Improving Catalytic Performance of Candida rugosa Lipase by

Article pubs.acs.org/IECR

Improving Catalytic Performance of Candida rugosa Lipase by Chemical Modification with Polyethylene Glycol Functional Ionic Liquids Xiujuan Li, Chuan Zhang, Shuang Li, He Huang,* and Yi Hu* State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 5 Xinmofan Road, Nanjing 210009, China

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on September 3, 2015 | http://pubs.acs.org Publication Date (Web): August 14, 2015 | doi: 10.1021/acs.iecr.5b01881

S Supporting Information *

ABSTRACT: Candida rugosa lipase was chemically modified with various polyethylene glycol functional ionic liquids to increase its catalytic performance. All tested polyethylene glycol functional ionic liquids improved lipase catalytic activity, thermostability, organic solvent tolerance, and adaptability to temperature and pH changes in olive oil hydrolysis reaction. In particular, lipase modified by [HOOCEPEG350Im][H2PO4] increased the catalytic activity by 1.7-fold, thermostability by 5.0-fold at 50 °C for 2.5 h, organic solvent tolerance by 13.3-fold in 60% dimethyl sulfoxide and 3.4-fold in 5% methanol compared with the native lipase. Investigations by ultraviolet, fluorescence, and circular dichroism spectroscopy revealed microenvironment changes, the increase in β-sheet and the decrease in α-helix content of secondary structures in the modified lipases. These changes were correlated with enzymatic properties alteration, including improved activity and stability of modified lipases. Compared with the results of previous reports, the novel modification with polyethylene glycol functional ionic liquids was more effective in improving the catalytic performance of lipase.

1. INTRODUCTION Nowadays, lipases are widely applied as biocatalysts to pharmaceuticals, cosmetic, textiles, and food industries.1,2 However, one of the main drawbacks for industrial application of lipases is their instability at high temperature and in strong polar organic solvents.3 The modification of protein surface characteristics by chemical binding with modifiers can improve biocatalyst performance.4 However, the activity and stability of lipase usually cannot be increased simultaneously with more significance when using routine modifiers. For example, Barbosa reported that the chemical modification of lipase B from Candida antarctica with 2,4,6-trinitrobenzensulfonic acid showed a 20% decrease on the activity and 60% decrease on the enzyme stability at 60 °C and pH 7.0.5 Forde reported that the chemical modification of lipase B from Candida antarctica with ethylene glycol bis(succinimidyl succinate) induced a slight increase in the hydrolytic activity, while the thermostability had a 7-fold increase compared with the native enzyme at 70 °C.6 Room temperature ionic liquids (ILs), as “green and designed” solvents, are attracting more and more attentions in the field of biocatalysis.7−9 In our previous studies, functional ILs, as new modifiers, were designed to improve the lipases performance. The results showed that different cations and anions of ILs had an important influence on the catalytic performance of lipase, and the influences of different modifiers on the various lipases were also different. For example, the enhancements of catalytic activity, stereoselectivity, thermostability and organic solvents tolerance were obtained for porcine pancreatic lipase modified by ionic liquid with a more kosmotropic cation and chaotropic anion, while the catalytic activities of most modified CRLs were slightly decreased and the enhancements of enantioselectivity were not obvious.10,11 © 2015 American Chemical Society

The effects of polyethylene glycol (PEG) on the enzyme performance has aroused wide interest because of the excellent water-solubility, nontoxicity, high hydration and flexibility of PEG. Chemical modification of proteins with PEG can enhance their performance and remove the drawback of unmodified enzymes to some extent. Li and Zhang reported that the attachment of PEG to the enzymes surface was considered to increase its organic solvents tolerance and then improved the enzymatic properties.12,13 Na and Hu also reported that the temperature stability and solubility were enhanced by the attachment of PEG to the proteins.14,15 Herein, we aim to introduce a PEG functional group in the imidazolium cation according to the designability of ILs, and expect that thus ILs could work together with a PEG functional group to greatly improve all the catalytic performance of lipase simultaneously with more significance compared with the previous reported chemical modification methods. In this study, CRL, a widely studied lipase, was modified by a series of PEG functional ILs composed of different cations and anions (Scheme 1). The catalytic activity, thermostability, organic solvent tolerance, and adaptability to temperature and pH changes of lipase were studied in an olive oil hydrolysis reaction. Ultraviolet, fluorescence, and circular dichroism (CD) spectroscopy were studied to correlate the structural changes and enzymatic properties alteration. Received: Revised: Accepted: Published: 8072

May 24, 2015 July 13, 2015 August 4, 2015 August 14, 2015 DOI: 10.1021/acs.iecr.5b01881 Ind. Eng. Chem. Res. 2015, 54, 8072−8079

Article

Industrial & Engineering Chemistry Research

banceB mean the absorbance of modified and native enzyme, respectively. The modified lipases were denoted as [C2OHmim]-CRL[H2PO4], [C2OHmim]-CRL-[Cl], [C2OHmim]-CRL-[BF4], [HOOCEPEG350Im]-CRL-[H2PO4], [HOOCEPEG350Im]CRL-[Cl], [HOOCEPEG350Im]-CRL-[BF4], [HOOCEPEG750Im]-CRL-[Cl], and [HOOCMMIm]-CRL-[Cl]. 2.4. Enzyme Activity. The enzymatic activity of CRL was measured by the method of olive oil hydrolysis.18 The olive oil (50 mL) was mixed with gum arabic solution (7%, w/v) to form the substrate solution. The substrate solution (5 mL) and phosphate buffer (5 mL, 0.025 M, pH 7.0) were put into the conical flask and then incubated at 40 °C for 5 min. The diluted CRL solution (100 μL, 100 μg/mL) was added to the mixture to start the hydrolysis reaction, and the reaction continued for 10 min under a stirring speed of 120 rpm/min. Finally, 10 mL acetone/ethanol (1/1, v/v) was added to end the hydrolysis reaction. The hydrolytic activity of the CRL was measured by titration with 0.05 M sodium hydroxide solution. One unit of CRL activity was defined as the amount of lipase used to produce 1 μmol of acid per minute. 2.5. Effect of Temperature on Enzyme Activity. The effect of temperature on the activity of native and modified CRLs was measured at pH 7.0 and temperatures ranging from 20 to 65 °C, at increasing intervals of 5 °C each. All data in the figure were an average of the triplicate of experiments. The relative activities of native and modified lipases were measured by defining the maximum activities as 100%. 2.6. Effect of pH on Enzyme Activity. The effect of pH (phosphate buffer, 6.0−8.0) on the activity of the CRL was measured at 40 °C through the described activity assay procedure. The maximum activities of the native and modified lipases were defined as 100%. 2.7. Enzyme Thermostability. The native and modified CRL solutions were incubated at 50 °C and pH 7.0 for 0.5, 1, 1.5, 2, 2.5, and 3 h. Then a certain amount of each solution was withdrawn at different times to measure the hydrolytic activity by the activity assay described above. The initial activity was defined as 100%. 2.8. The Organic Solvents Tolerance. The organic solvents used in the work were dimethyl sulfoxide (0−60%) and methanol (0−20%). The increasing percent volumes of organic solvent were added in phosphate buffer (0.025 M, pH 7.0). Then a certain amount of each solution was withdrawn at different times to measure the hydrolytic activity by the activity assay described above. The initial activity was defined as 100%. 2.9. Ultraviolet Spectroscopy. Ultraviolet spectra measurements of native and modified CRLs (25 μg/mL) were performed on aPerkinElmer-Lambda 25 instrument from 200 to 600 nm at room temperature. Distilled water was served as blank control group in the course of the experiment. Three spectra were accumulated and averaged for each sample. 2.10. Fluorescence Spectroscopy. Fluorescence spectra measurements were performed on a spectrofluorometer (PerkinElmer LS55, USA). The fluorescent emission spectra (300−400 nm) of native and modified CRLs (25 μg/mL) were collected with the excited wavelength of 270 nm. The excitation and emission slit widths were 5 nm.Three spectra were accumulated and averaged for each sample. 2.11. CD Spectroscopy. The CD spectra (190−260 nm) measurements were performed on a JASCO-J810 spectropolarimeter (Jasco Co., Japan) at room temperature that was continuously purged with N2. The measurements were carried


Scheme 1. Structures of Ionic Liquids Used for Modification on CRL

2. EXPERIMENTAL SECTION 2.1. Materials. 2,4,6-Trinitrobenzenesulfonic acid solution (5% w/v in H2O) and CRL (Type VII) were purchased from Sigma-Aldrich, China, Inc. A bisuccinic acid protein assay reagent kit was purchased from Pierce Chemical Co. (Rockford, IL, USA). All ionic liquids (99%, HPLC) used in this work were customized from Shanghai Cheng Jie Chemical Co. Ltd. and characterized by NMR (shown in Supporting Information). Carbonyldiimidazole (97%) was purchased from the Aladdin Chemistry Co. Ltd. All reagents used in our work are of analytical grade or higher. 2.2. Enzyme Preparation. The CRL powder (5 g) was solubilized in 50 mL of the distilled water to form the CRL solution which was then centrifuged at 4 °C for 30 min to collect the supernatant. Then, the excess salts in the supernatant were removed by dialysis with a 10 kDa dialysis membrane. Sodium dodecyl sulfate−polyacrylamide gel electrophoresis was used to test the purity of the enzyme. 2.3. Chemical Modification. IL (1 mmol) and carbonyldiimidazole (1 mmol) were solubilized respectively in anhydrous dimethyl sulfoxide (1 mL). They were mixed together and stirred for 4 h at room temperature to make the ILs activated. The activated ionic liquid (260 μL) was added into the CRL solution (15 mL, 5 mg/mL) with magnetic stirring at 4 °C for 24 h. The unreacted modifier molecules in the modified CRL solution were removed by dialysis. The 10 kDa membrane and deionized water were used in the process of dialysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to test the purity of the lipases (shown in Supporting Information). After modification, the purified lipases could be used directly or stored at 4 °C. In the measurements of protein concentrations, the bicinchoninic acid method was used.16 The trinitrobenzenesulfonic acid assay procedure was used to study the modification degree.17 CRL solution (0.5 mL, 100 μg/mL) and 0.01% TNBS (0.5 mL) were first incubated in 0.25 mL of phosphate buffer (pH 8.2, 0.025 M) at 37 °C for 2 h. Sodium dodecyl sulfate solution (0.5 mL, 10% w/v) and hydrochloric acid solution (0.25 mL, 1 M) were added to end the reaction, and then the absorbance (335 nm) was measured by the UV-1200 instrument. The degree of modification was calculated by the following equation: DM % = 1 − absorbanceA)/absorbanceB. The absorbanceA and absor8073

DOI: 10.1021/acs.iecr.5b01881 Ind. Eng. Chem. Res. 2015, 54, 8072−8079

Article

Industrial & Engineering Chemistry Research out for the diluted enzyme solution (25 μg/mL) using a circular cell (1.0 cm). The Jwsse32 software was used to calculate the secondary structure percentage of the native and modified enzymes.19

The same went for the modification of Interferon-α with MPEG 2000 and 5000, which was studied by Park.25 Therefore, it was not surprising that a higher modification degree of CRL was obtained when the cation of [C2OHmim] with lower steric hindrance was used. 3.2. Catalytic Activity. Table 1 showed that all modified CRLs in aqueous solution had a relative higher level of catalytic activity compared with native CRL. The same went for the CRLs modified with PEG functional ILs compared with traditional PEG modified CRLs. For example, the relative activities of MPEG350-CRL and MPEG750-CRL were much lower than most CRLs modified with PEG functional ILs. Moreover, the relative activities of ILs with a more kosmotropic anion and lower polymerization degree of PEG were conductive to enhance the catalytic activities of the lipases. Generally, chemical modification of CRL probably cause a decreased hydrolytic activity. Sanchez-Montero reported that CRL modified by polyethylene glycol caused more than 80% decrease of catalytic activity.26 Also, Park showed that the copolymer of polyethylene and maleic acid anhydride modification onto CRL lost nearly 20% catalytic activity under suitable conditions.27 By contrast, the modified CRLs in our study maintained a relative higher level of catalytic activity (Table 1). For the same cation, there was a strong correlation between the kosmotropic of anion and the activity, which was induced by the specific ion effect.28 We deduced that the higher modification and catalytic activity were obtained when more cations were grafted onto the surface of the enzyme by the facilitation of a more kosmotropic anion. And for the same anion, CRL chemically modified with all PEG functional ILs showed a higher catalytic activity compared with the lipase without the modification of PEG chain (e.g., [HOOCMMIm][Cl]), which was well studied in our previous work.10 Hydrophilic PEG derivates might keep some essential water molecules in the surface of an enzyme to a certain extent, which is beneficial to maintain the catalytically active conformation.29 Also, the lower molecular weight as compared to a higher molecular weight of PEG benefited the improvement of hydrolysis activity of CRL. The PEG might cause obstacles around the protein domain near the active site. Therefore, the enzyme probably reacted with difficultly with the substrate, decreasing the hydrolytic activity. Moreover, as the molecular weight increased, the chain of PEG molecule began to fold on itself, making it difficult to interact with enzymes.30 As a consequence, the lipase activity decreased when the larger molecular weights of PEG chains were used, as was also confirmed by the data shown in Table 1. 3.3. Effect of Temperature on Lipase Activity. The effects of temperature on CRLs hydrolysis reaction were studied within the range of 25−65 °C. As shown in Figure 1, the most suitable temperature was 40 °C for the native CRL and 45 °C for all the modified CRLs. Beyond the most suitable temperature, the activity of the native CRL declined rapidly, while the modified CRLs declined slowly. For example, the almost-modified CRL still maintained about 80% of its maximum activity at 50 °C. CRL modified with PEG functional ILs showed a better stability toward the high temperature compared with [HOOCMMIm][Cl], which was clearly shown in Figure 1-B. The results indicated that the modification of CRL with PEG functional ILs enhanced the adaptability to temperature.

3. RESULTS AND DISCUSSION 3.1. Modification Degree. As shown in Table 1, the modification degrees of CRLs are ordered as follows: Table 1. Modification Degrees and Catalytic Activities of Native and Modified CRLsa


sample CRL [C2OHmim]-CRL[H2PO4] [C2OHmim]-CRL-[Cl] [C2OHmim]-CRL[BF4] [HOOCEPEG350Im]CRL-[H2PO4] [HOOCEPEG350Im]CRL-[Cl] [HOOCEPEG350Im]CRL-[BF4] [HOOCEPEG750Im]CRL-[Cl] [HOOCMMIm]-CRL[Cl] MPEG350-CRL MPEG750-CRL

modification degree (%)

specific activity ( U/mg protein)

relative activity (%)b

35.6

784.4 1319.5

100 168.2

32.7 28.5

1203.2 1195.5

153.4 152.4

33.7

1331.7

169.8

31.6

1132.8

144.4

27.4

873.4

111.4

25.0

978.0

124.7

23.1

802.1

102.3

31.3 29.6

882.5 714.4

112.5 91.1

a

All the data in the table were the average of the triplicate of experiments. bRelative activity of unmodified CRL (784.4 U/mg protein) was defined as 100%.

[C2OHmim]-CRL-[H2PO4] > [HOOCEPEG350Im]-CRL[H2PO4] > [C2OHmim]-CRL-[Cl] > [HOOCEPEG350Im]CRL-[Cl] > [C2OHmim]-CRL-[BF4] > [HOOCEPEG350Im]CRL-[BF4] > [HOOCEPEG750Im]-CRL-[Cl] > [HOOCMMIm]-CRL-[Cl]. The kosmotropic ion could interact strongly with water molecules because of its high charge density, while the chaotropic ion is on the contrary.20,21 Generally, the kosmotropicity of the anions is following the order H2PO4− > Cl− > BF4−. Considering that the enzyme molecules are surrounded by a microaqueous phase, the functional ILs should access this aqueous layer in order to interact with the enzyme directly. The more kosmotropic anion was considered to have stronger interactions with water molecules, thus breaking the hydrated shell of the enzyme and allowing the activated cation to graft easily onto the enzyme.22,23 Therefore, for the same cation, a more kosmotropic anion has the advantage to enhance the modification degree. And for the same anion, the molecular weight of the PEG chains had a great influence on the modification degree, which was clearly shown in Table 1. The modification degree of the PEG functional IL was about 31.6% for [HOOCEPEG350Im]-CRL-[Cl], while only 25.0% for [HOOCEPEG750Im]-CRL-[Cl] was reached. Doumèche reported that the cation size and the alkyl chain flexibility of the functional ILs probably induced lower steric hindrance, which led to the higher modification degree to some extent.24 The chemical modification of lipase with high molecular weight PEG were tended to be limited compared with the low one. 8074


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Figure 1. Effect of temperature on the activity of CRLs. Enzyme activity was determined at pH 7.0 with the temperature ranging from 25 to 65 °C for 10 min.

Figure 2. Effect of pH on the activity of CRLs. Enzyme activity was determined at 40 °C within the pH ranging from 6.0 to 8.0 for 10 min.

3.4. Effect of pH on Lipase Activity. The relative activities of native and modified CRLs at different pH (6.0− 8.0) were shown in Figure 2, and the maximum activities of the native and modified CRLs were defined as 100%. The most suitable pH was 7.0 for all CRLs. As expected, the relative activities of all the lipases increased from pH 6.0 to 7.0, and then decreased rapidly, especially for the native CRL and [HOOCMMIm]-CRL-[Cl]. As shown in Figure 2, the lipases modified by imidazolium-based cations with PEG chains showed better pH adaptability, which might be attributed to the special structures of the positively charged imidazole ring and PEG functional group.31 3.5. Thermostability Assay. The thermostability of native and modified CRLs was studied by incubating them at 50 °C. As presented in Figure 3, the modified CRLs showed a better stability compared with the native CRL. Moreover, the CRLs modified with PEG functional ILs had a better thermostability compared with PEG-modified CRLs. For the same cation of modifiers, the thermostability of the modified CRL with a more kosmotropic anion had a greater improvement, which might be attributed to the higher modification degree. Hernaiz et al.32 also reported similar results: the thermal stability increased with the increase of modification degree. CRLs modified with imidazolium-based cations, when connected with PEG chains, showed a better thermostability than that modified with ILs without PEG functional group. For example, when the lipases were incubated at 50 °C for approximately 1.5 h, the relative activities of [HOOCEPEG 350 Im]-CRL-[H 2 PO 4 ], [HOOCEPEG 350 Im]-CRL-[Cl], [HOOCEPEG750Im]-CRL-[Cl], [HOOCEPEG350Im]-CRL[BF4], [HOOCMMIm]-CRL-[Cl] and the native CRL were 84.4%, 80.0%, 58.9%, 54.8%, 50.0%, 30.0%, respectively. Castellanos showed that PEG modification on alpha-chymo-

trypsin was an appropriate way to increase the heat resistance of the enzyme.33 Some researchers reported that PEG-bound molecules influenced the heat resistance of the enzyme when it solvated, and then reduced the unfolding rate of enzyme by limiting the mobility of the molecule.34 Besides, the hydrophobic, electrostatic properties and the ratio of positive-tonegative charged sites in an enzyme were deemed to have a great influence on the heat resistance of the enzymes.35,36 Likewise esterases from Bacillus stearothermophilus showed improved thermostability in the presence of short chain ionic liquids, which was due to electrostatic interaction between enzyme and ionic liquids leading to a rigid enzyme structure.37 Therefore, the improved thermostability of modified CRLs in our study could be attributed to the synergetic effect of imidazole ionic liquids and the PEG functional group. 3.6. Catalytic Activity in Strong Polar Organic Solvents. The variations of strong polar organic solvent tolerance for CRLs were shown in Figures 4 and 5. Generally, CRLs modified with PEG functional ILs had a better tolerance to dimethyl sulfoxide and methanol compared with the native lipase and PEG-modified CRLs. For the same cation, as the thermostability assay, the CRL modified with a more kosmotropic anion obtained a better stability in dimethyl sulfoxide and methanol compared with others. Moreover, the influence of methanol on the activities of CRLs was greater than that of dimethyl sulfoxide under the same conditions. For example, in 60% dimethyl sulfoxide the relative activity of [HOOCEPEG350Im]-CRL-[H2PO4] was 13.0-fold higher than the native CRL, while in 5% methanol, it was 3.4-fold. It was reported that the strong hydrogen bonds could induce deactivation of the enzyme, and the strong hydrogen bonds could be formed when the methanol (protic solvent) was 8075


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Figure 4. Effect of dimethyl sulfoxide (0−60%) on the activity of CRLs. Enzyme activity was determined at 40 °C and pH 7.0.

Figure 3. Thermostability of CRLs. Enzyme activity was determined at 40 °C and pH 7.0 after incubation at 50 °C for 0.5, 1, 2, and 3 h.

activity and stability showed a higher absorption intensity. As a consequence, [HOOCMMIm]-CRL-[Cl] with the relative poor modification effect showed the lowest absorption intensity. The results indicated that different ionic liquid modification probably induced different microenvironmental changes in the enzymes, as could be associated with conformational changes and a decrease in aromatic amino acid exposure.41 3.8. Fluorescence Spectroscopy Analysis. Fluorescence spectroscopy of proteins (mainly from tryptophan residues) is a powerful tool for the investigation of their conformational changes. The fluorescence analysis of tryptophan residues provided information on the enzyme-ligand interaction and ligand-induced conformational changes.42 Figure 7 showed that the tryptophan fluorescence emission blue-shifted from 350 nm of native CRL to 340−345 nm of modified CRLs, which could be attributed to the exposure of the fluorophore residues to a more hydrophobic microenvironment after modification.43 Also, the florescence intensities of the modified CRLs increased, which probably indicated that modification induced a more compact conformation.44 3.9. CD Spectroscopy Analysis. CD spectra were used to research the secondary structures (α-helice, β-sheet, β-turn, and random coil) of the native and modified CRLs. After modification, the structural changes of the CRLs could be obtained from far-UV CD spectra with the wavelength from 190 to 260 nm.45 As shown in Figure 8, the CRLs had minimum bands at 208 to 220 nm.46 However, there was a reduction in the CD spectra minima of the modified lipase with respect to the native enzyme, which was attributed to the loss in

used.38 In addition, the retention of the enzyme activity required some essential water molecules. The decreased enzyme activity was observed in a polar solvent, which could deprive enzyme molecules of the essential water.39 As shown in Figures 4B and 5B, the organic solvent tolerance of all CRLs with the modification of PEG functional ILs was better than that of ILs without the connection of PEG chains (e.g., [HOOCMMIm]-CRL-[Cl]), also was better than ILs with [C2OHmim] cation used in our work. According to Wen et al., PEG was water-soluble and the hydrogen bonds could be formed between PEG and water molecules, which were deemed to produce hydration shell and provided a hydrophilic microenvironment.40 Therefore, CRL modified by ionic liquids with a PEG functional group probably produced a more stable structure when exposed to strong polar organic solvents. 3.7. Ultraviolet Spectroscopy Analysis. To study the mechanism of the enhancement of the enzymatic properties of the modified lipases, the ultraviolet spectra of the native and modified CRLs were determined. Tryptophan and tyrosine are aromatic amino acids, which could induce protein ultraviolet absorption with the electronic excitation. After modification, the absorption spectra of these chromophores could be changed to some extent. As shown in Figure 6, the absorption peak of CRL was approximately 260 nm, with an absorption intensity of 0.46. Compared with the native CRL, the modified CRLs showed a slight red shift in their ultraviolet peaks and their absorbance values decreased, which agreed with our previous reports.10 Moreover, the modification with higher 8076


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Figure 6. Ultraviolet absorption spectra of native and modified CRLs. Figure 5. Effect of methanol (0−20%) on the activity of CRLs. Enzyme activity was determined at 40 °C and pH 7.0.

both secondary and tertiary structures.47 The secondary structures computed by Jwsse32 software were summarized in Table 2, the α-helix content was lower and β-sheet content was higher for almost all modified CRLs compared with the unmodified CRL, particularly for [C2OHmim]-CRL-[H2PO4] and [HOOCEPEG350Im]-CRL-[H2PO4], which possessed the highest β-sheet content. From the secondary structures contents of CRLs given in Table 2 and catalytic activity given in Table 1, it could be inferred that higher catalytic efficiency of modified lipases was related to decrease in α-helix and increase in β-sheet. Pavlidis reported the same phenomenon in the catalytic performance of lipases from Burkholderia cepacia, Chromobacterium viscosum, Candida rugosa, and Thermomyces lanuginosus.48,49 Jaeger et al.50 also reported that the tolerance of cellulases to ILs was related to the contents changes of αhelix and β-sheet by molecular dynamics. Decrease in α-helix influenced the active site of enzyme by stimulating higher tendency for the open conformation which allowed easier access of the substrate.51 As seen from the Table 2 and Figures 3, 4, and 5, the observed increase in β-sheet was related to the improvement of stability of modified CRLs, which could be attributed to the loss of hydrogen bonds between water molecules and α-helices as seen in the case of lyophilization leading to a more stable rigid structure of enzyme.52 These changes of secondary structures enhanced both activity and stability of modified CRLs.

Figure 7. Fluorescence spectra of native and modified CRLs.

4. CONCLUSIONS Various PEG functional ionic liquids were chemically modified onto CRLs and indicated that ILs with a more kosmotropic 8077


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All tested polyethylene glycol functional ionic liquids characterization by NMR (Bruker advanced III, Bruker), enzyme purity confirmation by SDS-PAGE (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Tel: +86-25-83172094. Fax: +86-25-83172094. E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.


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ACKNOWLEDGMENTS This research was financially supported by the Hi-Tech Research and Development Program of China (Grant No. 2011AA02A209), National Basic Research Program of China (Grant No. 2011CB710800), and National Science Foundation for Distinguished Young Scholars of China (Grant No. 21225626).

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(1) Reetz, M. T. Biocatalysis in organic chemistry and biotechnology: past, present, and future. J. Am. Chem. Soc. 2013, 135, 12480. (2) Busto, E.; Gotor-Fernandez, V.; Gotor, V. Hydrolases: catalytically promiscuous enzymes for non-conventional reactions in organic synthesis. Chem. Soc. Rev. 2010, 39, 4504. (3) Strohmeier, G. A.; Pichler, H.; May, O.; Gruber-Khadjawi, M. Application of designed enzymes in organic synthesis. Chem. Rev. 2011, 111, 4141. (4) Diaz-Rodriguez, A.; Davis, B. G. Chemical modification in the creation of novel biocatalysts. Curr. Opin. Chem. Biol. 2011, 15, 211. (5) Barbosa, O.; Ruiz, M.; Ortiz, C.; Fernandez, M. Modulation of the properties of immobilized CALB by chemical modification with 2,3,4-trinitrobenzenesulfonate or ethylendiamine. Advantages of using adsorbed lipases on hydrophobic supports. Process Biochem. 2012, 47, 867. (6) Forde, J.; Vakurov, A.; Gibson, T. D.; Millner, P. Chemical modification and immobilisation of lipase B from Candida antarctica onto mesoporous silicates. J. Mol. Catal. B: Enzym. 2010, 66, 203. (7) Van Rantwijk, F.; Sheldon, R. A. Biocatalysis in ionic liquids. Chem. Rev. 2007, 107, 2757. (8) Goldfeder, M.; Fishman, A. Modulating enzyme activity using ionic liquids or surfactants. Appl. Microbiol. Biotechnol. 2014, 98, 545. (9) Bekhouche, M.; Blum, L. J.; Doumeche, B. Ionic liquid-inspired cations covalently bound to formate dehydrogenase improve its stability and activity in ionic liquids. ChemCatChem 2011, 3, 875. (10) Jia, R.; Hu, Y.; Liu, L.; Jiang, L.; Zou, B.; Huang, H. Enhancing catalytic performance of porcine pancreatic lipase by covalent modification using functional ionic liquids. ACS Catal. 2013, 3, 1976. (11) Hu, Y.; Yang, J.; Jia, R.; Ding, Y.; Li, S.; Huang, H. Chemical modification with functionalized ionic liquids: a novel method to improve the enzymatic properties of Candida rugosa lipase. Bioprocess Biosyst. Eng. 2014, 37, 1617. (12) Li, L.; Xie, J.; Yu, S. T.; Su, Z. L. N-terminal PEGylated cellulase: a high stability enzyme in 1-butyl-3-methylimidazolium chloride. Green Chem. 2013, 15, 1624. (13) Zhang, Y. X.; Patil, A. J.; Perriman, A. W.; Mann, S. Enhanced catalytic activity in organic solvents using molecularly dispersed haemoglobin-polymer surfactant constructs. Chem. Commun. 2013, 49, 9561. (14) Na, D. H.; Youn, Y. S.; Park, E. J.; Lee, J. M. Stability of PEGylated salmon calcitonin in nasal mucosa. J. Pharm. Sci. 2004, 93, 256. (15) Hu, R. G.; Zhai, Q. W.; He, W.; Mei, L. Bioactivities of ricin retained and its immunoreactivity to anti-ricin polyclonal antibodies alleviated through pegylation. Int. J. Biochem. Cell Biol. 2002, 34, 396.

Figure 8. Far-UV CD spectra of native and modified CRLs.

Table 2. Secondary Structures of Native and Modified CRLs samples CRL [C2OHmim]-CRL-[H2PO4] [C2OHmim]-CRL-[Cl] [C2OHmim]-CRL-[BF4] [HOOCEPEG350Im]-CRL[H2PO4] [HOOCEPEG350Im]-CRL[Cl] [HOOCEPEG350Im]-CRL[BF4] [HOOCEPEG750Im]-CRL[Cl] [HOOCMMIm]-CRL-[Cl]

α-helix (%)

β-sheet (%)

β-turn (%)

random (%)

29.7 21.2 21.9 25.3 17.2

15.3 31.7 28.1 5.1 30.1

31.6 18.0 17.0 29.1 18.9

23.4 29.1 33.0 40.5 33.8

15.6

25.8

21.2

37.4

20.2

15.8

23.5

40.6

19.3

25.0

21.7

33.9

27.0

3.6

31.4

38.0

anion and a shorter PEG chain grafted on CRL simultaneously improved the catalytic activity, thermostability, organic solvents tolerance, adaptability to temperature, and pH changes with more significance, especially for [HOOCEPEG350Im][H2PO4]. The improved catalytic activities and stabilities of modified CRLs were related to the structural changes. For example, the decrease of α-helix and increase of β-sheet in the modified CRLs probably made the substrate interact easily with the lipase active site and formed a more stable rigid structure of enzyme. Just as our expectation, polyethylene glycol functional ILs showed a better modification effect of improved enzymatic properties compared with the previous reports.

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REFERENCES

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.iecr.5b01881. 8078


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Improving Catalytic Performance of Candida rugosa Lipase by

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