Immobilization of Lipases on Magnetic Collagen Fibers and Its Applications for Short-Chain Ester Synthesis

Magnetic nanoparticles (MNp) Fe3O4 were prepared by chemical coprecipitation, and introduced onto collagen fibers to form magnetic collagen support (MNp-Col) for enzyme immobilization. Candida rugosa lipase has been successfully immobilized on MNp-Col supports by a covalent bond cross-linking agent, glutaraldehyde. The characteristics of MNp-Col and the immobilized lipase were investigated. The immobilized lipase displayed sound magnetic separation abilities in both aqueous and organic media. The activity of the immobilized lipase reached 2390 U/g under optimal conditions. The MNp-Col immobilized lipase shows broadened temperature and pH ranges for hydrolysis of olive oil emulsion. For synthesis of butyrate esters in an n-hexane medium, the yield changes through use of different alcohols, among which, butyric butyrate showed the highest yield. The prepared magnetic collagen fiber provides separation support for enzyme immobilization and has the potential to be used in other biotechnology fields.


Introduction
Enzymes are broadly used as 'green' biocatalysts in many fields because enzymatic reactions are normally carried out at mild conditions with high efficiency and specificity [1].However, several shortcomings such as high cost, unstability to thermal or chemical, and non-reusability have restricted the application of free enzymes [2][3][4].Enzyme immobilization has been reported as a valuable alternative since it not only permits the reuse of enzymes, but also improves some other critical properties like activity, inhibition by reaction products, and selectivity [5].
Collagen fibers are bioresources that are inexpensive and readily available in animal skins and bones.Made up of spongy characteristics and the existence of functional groups such as carboxyl, amino, and hydroxyl groups, collagen fibers have been proposed by many as supports for enzyme immobilization [13][14][15].In our previously study, collagen fibers were developed as supports for The results of N 2 adsorption-desorption of native collagen fiber, MNp-Col supports, and MNp-Col-IL are shown in Table 1.The total pore volume and Brunauer, Emmett, Teller (BET) surface area of native collagen fiber were only 0.0049 m 3 /g and 2.10 m 2 /g, respectively.This is relatively low compared with other porous supports [19][20][21].However, after grafting with Fe 3 O 4 particles, the BET surface area of MNp-Col is 11.59 m 2 /g, nearly 5.5 times bigger than native collagen.While immobilized by lipase, the BET surface area of the support shifted to 7.63 m 2 /g, which is still 3.6 times bigger than native collagen.The total pore volume of MNp-Col supports is 0.042 m 3 /g, which is approximately 10 times bigger than native collagen.After immobilization, the total pore volume was maintained at 0.038 m 3 /g.This indicates that the introduction of Fe 3 O 4 particles led to an increase of pore volume and a rougher surface of collagen fiber, while the immobilizing of lipase onto the magnetic supports caused a slight decrease of pore volume.The average pore sizes of native collagen, MNp-Col and MNp-Col-IL, are approximately 13.70, 10.47, and 14.65 nm, respectively.This shows that the pores in collagen fiber supports and MNp-Col-IL fall into the mesopore size range [21].The average pore size of native collagen decrease when magnetic particles are added, and then increases again upon lipase immobilization.This should be due to GA used for cross-linking the enzyme.SEM images of MNp-Col-IL are shown in Figure 1.The magnetic particles were dispersed uniformly on the surface of collagen fiber.The physical adsorption may be the main cause in stabilizing the magnetic nanoparticles onto the collagen fiber.The surface of MNp-Col-IL appeared to be of irregular morphology, and layers of Fe  MNp-Col-IL showed positive dispersion ability in both aqueous and organic media, especially in aqueous (Figure 2).On the other hand, it could be separated from the medium within 10 s by an imposed magnetic field.This illustrates that the prepared magnetic collagen could be a potential support for enzyme immobilization that can be easily separated from the reaction medium.MNp-Col-IL showed positive dispersion ability in both aqueous and organic media, especially in aqueous (Figure 2).On the other hand, it could be separated from the medium within 10 s by an imposed magnetic field.This illustrates that the prepared magnetic collagen could be a potential support for enzyme immobilization that can be easily separated from the reaction medium.MNp-Col-IL showed positive dispersion ability in both aqueous and organic media, especially in aqueous (Figure 2).On the other hand, it could be separated from the medium within 10 s by an imposed magnetic field.This illustrates that the prepared magnetic collagen could be a potential support for enzyme immobilization that can be easily separated from the reaction medium.The denaturation temperature (Td) of the MNp-Col supports and immobilized lipase was investigated by differential scanning calorimeter (DSC) (Figure 3A).The Td of native collagen fiber is 57.9 • C, while the Td of MNp-Col and MNp-Col-IL were observed at 69.9 • C and 69.2 • C, respectively.This is an increase of approximately 12 • C compared to the native collagen.Glutaraldehyde (GA), the widely used cross-linking agent for collagen fibers, could improve the thermal stability of collagen fibers due to the forming of chemically stable covalent bonds between the side chain amino groups of the residues by formation of Schiff bases [22].The enhancement of Td by GA implies a broader processing temperature range of the magnetic collagen support, which is beneficial to enzyme immobilization.
The denaturation temperature (Td) of the MNp-Col supports and immobilized lipase was investigated by differential scanning calorimeter (DSC) (Figure 3A).The Td of native collagen fiber is 57.9 °C, while the Td of MNp-Col and MNp-Col-IL were observed at 69.9 °C and 69.2 °C, respectively.This is an increase of approximately 12 °C compared to the native collagen.Glutaraldehyde (GA), the widely used cross-linking agent for collagen fibers, could improve the thermal stability of collagen fibers due to the forming of chemically stable covalent bonds between the side chain amino groups of the residues by formation of Schiff bases [22].The enhancement of Td by GA implies a broader processing temperature range of the magnetic collagen support, which is beneficial to enzyme immobilization.
The thermogravimetry (TG) profiles showed that the major decomposition temperature for native collagen fiber was from 280 to 400 °C, and the major weight loss was 65%.The major decomposition temperature for MNp-Col and MNp-Col-IL was from 300 to 500 °C, and the major weight loss was only 37% (Figure 3B).This may be due to the dilution effect of Fe3O4 particles.The improvement of the heat decomposition resistance of MNp-Col and MNp-Col-IL can be contributed to the cross-linking by GA [23].

Initial Concentration of Lipase Solution
The immobilization of C. rugosa lipase onto MNp-Col support was performed at 20 °C and pH 7.5 for six hours by varying the initial concentration of lipase from 5 milligrams/milliliter to 50 mg/mL.The results were shown in Figure 4A.The activity of MNp-Col-IL increases as the initial concentration rises 5-30 mg/mL, and reaches a peak value 2390 U/g at 30 mg/mL, in which 70.4% of the expressed activity (observed activity/expected activity considering immobilized enzyme) was recovered.The results showed that the activity of lipase decreased at too high or too low enzyme concentration.The immobilization reaction occurs by covalent bonds between free aldehyde groups on the support and primary amino groups on the side chain of lipase [22,23].When lipase concentrations were higher than 30 mg/mL, the observed activity of MNp-Col-IL was lower, indicating that excessive immobilized enzymes on the support could hamper their transformation into active conformations for catalysis of the hydrolysis reaction.Similar results were observed by others [21,24].Furthermore, the immobilization procedure may cause secondary structural changes to the enzyme molecules, especially when some multipoint covalent bonds are built between the support and the enzyme [25].The thermogravimetry (TG) profiles showed that the major decomposition temperature for native collagen fiber was from 280 to 400 • C, and the major weight loss was 65%.The major decomposition temperature for MNp-Col and MNp-Col-IL was from 300 to 500 • C, and the major weight loss was only 37% (Figure 3B).This may be due to the dilution effect of Fe 3 O 4 particles.The improvement of the heat decomposition resistance of MNp-Col and MNp-Col-IL can be contributed to the cross-linking by GA [23].

Initial Concentration of Lipase Solution
The immobilization of C. rugosa lipase onto MNp-Col support was performed at 20 • C and pH 7.5 for six hours by varying the initial concentration of lipase from 5 milligrams/milliliter to 50 mg/mL.The results were shown in Figure 4A.The activity of MNp-Col-IL increases as the initial concentration rises 5-30 mg/mL, and reaches a peak value 2390 U/g at 30 mg/mL, in which 70.4% of the expressed activity (observed activity/expected activity considering immobilized enzyme) was recovered.The results showed that the activity of lipase decreased at too high or too low enzyme concentration.The immobilization reaction occurs by covalent bonds between free aldehyde groups on the support and primary amino groups on the side chain of lipase [22,23].When lipase concentrations were higher than 30 mg/mL, the observed activity of MNp-Col-IL was lower, indicating that excessive immobilized enzymes on the support could hamper their transformation into active conformations for catalysis of the hydrolysis reaction.Similar results were observed by others [21,24].Furthermore, the immobilization procedure may cause secondary structural changes to the enzyme molecules, especially when some multipoint covalent bonds are built between the support and the enzyme [25].

Incubation Time
The effect of incubation time was investigated at pH 7.5 and 20 °C with the initial concentration of lipase at 30 mg/mL by varying incubation duration from 1 hour to 10 h.The results were shown in Figure 4B.The activity of MNp-Col-IL increased gradually in the first six hours, exhibited maximal activity at six hours, and the observed activity remained stable afterwards.

Temperature
The immobilization processes were operated with the initial concentration of lipase at 30 mg/mL at temperatures varying from 5 to 35 °C.The activities of MNp-Col-IL were determined and the results were shown in Figure 4C.The optimal immobilization temperature was 20 °C.The activity decreases noticeably once the process temperature rises above 20 °C.This result may indicate that the conformation of the enzyme may be loosened at a high temperature, and the cross-linking reaction during immobilization will hinder the right conformation [26].

Incubation Time
The effect of incubation time was investigated at pH 7.5 and 20 • C with the initial concentration of lipase at 30 mg/mL by varying incubation duration from 1 hour to 10 h.The results were shown in Figure 4B.The activity of MNp-Col-IL increased gradually in the first six hours, exhibited maximal activity at six hours, and the observed activity remained stable afterwards.

Temperature
The immobilization processes were operated with the initial concentration of lipase at 30 mg/mL at temperatures varying from 5 to 35 • C. The activities of MNp-Col-IL were determined and the results were shown in Figure 4C.The optimal immobilization temperature was 20 • C. The activity decreases noticeably once the process temperature rises above 20 • C.This result may indicate that the conformation of the enzyme may be loosened at a high temperature, and the cross-linking reaction during immobilization will hinder the right conformation [26].The optimal activity for both free lipase and MNp-Col-IL are at 55 °C (Figure 5A).However, MNp-Col-IL maintains high activity at a broader range of temperature than the free lipase.Between 40 °C and 65 °C, more than 90% of the relative activity could be maintained.At 70 °C, the maintained relative activity for MNp-Col-IL is three times higher than that of the free lipase.The multi-interactions between the support and lipase may play a vital role in the tolerance to temperature of immobilized lipase.

Effect of pH
Olive oil emulsion was used as a substrate to investigate the effect of pH on the activity of free lipase and MNp-Col-IL by varying the pH range from 4.0 to 9.5.The optimal pH of MNp-Col-IL is 9.0 (Figure 5B), which is one unit higher compared to free enzyme (optimal pH 8.0).Interestingly, MNp-Col-IL shows a broadened pH range compared to the free lipase.Such a change and widening of the optimal pH value may be caused by diffusion resistance to the fatty acid in the hydrolyzed emulsion transport on the surface of the support [27].The stabilization of MNp-Col-IL by covalent bonding on the support may limit the active conformation of lipase against the pH change of the medium [28].

The Kinetic Parameters
The kinetic parameters of Km and Vmax are commonly used to evaluate the catalytic efficiency of immobilized verses of free enzymes.The kinetic parameters, Km and Vmax, of free and immobilized enzymes were evaluated by Lineweaver-Burk plot (Figure 6).The Km value of the free lipase and MNp-Col-IL is at 27.54 mM and 36.11mM respectively (Table 2).The Km of MNp-Col-IL is higher than that of the free lipase, suggesting that the immobilized lipase acting at half of maximum velocity needs a higher concentration of substrate.Furthermore, MNp-Col-IL has weaker affinity than free lipases [29].Such a decrease in affinity is apparent, which may due to the mass transfer limitations that result of immobilization.Lower Km value decreases of the MNp-Col-IL compared with Col-GA-IL (56.68 mM) may be due to the introduction of Fe3O4 particles.This enhanced the hydrophobicity of immobilized enzymes and improved the binding efficiency of substrate.The Vmax value of MNp-Col-IL was 38.5% that of free lipase.The decrease in Vmax might be due to generous secondary conformational perturbation, and changes on the lipase surface upon immobilization.The catalytic efficiency (Vmax/Km) of MNp-Col-IL (0.11) is lower than that of free lipase (0.37), illustrating that MNp-Col-IL has a relatively low catalytic efficiency [30,31].The optimal activity for both free lipase and MNp-Col-IL are at 55 • C (Figure 5A).However, MNp-Col-IL maintains high activity at a broader range of temperature than the free lipase.Between 40 • C and 65 • C, more than 90% of the relative activity could be maintained.At 70 • C, the maintained relative activity for MNp-Col-IL is three times higher than that of the free lipase.The multi-interactions between the support and lipase may play a vital role in the tolerance to temperature of immobilized lipase.

Effect of pH
Olive oil emulsion was used as a substrate to investigate the effect of pH on the activity of free lipase and MNp-Col-IL by varying the pH range from 4.0 to 9.5.The optimal pH of MNp-Col-IL is 9.0 (Figure 5B), which is one unit higher compared to free enzyme (optimal pH 8.0).Interestingly, MNp-Col-IL shows a broadened pH range compared to the free lipase.Such a change and widening of the optimal pH value may be caused by diffusion resistance to the fatty acid in the hydrolyzed emulsion transport on the surface of the support [27].The stabilization of MNp-Col-IL by covalent bonding on the support may limit the active conformation of lipase against the pH change of the medium [28].

The Kinetic Parameters
The kinetic parameters of K m and V max are commonly used to evaluate the catalytic efficiency of immobilized verses of free enzymes.The kinetic parameters, K m and V max , of free and immobilized enzymes were evaluated by Lineweaver-Burk plot (Figure 6).The K m value of the free lipase and MNp-Col-IL is at 27.54 mM and 36.11mM respectively (Table 2).The K m of MNp-Col-IL is higher than that of the free lipase, suggesting that the immobilized lipase acting at half of maximum velocity needs a higher concentration of substrate.Furthermore, MNp-Col-IL has weaker affinity than free lipases [29].Such a decrease in affinity is apparent, which may due to the mass transfer limitations that result of immobilization.Lower K m value decreases of the MNp-Col-IL compared with Col-GA-IL (56.68 mM) may be due to the introduction of Fe 3 O 4 particles.This enhanced the hydrophobicity of immobilized enzymes and improved the binding efficiency of substrate.The V max value of MNp-Col-IL was 38.5% that of free lipase.The decrease in V max might be due to generous secondary conformational perturbation, and changes on the lipase surface upon immobilization.The catalytic efficiency (V max /K m ) of MNp-Col-IL (0.11) is lower than that of free lipase (0.37), illustrating that MNp-Col-IL has a relatively low catalytic efficiency [30,31]

Storage Stability and Reusability Examinations
The storage stability of immobilized enzymes is an essential aspect for their industrial applications.In the storage stability study, MNp-Col-IL and free lipases were stored at 4 °C and their activity was determined at five day intervals.The activity of free lipase decreased to 60% of the initial activity after only five days, while the immobilized lipase reached the same loss of activity after 25 days (Figure 7).At 30 days of storage, MNp-Col-IL still preserved 50% of the initial activity compared to only 8% for free lipases.This suggests that MNp-Col-IL has significantly better storage stability than the free lipase.The significant loss of activity for the free lipase during storage might be due to its higher susceptibility to autolysis.A similar result was reported in the immobilization of C. rugosa lipase after being stored 30 days at 4 °C while the free lipase retained only 10% of its initial activity [20].
For immobilized enzymes, reusability is another character.The used MNp-Col-IL was recovered and used again for a hydrolytic activity test.As shown in Figure 8, at least 75% of the initial activity kept after MNp-Col-IL was reused for 10 cycles.A small loss of activity during the recovery/reuse cycles is possibly since the conformation of lipase was influenced by organic solvent [32,33].This result demonstrates that MNp-Col has a positive potential for lipase immobilization and related bio-catalytic applications.

Storage Stability and Reusability Examinations
The storage stability of immobilized enzymes is an essential aspect for their industrial applications.In the storage stability study, MNp-Col-IL and free lipases were stored at 4 • C and their activity was determined at five day intervals.The activity of free lipase decreased to 60% of the initial activity after only five days, while the immobilized lipase reached the same loss of activity after 25 days (Figure 7).At 30 days of storage, MNp-Col-IL still preserved 50% of the initial activity compared to only 8% for free lipases.This suggests that MNp-Col-IL has significantly better storage stability than the free lipase.The significant loss of activity for the free lipase during storage might be due to its higher susceptibility to autolysis.A similar result was reported in the immobilization of C. rugosa lipase after being stored 30 days at 4 • C while the free lipase retained only 10% of its initial activity [20].
For immobilized enzymes, reusability is another character.The used MNp-Col-IL was recovered and used again for a hydrolytic activity test.As shown in Figure 8, at least 75% of the initial activity kept after MNp-Col-IL was reused for 10 cycles.A small loss of activity during the recovery/reuse cycles is possibly since the conformation of lipase was influenced by organic solvent [32,33].This result demonstrates that MNp-Col has a positive potential for lipase immobilization and related bio-catalytic applications.

Synthesis of Butyrate by MNp-Col-IL
Short chain esters are a kind of important flavoring and fragrance agents [34][35][36].Lipases are often used as the catalyst of choice because the reactions can be carried out at mild temperatures, and the processes using these catalysts are considered clean.The MNp-Col-IL catalyzed synthesis of butyric acid esters was investigated.
Table 3 shows the yields for the synthesis of various esters.It showed that MNp-Col-IL effectively catalyzed the synthesis of several short-chain flavoring esters.The yield of butyl butyrate was observed with a peak value of 82.7%.While other butyrate esters displayed less yield, such as the yield of methyl butyrate and n-hexyl butyrate observed at 39.8% and 36.2%,respectively.The alcohol length and position

Synthesis of Butyrate by MNp-Col-IL
Short chain esters are a kind of important flavoring and fragrance agents [34][35][36].Lipases are often used as the catalyst of choice because the reactions can be carried out at mild temperatures, and the processes using these catalysts are considered clean.The MNp-Col-IL catalyzed synthesis of butyric acid esters was investigated.
Table 3 shows the yields for the synthesis of various esters.It showed that MNp-Col-IL effectively catalyzed the synthesis of several short-chain flavoring esters.The yield of butyl butyrate was observed with a peak value of 82.7%.While other butyrate esters displayed less yield, such as the yield of methyl butyrate and n-hexyl butyrate observed at 39.8% and 36.2%,respectively.The alcohol length and position

Synthesis of Butyrate by MNp-Col-IL
Short chain esters are a kind of important flavoring and fragrance agents [34][35][36].Lipases are often used as the catalyst of choice because the reactions can be carried out at mild temperatures, and the processes using these catalysts are considered clean.The MNp-Col-IL catalyzed synthesis of butyric acid esters was investigated.
Table 3 shows the yields for the synthesis of various esters.It showed that MNp-Col-IL effectively catalyzed the synthesis of several short-chain flavoring esters.The yield of butyl butyrate was observed with a peak value of 82.7%.While other butyrate esters displayed less yield, such as the yield of methyl butyrate and n-hexyl butyrate observed at 39.8% and 36.2%,respectively.The alcohol length and position of the hydroxyl groups have important effects on the yields [34,35].During the esterification process, a tetrahedral complex will be formed between the enzyme and acid by losing water, and then the alcohol binds to the acyl enzyme intermediate, acting as a nucleophile, thus forming a second tetrahedral complex.Resolution of this tetrahedral complex yields the ester and the free enzyme [36].Therefore, the diffusion rate of the alcohol to the active site should play a key role to the reaction.The efficiency and specificity of lipase catalyzed esterification is governed by the chain length of acids and alcohols [37].Shinetre et al. [38] reported that the initial rate of reaction increases linearly with increasing the chain length of fatty acid.Interestingly, Table 3 also showed that different alcohol isomers exhibited different yields, with the following order: n-alcohol > iso-alcohol > tert-alcohol.This could be explained by the influence of steric effects [36,39].The conversion of iso-butanol (61.9%) and tert-butanol (6.2%) was significantly lower compared with n-butanol (82.7%).This is probably due to progressively higher steric hindrance by the methyl groups in the proximity of the hydroxyl group.Results consistent with our work have been observed in previous studies focusing on the effects of the structures of alcohols on ester synthesis catalyzed by lipases [39][40][41].In general, Fe 3 O 4 -collagen immoblilzed lipases displayed high activity and specificity for butyrate synthesis in an n-hexane medium.

Preparation of Magnetic Fe 3 O 4 Particles
Preparation of Fe 3 O 4 magnetic particles was carried out according to the chemical coprecipitation method reported by others [16].Briefly, 5.4 g of FeCl 3 •6H 2 O and 2.0 g of FeCl 2 •4H 2 O were dissolved together in 100 mL water at 25 • C with vigorous stirring.Then 20% of NH 3 •H 2 O was added to adjust the pH value to 10 with vigorous stirring.The formed particles were black, and exhibited a strong response to an imposed magnetic field 1.0 mL of oleic acid was added and the mixture was incubated at 60 • C for 30 min.The synthesized Fe 3 O 4 were then filtered, rinsed, with deionized water and ethanol for several times, and used directly for the preparation of magnetic collagen support (MNp-Col).

Preparation of Magnetic Collagen Support and Enzyme Immobilization
The magnetic collagen supports were prepared by mixing Fe 3 O 4 magnetic particles with collagen fiber dispersion.Briefly, 5.0 g of collagen fiber and 100 mg Fe 3 O 4 magnetic particles were dispersed in 10 mL of 0.1 M acetic acid (HAc) with vigorous stirring.Subsequently, 10 mL of 45% glutaraldehyde was added for covalent bonding.The mixture was stirred for one hour at 30 • C before the pH value was adjusted to 7.5 by 0.1 M sodium bicarbonate and the mixture incubated for another hour.The obtained magnetic collagen supports (MNp-Col) were separated by an imposed magnetic field, and thoroughly rinsed with deionized water.
3 O 4 particles could be observed.It demonstrated that Fe 3 O 4 composite particles play a significant role in the enlargement of the surface area of MNp-Col and MNp-Col-IL.

Figure 2 .
Figure 2. Pictures of the dispersion and magnetic separation effect of MNp-Col-IL in media.(A) aqueous, and (B) olive oil.

Figure 2 .
Figure 2. Pictures of the dispersion and magnetic separation effect of MNp-Col-IL in media.(A) aqueous, and (B) olive oil.

Figure 2 .
Figure 2. Pictures of the dispersion and magnetic separation effect of MNp-Col-IL in media.(A) aqueous, and (B) olive oil.

Figure 4 .
Figure 4. Effect of the parameters on the lipase immobilization.(A) lipase concentration, (B) time, and (C) temperature.

2. 3
.1.Temperature The mixture of free lipase or MNp-Col-IL with substrate emulsion (pH 8.0) was incubated at temperatures varying from 15 to 70 °C.The activities were measured periodically.The results are shown in Figure 5A.

Figure 4 .
Figure 4. Effect of the parameters on the lipase immobilization.(A) lipase concentration, (B) time, and (C) temperature.

2. 3
.1.Temperature The mixture of free lipase or MNp-Col-IL with substrate emulsion (pH 8.0) was incubated at temperatures varying from 15 to 70 • C. The activities were measured periodically.The results are shown in Figure 5A.

Figure 5 .
Figure 5.Effect of Temperature and pH on the relative activity.(A) temperature, and (B) pH.

Figure 5 .
Figure 5.Effect of Temperature and pH on the relative activity.(A) temperature, and (B) pH.

Figure 7 .
Figure 7. Storage stability of free and immobilized lipase.

Figure 8 .
Figure 8. Reusability potential of MNp-Col immobilized lipase for hydrolysis of olive oil emulsion.

Figure 7 .
Figure 7. Storage stability of free and immobilized lipase.

Figure 7 .
Figure 7. Storage stability of free and immobilized lipase.

Figure 8 .
Figure 8. Reusability potential of MNp-Col immobilized lipase for hydrolysis of olive oil emulsion.

Figure 8 .
Figure 8. Reusability potential of MNp-Col immobilized lipase for hydrolysis of olive oil emulsion.

Table 1 .
The pore size of magnetic collagen support and immobilized lipase.

Table 2 .
The kinetic parameters.

Table 2 .
The kinetic parameters.

Table 3 .
The yield of butyrate synthesis catalyzed by MNp-Col-IL.