thermal stability of glyoxyl derivatives was studied with an enzymatic hydrolytic 211

assay modified by adding 0.3% of Triton X-100 in the cuvette. The experiments were 212

carried out in triplicate and error was never more than 5%. 213

214

Stability of different RML immobilized preparations in organic solvents 215

The different RML preparations were incubated in 25 mM sodium phosphate 216

at pH 7.0 and 50% of diglyme or isopropanol at 25 ºC. Samples were periodically 217

withdrawn and their activities were checked with the enzymatic activity assay 218

described above. The stability of glyoxyl derivatives was studied with an enzymatic 219

hydrolytic assay modified by adding 0.5% of sucrose laurate in the cuvette. The 220

experiments were carried out in triplicate and the error was never more than 5%. 221

222

Hydrolysis of Fish Oil by immobilized lipases 223

Hydrolysis of fish oil was performed in a water-organic two-phase system. The 224

procedure was as follows: 4.5 ml of cyclohexane, 5 ml of phosphate buffer (0.1 M) at 225

pH 6, and 0.5 ml of sardine oil were placed in a reactor and preincubated for 30 min. 226

The reaction was then initiated by adding 0.3 g of lipase derivative and shaking at 150 227

rpm. A Mettler Toledo DL50 Graphix titrator was used to maintain a constant pH value 228

during the reactions. The concentration of polyunsaturated free fatty acids was 229

determined at various times by HPLC assay. 230

231

Analysis of Polyunsaturated free fatty acids (PUFAS) by HPLC 232

After 10 minutes, 0.1 ml aliquots of the organic phase were withdrawn and dissolved 233

in 0.8 ml of acetonitrile. The unsaturated fatty acids produced were analyzed by RP-234

HPLC (Spectra Physic SP 100 coupled with an UV detector Spectra Physic SP 8450) 235

using a Kromasil C8 column (15 cm × 0.4 cm). Products were eluted at a flow rate of 236

1.0 ml/min using an acetonitrile-10 mM ammonium Tris buffer at pH 8 (70:30, v/v), 237

and UV detection was performed at 215 nm. The retention times for the unsaturated 238

fatty acids were: 9.4 min for EPA and 13.5 min for DHA. 239

240

Determination of Hydrolysis Conversion 241

The percent hydrolysis was computed as the amount of EPA and DHA (PUFAS) released 242

as a percentage of their original content in the oil, considering that the PUFAS content 243

in the fish oil was 30%. The percent of peak area in an experiment was assumed to 244

indicate the percent content of the corresponding compound. PUFAS productivity (%) 245

was calculated according to the following equation: 246

PUFAS (g/min)= PUFAS (%) X fish oil (g/ml), 247

In which fish oil content was the weight of the fish present in the substrate mixture. 248

249

RESULTS 250

251

1. Hydrolysis of a small substrate (pNPB) by soluble RML in the presence of 252

detergents. 253

The presence of several detergents during the hydrolytic assay exerts very different 254

effects on the catalytic activity of soluble RML (Figure 1). Some detergents (e.g., CTAB) 255

promote a strong inhibition even at very low concentrations (0.001%). The presence 256

of TRITON X-100 promotes a small hyperactivation at very low concentrations (0.01%) 257

but exerts inhibitory effects at higher concentrations (0.1% or higher). Sucrose laurate 258

exerts a remarkable hyperactivating effect: the enzyme is 10-fold more active in the 259

presence of 0.4% of this detergent than in the reference assay in the absence of 260

detergents. This hyperactivation of RML is similar to that obtained for the same 261

enzyme adsorbed on octyl agarose (8-10 fold more active than soluble RML) (19). 262

2. Long-term Incubation of soluble RML in the presence of detergents. 263

In order to study the kinetics of hyperactivation as well as to avoid possible inhibiting 264

effects of the presence of high concentrations of detergents in the enzymatic assay, 265

the hyperactivation of RML was also studied by incubating the soluble enzyme in the 266

presence of detergents outside the reaction cell. The catalytic activity of soluble RML 267

increased over 1 hour (figure 2), at which point the concentration of detergent in the 268

assay mixture was almost negligible (lower than 0.01%). The observed activities were 269

constant for 5-10 minutes. Hyperactivation was faster and more intense when using 270

high concentrations of detergent. RML incubated In the presence of 0.5 % sucrose 271

laurate for 30 minutes exhibits a 25-fold increase in activity. This hyperactivation was 272

much greater than that observed in the reaction cell. The enzyme was then 273

hyperactivated for longer incubation times and was assayed in the absence of 274

detergent. The hyperactivation was moderately slow, stable and very high. Similar 275

experiments were conducted with TRITON X-100, in which a 7-fold hyperactivation was 276

observed, also much greater than the hyperactivation previously observed when 277

detergent was added to the reaction cell. 278

279

3. Hyperactivation of RML immobilized on CNBr activated agarose. 280

The mild immobilization of enzyme on CNBr activated agarose conducted here (for 15 281

minutes at 4 ºC) strongly reduces any possibility of multipoint covalent attachment. In 282

fact, these immobilized derivatives of a number of enzymes have activity-stability 283

properties that are very similar to those of the corresponding pure and diluted soluble 284

enzymes in the absence of stirring (17). However, one-point covalently immobilized 285

derivatives fully dispersed on the internal support surface cannot undergo interactions 286

with hydrophobic interfaces (air, oil, solvents) and cannot undergo any kind of 287

intermolecular process (aggregation, proteolysis, etc.) (20). These RML derivatives 288

were hyperactivated similarly to soluble RML (Figure 3). These derivatives were 289

hyperactivated 20-fold in the presence of 0.5 % sucrose laurate. The subsequent 290

washing of the derivatives, done in order to remove the detergent, promoted a slow 291

and complete loss of hyperactivation. This slow and high level of hyperactivation 292

seems to be an intramolecular process that is completely reversible. 293

294

4. Sucrose laurate hyperactivation of RML adsorbed on octyl-agarose. 295

RML was selectively adsorbed and hyperactivated by adsorption on octyl-agarose (an 296

8-10 fold hyperactivation). The adsorbed enzyme could be desorbed away from the 297

support by washing the derivatives with 0.5 % sucrose laurate. The desorbed enzyme 298

was immediately hyperactivated by up to a factor of 20 (very similar to the slow 299

hyperactivation of the soluble enzyme). Hyperactivation by detergent is higher but 300

compatible with the hyperactivation on hydrophobic supports, but full hyperactivation 301

is much more rapid. 302

303

304

5. Immobilization of hyperactivated RML on different activated supports. 305

Hyperactivated soluble RML (purified on octyl-agarose and desorbed away with 0.5 % 306

sucrose laurate) was immobilized using 3 different immobilization protocols (TABLE 1). 307

In protocol a, mild immobilization was performed on CNBr-activated agarose (very 308

likely a one-point immobilization through the most reactive amino group, e.g., the 309

terminal amino group). The enzyme preserved its hyperactivation following 310

immobilization, but this hyperactivation was completely lost when the derivative was 311

washed and the detergent was removed at the end of immobilization. In protocol b, 312

immobilization was performed on highly activated glyoxyl agarose at pH 10 (multipoint 313

covalent attachment through the enzyme region with the highest amount of Lys 314

residues (18). The enzyme lost 50% of its hyperactivation during immobilization and 315

lost 100% after the washing away of derivatives and removal of the detergent at the 316

end of immobilization. In protocol c, the enzyme was physically adsorbed on 317

Sepharose Q (multipoint anion exchange via the enzyme region having the highest 318

density of net negative charge). The enzyme preserved more than 90% of its 319

hyperactivation, and this level of hyperactivation was maintained even after the 320

complete removal of the detergent and after incubation of the derivative for several 321

hours in the absence of detergent. This multipoint anionic exchange allows the 322

fixation, in the absence of detergent, of the hyperactivated form of the enzyme that 323

was previously activated in the presence of 0.5 % sucrose laurate. 324

6. Immobilization of hyperactivated RML on different anion exchangers. 325

A similar fixation of the hyperactivated structure of the enzyme was also attempted 326

using different anion exchangers: ionic exchangers containing a very high 327

concentration of ionized primary and secondary amino groups (MANAE-agarose) (11) 328

and very highly activated polymeric exchangers obtained by chemical modification of 329

aldehyde-agarose with polyethyleneimine (PEI-agarose) (12). In both cases, the 330

hyperactivation was partially maintained (at 50%) and the final results of 331

hyperactivation, evaluated in the absence of detergent, were similar to those obtained 332

by adsorption on hydrophobic octyl-agarose (Table 2). 333

7. Stability of derivatives of hyperactivated RML. 334

The three derivatives where the hyperactivated form of RML was fixed were very 335

stable under mild experimental conditions. For example, they maintained 100% of 336

their activity after incubation for 3 weeks at 25 ºC and pH 7.0. The stability of the 337

derivatives was different under more severe conditions. At 37 ºC, the most stable 338

derivative was RML-Sepharose Q (Figure 4), which preserved 80 % of its activity (20-339

fold enhanced compared with soluble enzyme) after a 24 hr incubation. On the 340

contrary, in the presence of cosolvents (e.g., 30 % of diglyme), RML-Sepharose Q was 341

the least stable. 342

343

8. Hydrolysis of fish oil by different derivatives of RML. 344

The hydrolysis of sardine oil was performed using RML-CNBr-Sepharose, RML-octyl 345

Sepharose and hyperactivated RML-Sepharose Q derivatives (TABLE 3). RML 346

hyperactivated on octyl-Sepharose was 2-fold more active than non-hyperactivated 347

RML immobilized on CNBr Sepharose. This increase was 9.5-fold for the hydrolysis of 348

small substrates (e.g., pNPB). The hyperactivated RML-Sepharose Q was 7-fold more 349

active for oil hydrolysis than non hyperactivated RML-CNBr-agarose and 3.5-fold more 350

active than RML hyperactivated on hydrophobic supports. In this case, the difference 351

was also lower than that obtained using small substrates (an 18-20-fold increase for 352

pNPB when using CNBr agarose derivatives). The EPA/DHA selectivity of these new 353

hyperactivated derivatives could make them attractive for industrial application, for 354

example, allowing the production of 3:1 EPA:DHA mixtures from sardine oil hydrolysis. 355

This hyperactivation of RML is especially important given that once it is immobilized 356

inside porous supports, RML cannot undergo interfacial activation towards drops of oil 357

or drops of solvents containing oil because these drops are unable to penetrate inside 358

the porous structure of the catalyst. 359

360

DISCUSSION 361

The 20-25-fold hyperactivation obtained for soluble or immobilized RML in the 362

presence of high concentrations of sucrose laurate is one of the highest 363

hyperactivations obtained for lipases via non-natural methods (different from 364

interfacial activation on drops of oil): TLL was hyperactivated 20-fold by adsorption on 365

hydrophobic supports (21), BTL was hyperactivated 2-fold in the presence of 366

detergents (22), BTL was hyperactivated 3-fold in the presence of cosolvents (22), and 367

BTL was hyperactivated 3-fold via site-directed chemical modification (23). 368

Furthermore, BTL was hyperactivated 18-fold by the additive effects of chemical 369

modification, detergents and cosolvents (22), etc. However, the experiments reported 370

in this paper represent the first trial in which hyperactivation by detergents was 371

induced outside of the reaction cell and was studied over a long period of time. Similar 372

experiments could likely give similar interesting results with other lipases. However, at 373

the moment, an additional hyperactivation during the desorption by detergents of 374

lipases adsorbed and hyperactivated on hydrophobic supports had never been 375

observed. Hyperactivations on both hydrophobic supports and in the presence of 376

detergents seem to be related to the opening of the lipase active center. This large 377

hydrophobic pocket of the open form of lipases may be stabilized by adsorption on 378

hydrophobic supports and by interaction with the hydrophobic moiety of the 379

detergent. However, in the case of RML, the detergent seems to be more effective 380

than hydrophobic surfaces, and the new active structure of RML could be slightly 381

different from the structure formed on hydrophobic interfaces. 382

383

The fixation of the hyperactivated form of RML by multipoint anion exchange of 384

Sepharose Q is quite interesting. The immobilized enzyme remains hyperactivated 385

after removing the hyperactivating agent (the sucrose laurate). A similar fixation is not 386

possible by very mild (possibly one-point) covalent immobilization on CNBr-activated 387

agarose. Furthermore, the hyperactivated form of the enzyme is not fixed by covalent 388

immobilization on glyoxyl-agarose (multipoint attachment through the enzyme region 389

with the highest amount of Lys). This region seems to be unaltered between the 390

closes and hyperactivated forms of the enzyme. On the contrary, the region with the 391

highest density of net negative charge (involved in the adsorption on Sepharose Q and 392

other anionic exchangers) seems to be dramatically changed when the closed form of 393

the lipase in transformed into the hyperactivated open form. Logically, this region 394

with the highest negative charge is different from the enzyme active center, which is in 395

a large hydrophobic pocket. One important finding from this work is that a complex 396

change in the active center may be associated with a dramatic change in other regions 397

of the enzyme, and the fixation of the associated change preserves the first and most 398

important change in the active center. This type of long-distance stabilization of 399

conformational changes in active centers of enzymes (namely lipases) has not been 400

previously reported in the literature. 401

These results also showed a 7-fold improvement of the hydrolytic activity of 402

RML towards fish oil with a significant selectivity towards EPA versus DHA. This allows 403

for the rapid production of mixtures of omega-3 acids fairly enriched in EPA via 404

enzymatic methods. This system would allow for the exploitation of all of the 405

advantages of lipases immobilized on porous supports: re-use of the biocatalyst, 406

resistance to strongly stirred reactors because of the lack of interaction of hydrophobic 407

interfaces with air, solvents, oils, etc., and resistance to intermolecular phenomena 408

such as aggregation and proteolysis. Additionally, the main drawback of these reaction 409

systems, the absence of interfacial activation on oil drops, is compensated for by the 410

hyperactivation of immobilized derivatives. 411

The hyperactivated RML derivatives are very stable under the mild 412

experimental conditions (e.g., pH 7-0 and 5 ºC) required to hydrolyze fish oil in the 413

absence of undesirable oxidations of omega 3. Therefore, these hyperactivated 414

derivatives could be used for fish oil hydrolysis for a number of reaction cycles. Under 415

more harsh experimental conditions, the derivatives are less stable. The presence of a 416

high concentration of quaternary amino groups on the supports may fix distorted 417

enzyme structures formed under distorting conditions (high temperatures, cosolvents, 418

etc.). The distorted enzyme could interact more with the highly activated support. 419

420

CONCLUSIONS 421

RML was 20-fold hyperactivated (towards small substrates) by incubation in the 422

presence of high concentrations (0.4%) of sucrose laurate. Hyperactivation was 423

intermolecular, slow, moderately stable and completely reversible. Hyperactivation is 424

likely related to the stabilization of the large hydrophobic pocket exposed to the 425

medium in the hyperactivated form of RML. 426

The hyperactivated form of RML was successfully fixed by multipoint adsorption on 427

anionic exchangers and is stabilized even after removing hyperactivating agents (the 428

detergent). The fixed region is not the hydrophobic active center. A clear correlation 429

between the hydrophobic active center and the region with the highest density in net 430

negative charge may be responsible for the stabilization of the hyperactivated form of 431

RML. 432

RML immobilized inside porous supports becomes very active, very stable and fairly 433

selective for the mild hydrolysis of fish oils (25 ºC and pH 7.0) in the absence of 434

undesirable phenomena such as interaction with interfaces and aggregation 435

phenomena. Now, interfacial activation of soluble lipases on drops of oil is not 436

necessary since a similar activation was achieved via a careful non-natural 437

hyperactivation of the immobilized enzyme. 438

439

Acnowledgements 440

This work has been sponsored by the Spanish Ministry of Science and 441

Innovation (project AGL-2009-07625) and the Comunidad Autónoma de Madrid 442

(Project S0505/PPQ/03449). We gratefully recognize to Spanish Ministry of Science 443

and Innovation for “Ramón y Cajal” contract to Dr. Fernandez-Lorente.. 444

445

446

FIGURE LEGENDS 447

Figure 1. Hydrolysis of p-nitrophenyl butyrate in the presence of different 448

concentrations of detergents by soluble RML. Experiments were carried out as 449

described in the Methods section. The activity of the enzyme assayed in the absence 450

of detergents is assigned as 100%. Squares: Hexadecyltrimethylammonium bromide 451

(CTAB); triangles: TRITON X-100; circles: sucrose laurate. 452

453

Figure 2. Time-courses of the hyperactivation of soluble RML incubated in the 454

presence of different concentrations of sucrose laurate. Experiments were carried 455

out as described in the Methods section. The enzyme was incubated in the presence of 456

detergents and aliquots of the incubation mixtures were assayed in the presence of 457

minimal traces of detergent (less than 0.01%). The activity of the enzyme incubated in 458

the absence of detergent was assigned as 100%. Triangles: RML incubated in 0.1 % 459

sucrose laurate; squares: RML incubated in 0.2 % sucrose laurate; circles: RML 460

incubated in 0.5 % sucrose laurate. 461

462

Figure 3. Reversible hyperactivation of immobilized RML incubated in the presence 463

of 0.5% sucrose laurate. RML was very mildly immobilized on CNBr-activated agarose 464

gels. Column 1: activity of immobilized RML before incubation in the presence of 465

detergent; Column 2: activity of immobilized RML after incubation for 1 hour in the 466

presence of 0.5 % sucrose laurate; Column 3: activity of immobilized RML after a final 467

buffer wash that was performed in order to remove the detergent. 468

Figure 4. Time-courses of the thermal inactivation of different RML derivatives 469

(hyperactivated enzyme adsorbed on different anion exchangers). Derivatives were 470

incubated at 37 ºC and pH 7.0. Squares: hyperactivated RML adsorbed on MANAE-471

agarose; triangles: hyperactivated RML adsorbed on PEI-agarose; circles: 472

hyperactivated RML adsorbed on Sepharose Q. 473

474

Figure 5. Time-course of cosolvent inactivation of different RML derivatives 475

(hyperactivated enzyme adsorbed on different anion exchangers Derivatives were 476

incubated in the presence of 30% of diglyme at pH 7.0 and 25 ºC. Squares: 477

hyperactivated RML adsorbed on MANA-agarose; triangles: hyperactivated RML 478

adsorbed on PEI-agarose; circles: hyperactivated RML adsorbed on Sepharose Q. 479

480

481

REFERENCES 482

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Submitted. 485

486

2. Brady L, Brzozowski AM, Derewenda ZS, Dodson E, Dodson G, Tolley S, Turkenburg, 487

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490

3. Brzozowski, AM, Derewenda, U., Derewenda, Z.S., Dodson, G.G., Lawson, 491

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activation in lipases from the structure of a fungal lipase-inhibitor 493

complex. Nature 351: 491-494. 494

495

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interface: The anatomy of a conformational change in a triglyceride lipase. 497

Biochemistry 31: 1532-1541. 498

499

5. Fernández-Lorente G, Palomo JM, Cabrera Z, Fernandez-Lafuente R, Guisán JM 500

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504

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12. Guisan JM, Sabuquillo P, Fernandez-Lafuente R, Fernandez-Lorente G, Mateo C, 528

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13. Wilson L, Palomo - -533

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the lipase properties. Enzyme Microb Technol 36: 447-454. 543

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578

579

580

581

582

Table 1.- Immobilization of hiperactivated RML on different activated 583

supports. Experiments are described in Methods. Relative activities are 584

reported using 100% as the activity of the soluble enzyme before 585

hyperactivation and immobilization. 586

587

588

589

590

591

592

593

594

595

596

Steps Relative activity (%)

Soluble enzyme 100

Adsoption to Octyl-sepharose 950

Desortion with 0.5% lauryl

sucrose 2000

CNBr-

Sepharose

Q-

Sepharose

Glyoxyl-

agarose

Immobilization on different

supports 1800 1880 1200

Immobilized derivatives after

washing and removing the

detergent

100 1800 100

597

598

Table 2.- Immobilization of hiperactivated RML on different anion-599

exchange supports. Experiments are described in Methods. Relative 600

activities are reported using 100% as the activity of the soluble enzyme 601

before hyperactivation and immobilization. 602

603

604

605

606

607

608

Steps Relative activity (%)

Soluble enzyme 100

Adsoption to Octyl-sepharose 950

Desortion with 0.5% lauryl sucrose 2000

Sepharose-

Q MANAE PEI

Adsorption on Anion Exchange

Resins 1880 900 800

Immobilized derivatives after

washing and removing the

detergent

1800 900 800

Table 3.- Hydrolysis of sardine oil catalyzed by different derivatives of 609

RML. Experiments were carried out as described in Methods. 610

611

Activitya represents the rate of hydrolysis of a small synthetic substrate (p-612

nitrophenyl butyrate). Activityb represents the rate of hydrolysis of sardine 613

oil. In both cases, 100% is the activity of derivative obtained by very mildly 614

immobilization on CNBr-activated Sepharose. EPA/DHA is the ration 615

between both omega 3 released in the first stages of hydrolysis of sardine 616

oil (up to a 10% of conversion). ω-3/oleic plus palmitic acid is the ration 617

between both omega3 and saturated and monounsaturated acids 618

obtained in the first stages of hydrolysis of sardine (up to a 10% of 619

conversion) 620

621

622

623

624

625

626

627

628

629

Derivative Activitya (%) Activity b 10-3 EPA/DHA

-3/Oleic

plus palimitic

acids

CNBr- 100 100 2.6 1.4

OCTIL 950 200 3.45 0.89

Q 2000 700 4.2 1.06

0

200

400

600

800

1000

1200

0 0,1 0,2 0,3 0,4 0,5

Re

lati

veac

tivi

ty(%

)

Detergent (%)

Figure 1

630 631

632

633

634

635

636

637

638

639

640

641

642

643

644

645

646

647

Figure 2

648 649

650

651

652

653

654

655

656

657

658

659

660

661

0

200

400

600

800

1000

1200

1 2 3

Re

lati

ve a

ctiv

ity

(%)

662

Figure 3 663

664

665

666

667

668

669

670

671

Figure 4

672 673

674

675

676

677

678

679

680

681

682

683

684

685

686

687

688

689

Figure 5

690 691