Improved stability of immobilized lipases via modification with polyethylenimine and glutaraldehyde

Accepted Manuscript Title: Improved stability of immobilized lipases via modification with polyethylenimine and glutaraldehyde. Authors: Hadjer Zaak, Laura Fernandez-Lopez, Cristina Otero, Mohamed Sassi, Roberto Fernandez-Lafuente PII: DOI: Reference:

S0141-0229(17)30128-X http://dx.doi.org/doi:10.1016/j.enzmictec.2017.07.001 EMT 9102

To appear in:

Enzyme and Microbial Technology

Received date: Revised date: Accepted date:

12-5-2017 30-6-2017 5-7-2017

Please cite this article as: Zaak Hadjer, Fernandez-Lopez Laura, Otero Cristina, Sassi Mohamed, Fernandez-Lafuente Roberto.Improved stability of immobilized lipases via modification with polyethylenimine and glutaraldehyde.Enzyme and Microbial Technology http://dx.doi.org/10.1016/j.enzmictec.2017.07.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Improved stability of immobilized lipases via modification with polyethylenimine and glutaraldehyde.

Hadjer Zaak +,a,b,c, Laura Fernandez-Lopez+,a, Cristina Oteroa, Mohamed Sassic, Roberto Fernandez-Lafuentea,*

a

Departamento de Biocatálisis. ICP-CSIC, Campus UAM-CSIC, Madrid, Spain.

b

Food Biotechnology Division, Biotechnology Research Center (CRBt),Constantine,

Algeria. c

Faculty of Nature and Life Sciences, Ibn Khalboun University, Tiaret, Algeria.

* Corresponding author: Roberto Fernandez-Lafuente. ICP-CSIC, C/ Marie Curie 2, Campus UAM-CSIC, Cantoblanco, 28049 Madrid, Spain. E-mail address: [email protected].

Graphical abstract

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Highlights      

LU and TLL was immobilized on OC supports at two different loadings The loading affected the enzyme stability, LU highly loaded was always more stable than lowly loaded preparations All preparations were modified with PEI and/or Glu with scarce effect on enzyme activity Highly loaded preparations were much stabilized by the modifications that lowly loaded preparations The loading effect was mainly observed using Glu modification Stabilization using PEI was very significant using highly loaded preparations in organic solvents.

Abstract Phospholipase Lecitase Ultra (LU) and lipase from Thermomyces lanuginosus (TLL) have been immobilized under conditions that favor either enzyme crowding or enzyme dispersion. Highly loaded LU was more stable than low loaded biocatalyst under all studied conditions. Using TLL, the results depended on the inactivation conditions, e.g., crowding was positive at pH 5 and negative at pH 7. Then, all preparations were treated with glutaraldehyde (Glu), polyethyleneimine (PEI) or sequentially with Glu and PEI. These treatments may permit to stabilize the physically immobilized lipases by avoiding enzyme desorption via intermolecular crosslinking. Moreover, immobilizing a second enzyme on the lipase-glutaraldehyde-PEI has been proposed as a strategy without risks of PEI desorption by incubation in high ion strength solutions. The treatments altered the enzyme activity slightly but produced significant enzyme stabilization. This enzyme stabilization was more significant when using the highly loaded preparations, where intermolecular crosslinking was easier to obtain. SDS-PAGE analyses confirmed that crowded enzyme preparations were intermolecular crosslinked using Glu plus PEI, but

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some molecules still remained non-crosslinked. In general, PEI treatment was the most effective in increasing enzyme stability, while glutaraldehyde had a milder stabilization effect.

Key words: physical modification with ionic polymer, chemical modification with glutaraldehyde, enzyme desorption, intermolecular crosslinking, enzyme stabilization, coimmobilization of enzymes. 1.- Introduction Lipases are among the most used enzymes due to their high activity and stability together with the wide range of substrates that they can recognize [1-4]. The industrial use of these biocatalysts may be favored if they are in an immobilized form [5] to permit their reuse and easy separation from the reaction mixture. Immobilization may be also utilized to improve other enzyme properties like stability or activity, purity or selectivity, etc. [6-12]. Immobilization of lipases on hydrophobic supports has proven to be an efficient method for their one step immobilization, purification, and stabilization of the lipase open conformation [13]. The lipase immobilization on these supports is based on the lipase interfacial activation on the support hydrophobic surface, leaving the open form of the lipase stabilized [14]. This physically immobilized lipase form is more stable than even multipoint covalently attached lipases [15-16]. This has been explained because this adsorbed open form of the lipase is more stable than the lipase in the opening/closing conformational equilibrium [17-19]. Many different hydrophobic supports have been successfully utilized to immobilize lipases [20-26]. 3

This simple and efficient lipase immobilization method is therefore very useful but it has a problem: although the immobilization is quite strong, some enzyme molecules are released from the support under drastic conditions, e.g., high temperatures, presence of high concentrations of organic solvents or detergents, etc. [27]. One additional problem that has been recently pointed out is that many substrates/products of lipases have some detergent character (e.g., free fatty acids, but also apparently simpler substances like dibutyrin or diacetin), and may facilitate undesired lipase desorption during operation [28-30]. This way, the medium is contaminated by the lipase and the biocatalyst loses activity over each operational cycle, reducing the interest of this immobilization method. Thus, many efforts are being made to maintain the advantages of this immobilization protocol while preventing enzyme desorption, like chemical crosslinking [31] or the use of heterofunctional supports able to give covalent bonds after enzyme immobilization via interfacial activation [27, 32-40]. Recently, the physical treatment with polyethylenimine (PEI) of lipases immobilized on octyl agarose beads has shown to be quite efficient to decrease the relevance of this problem [41-43]. The positive results were explained considering that PEI was able to produce physical intermolecular crosslinkings, forming large enzyme aggregates, and thus avoiding enzyme release from the support. However, due to the use of only ion bonds to fix the enzyme to the enzyme molecules, this could not be fully confirmed. PEI may be considered a good reagent to modify enzymes, as it may improve many enzyme properties, like stabilization versus organic solvents or oxygen, radical scavenger, or stabilization of multimeric structures [44-52]. PEI coating of immobilized enzymes may have some more complex applications. Recently, the coating of

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immobilized enzymes with PEI has been shown to be very useful to “co-immobilize” enzymes and cofactors [53]. Another use of immobilized enzymes coated with PEI is the production of multilayers of enzymes [54-55] and recently, to co-immobilize lipases and other enzymes [59]. This strategy was designed to permit the reuse of the immobilized lipase on octyl agarose beads when the second enzyme, immobilized via ion exchange on the immobilized lipase coated with PEI, was inactivated during operation, provided that the immobilized lipase was more stable than the second immobilized enzyme [56]. However, the reuse of the immobilized lipase coated with PEI required the incubation of the immobilized lipase with fresh PEI, because PEI molecules were desorbed from the immobilized lipase when desorbing the inactivated second enzyme [56], which became very strongly adsorbed on the polymer after its inactivation [57-58]. This is an irrelevant problem at laboratory scale, but at industrial level, it may become an issue. On the other hand, the modification with glutaraldehyde of immobilized lipases may produce some positive effects on enzyme properties, like covalent inter (also reducing enzyme leakage from the support), - or intra-molecular-crosslinking [59]. It may be also used to modulate lipase properties, usually with moderate (or even positive) effect on enzyme activity [60-61]. Enzyme crowding on the immobilized enzyme molecules has been shown to alter enzyme stability. Moreover, the proximity of the enzyme molecules may greatly alter the possibilities of obtaining intermolecular crosslinking, much more significantly using glutaraldehyde than PEI.

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Thus, in this paper, we have analyzed the effects of the octyl agarose immobilized lipase (using crowded or diluted immobilized biocatalyst) modification with glutaraldehyde and their further coating with PEI with several different objectives. First, this new strategy may combine the positive effects of two stabilization strategies, enhancing the individual results. Moreover, now the PEI-enzyme bonds will be converted in covalent bonds by the glutaraldehyde treatment. That way, we can fully confirm the relevance of the enzyme intermolecular crosslinking on the enzyme stabilization produced by the PEI coating. Second, this composite will not permit PEI desorption at high ion strength [62], making this kind of composite much more interesting that the use of just PEI as glue to coimmobilize two enzymes [59]. Now, the desorption of the inactivated second enzyme immobilized on PEI will not be accompanied by PEI release, and it will not be necessary to re-incubate the immobilized enzyme with PEI before immobilization of the second enzyme [62]. This strategy will be more interesting if it has some positive effects on the performance of the immobilized lipases, as for example enzyme stability (only if immobilized lipases are much more stable than the other enzyme, their reuse will be possible). The effect of enzyme crowding will also be analyzed, both on the initial stability of the enzymes, and on the stabilization achieved after the modifications as this may condition the possibilities of intermolecular crosslinking. For this purpose, two different lipase loadings of the supports have been employed. One biocatalyst with low loading, prepared using diluted enzymes solution, to facilitate a 6

certain diffusion of the enzyme inside the pores of the support before its immobilization. The other biocatalyst using maximum support loading, was prepared using high concentrations of the enzyme, to rapidly immobilize the enzyme molecules and favor their proximity [63-64]. As model lipases, we have selected the lipase from Thermomyces lanuginosus [65] and the commercial artificial chimeric phospholipase Lecitase ultra [6670]. 2. Materials and Methods 2.1. Materials Liquid solutions of the phospholipase Lecitase Ultra (17 mg of protein per mL) and the lipase from Thermomyces lanuginosus (TLL) (26 mg of protein per mL) were gentle donations from Novozymes (Spain). Polyethyleneimine (PEI) (MW 25,000), glutaraldehyde and p-nitrophenyl butyrate (p-NPB) were acquired from Sigma-Aldrich (St. Louis, USA). Octyl Sepharose CL-4B beads were from GE Healthcare. All other chemicals and solvents were of analytical grade. 2.2. Determination of lipase activity The lipase activity was determined by recording the increase in absorbance at 348 nm produced by the released p-nitrophenol in the hydrolysis of 0.4 mM p-NPB in 25 mM sodium phosphate buffer at pH 7.0 and 25°C (ɛ under these conditions is 5150 M-1 cm-1). 50–100 µL of lipase solution or suspension were added to 2.5 mL of substrate solution to initialize the reaction. One international unit of activity (U) was defined as the amount of enzyme that hydrolyzes 1 µmol of p-NPB per minute under the described conditions. In some instances, some additional acetonitrile was added to the assay. 7

2.3. Immobilization of enzymes on octyl (OC) support The immobilization experiments were performed using 1 or 16 mg of protein per g of wet support for LU and 1 mg or 13 mg of protein for TLL. The highest amount of enzyme doubled the loading capacity of the OC support [71] and permitted a closed packing of the enzyme molecules, with some effects on enzyme stability [63-64]. Lowly loaded derivatives were prepared using 0.1 mg enzyme/ml, while highly loaded biocatalyst was prepared using 1 mg of enzyme/ml of suspension. The commercial samples of the enzymes were diluted in the corresponding volume of 5 mM sodium phosphate at pH 7. Then, the support was added. The activity of both supernatant and suspension was followed using pNPB. After immobilization the suspension was filtered and the supported enzyme was washed several times with distilled water. Overloaded biocatalyst had an immobilization yield of 45%, while for low loading the immobilization yield was higher than 95%. 2.4. Glutaraldehyde modification of immobilized lipases on OC support The enzyme octyl-agarose derivatives prepared as previously described were incubated in a solution of 0.1% (v/v) glutaraldehyde in 25 mM sodium phosphate buffer at 25°C and pH 7 for 1 hour, under continuous mild stirring. This range of conditions permitted to modify the primary amino groups of the enzyme with just one glutaraldehyde molecule [72-74]. The suspension was then filtered and washed with water to remove the excess of glutaraldehyde. 2.5. Reduction with sodium borohydride A mass of 500 mg of immobilized and modified lipases were added to 20 mL of 0.1 M sodium borate containing 1 mg/mL of NaBH4 at pH 10.0 and 4°C; the reaction mixture was 8

continuously stirred for 30 min. The reduced biocatalysts were filtered and washed several times with 100 mM phosphate at pH 7 and finally with distilled water. This treatment was performed for the SDS-PAGE samples, before after boiling in SDS. 2.6. Coating of immobilized lipases with PEI A 100 mL solution of 10% PEI (w/v) was prepared and the pH was adjusted at pH 7. Then, 3 g of immobilized lipase derivatives were suspended and submitted to gentle stirring for 24 h. Afterwards, the modified enzymes were washed with an excess of distilled water to eliminate the free PEI. 2.7. Thermal inactivation of different enzyme immobilized preparations We performed stress inactivations selecting the temperatures where the inactivation rate presented a reasonable speed to compare all preparations. 1 g of immobilized enzyme was suspended in 30 ml of 25 mM of sodium acetate at pH 5, sodium phosphate at pH 7 or sodium carbonate at pH 9 at different temperatures. Periodically, samples were withdrawn and the activity was measured using pNPB. Half-lives were calculated from the observed inactivation courses. 2.8. Inactivation of different enzyme immobilized preparations in the presence of organic solvents Enzyme derivatives were incubated in mixtures of acetonitrile (ACN) (LU) or dioxane (TLL) in 25 mM tris-HCl pH 7 at 25ºC. Periodically, samples were withdrawn and the residual activity was measured using pNPB. Half-lives were calculated from the observed inactivation courses. 2.9. SDS-PAGE analyses 9

SDS-polyacrylamide gel electrophoresis of the proteins was performed on 12% resolving gel with 5% stacking gel according to Laemmli [75]. To analyze the amount of proteins that remains adsorbed to the octyl-agarose, 100 mg of the derivative were suspended in 0.5 mL of rupture buffer (4% SDS and 10% mercaptoethanol). The samples were incubated in boiling water for 8 min and a 10-µL (low-load) or 20 µL (high load) aliquot of the supernatant was loaded to the sample well. The samples were run at 80 volts until the lowest marker reached the lower edge of the gel. Gels were stained with Coomassie brilliant blue. A low molecular weight calibration kit for SDS electrophoresis (GE Healthcare) was used as a molecular weight marker (14.4–97 kDa). 3. Results 3.1.- Effect of the modifications on the activity of the different preparations Table 1 shows the changes in activity of the highly and lowly loaded preparations of both enzymes. Lowly loaded LU-OC physically coated with PEI increased the enzyme activity by 10%, in agreement with previous reports [52]. The modification with glutaraldehyde decreased the enzyme activity by around 40%. In fact, after 1 h of modification with glutaraldehyde (before washing), the activity increased by 10%. However, the activity decreased over time after eliminating the excess of glutaraldehyde [76]. When the enzyme modified with glutaraldehyde was treated with PEI, a lower decrease in the activity was observed. Similar results were obtained using the highly loaded preparations, but with a smaller decrease in enzyme activity by glutaraldehyde treatment (20%) and a negligible effect of the PEI on enzyme activity (perhaps because the polymer, together with the positive effects, may also increase the diffusion problems of the substrate in this crowding preparation). In the case of TLL, the treatment with PEI did not 10

produce any significant change in TLL activity. The treatment with glutaraldehyde produced a slight decrease in enzyme activity after 24 h (although initially it also produced an increase in activity), more significant for the highly loaded preparation (around 20%). The sequential modification Glu plus PEI produced an increase in enzyme activity compared to the only glutaraldehyde treated enzyme. Glu can produce several kinds of modifications on the enzyme. First, primary amino groups will be modified after the first incubation. This modification did not affect the ionization of the amino group, and just produced a certain hydrophobization on the enzyme. This may have positive or negative effects on enzyme stability/activity [54-61]. After eliminating the remaining Glu, in the cases where other Glu modified amino group is near enough, crosslinkings may be achieved [72], which may be intramolecular if both amino-Glu groups are located in the same molecule, or intermolecular if they are located in different enzyme molecules. This should produce a certain rigidification of the enzyme molecule, giving some stabilization. When PEI is added, its primary amino groups will compete with these reactions, reducing the effects on enzyme activity of the glutaraldehyde modification. In any case, the modifications have no dramatic effects on the enzymes activity, an even in some cases they are slightly positive.

3.2.- Effect of the modifications on the thermostability of the immobilized enzymes

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Figures 1-3 show the inactivations of both immobilized enzymes using lowly and highly loaded preparations. Starting with LU, one outstanding point is that the highly loaded octyl preparation is much more stable than the lowly loaded preparation. An effect on enzyme stability of enzyme crowding has been previously described using other enzymes [64-65], and it seems that the difference in the LU immobilization rates on OC under the two utilized conditions has permitted to observe these effects. Moreover, it is also clear that the modification of LU-OC with PEI and glutaraldehyde had different stability effects depending on the LU load of the biocatalyst. At pH 5, the low-load preparation retained its stability after glutaraldehyde modification (the improvement was marginal), while the highly loaded one showed a significant improved stability. This could be related to an easier intermolecular crosslinking of the highly loaded preparation, where enzyme molecules should be more closely immobilized [54, 64, 65]. The treatment with PEI improved the stability of the low-load LU-OC, giving similar values when the enzyme was previously activated with glutaraldehyde. PEI may have direct positive effects on enzyme stability by generating favorable environments or just prevent enzyme desorption from the OC support by physical intermolecular crosslinking [41-50]. The large size of the polymer may permit to crosslink relatively distant enzyme molecules. Using the high-load preparation, stability was also improved using PEI for lipase preparations, treated or not with glutaraldehyde. However, while the

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initial inactivation course is similar for LU-OC-PEI and LU-OC-Glu-PEI, after a certain percentage of inactivation the last preparation exhibited a significantly higher stability. In inactivations at pH 7, the modifications of the lowly loaded preparations produced similar effects to that found at pH 5 (glutaraldehyde almost did not affect enzyme stability, while PEI treatment improved LU-OC and LU-OC-Glu stability at similar levels). When using the highly loaded preparations, the main difference was that LU-OC-PEI and LU-OC-Glu-PEI have similar stability. Although the final stability of the modified highly loaded biocatalyst remains higher than that of the lowly loaded ones after modifications (because they started from a very low point), the stabilization achieved by the modifications is higher using the lowly loaded ones. When inactivations were performed at pH 9, the situation was quite different, LUOC-PEI was the biocatalyst with the lowest stability, the treatment with glutaraldehyde yielding the most stable enzymes, both using high and low loads of enzyme. The stabilization is much more significant using the highly loaded enzymes preparations than the lowly loaded preparations, in opposition to the results when the inactivations were performed at pH 5 or 7. The pH may alter the interactions between enzyme and polymer; at pH 9 the polymer will lose some ionized cationic groups, and become more hydrophobic. It may also be considered that PEI primary groups may be oxidized to nitro groups under this pH value, again changing the enzyme-support interactions. However, PEI desorption was not detected. Regarding TLL, the effect of the enzyme loading is dependent on the pH as previously described [77], even though now lower enzyme concentrations during the

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enzyme immobilization were employed and the effects are smaller. The highly loaded TLL biocatalyst is more stable at pH 5, less stable at pH 7 and similarly stable at pH 9 than the lowly loaded immobilized enzyme. Analyzing the effects of the modifications, at pH 5 the most positive modification is with PEI, glutaraldehyde having a marginal positive effect using the lowly loaded preparation and a very significant one using the highly loaded one. The PEI coating of the enzyme modified with glutaraldehyde presented a stability lower than that of the enzyme just modified with PEI. The different modified overloaded preparations remained more stable than the equivalent lowly loaded at pH 5. When the inactivation was performed at pH 7, glutaraldehyde modification presented scarce effect for the low loading preparation while having a high stabilizing effect using the highly loaded preparation. PEI coating of this glutaraldehyde modified preparation offers the highest stabilization using the low protein loaded preparation, while it even has a negative effect using the highly loaded preparation (perhaps by reducing the number of glutaraldehyde intermolecular croslsinkings). PEI modification of the OC-TLL gave the highest stabilization of the high loaded preparation. It is remarkable that while the unmodified high loaded preparation was less stable at this pH than the low loaded, all modified high loaded preparations became more stable than their corresponding low loaded preparations; that means a higher stabilization of these last preparations. In inactivations at pH 9, the stabilizations obtained by the different treatments were short. Using the low loaded preparations, the most stable ones were both PEI treated preparations, while glutaraldehyde has a low effect. The stabilization effects were higher

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using the highly loaded preparations, initially the PEI treatment seemed the most effective one to stabilize the enzymes. The last fraction of enzyme activity of the biocatalyst treated with glutaraldehyde, however, presented a higher stability.

3.3.- Effect of the modifications on the enzyme performance in the presence of organic solvents. Figure 4 shows the inactivation of LU preparations in the presence of acetonitrile at pH 7. Again the stability of the highly loaded LU-OC is clearly higher than that of the lowly loaded preparation. Thus, in the case of LU the crowding of the enzyme molecules on the support surface had positive effects on enzyme stability under all studied conditions, differently to the results reported in other examples [64-65]. The treatment of the lowly loaded LU preparation with glutaraldehyde has not a significant effect in enzyme stability under these conditions, while it is very significant using the highly loaded enzyme preparation. This could be again related to the higher possibilities of intramolecular crosslinking using the highly loaded preparations [54]. PEI offered a significant stabilization for preparations at both enzyme loads and this effect was higher using the preparations previously modified with glutaraldehyde. Using the high load preparation, the effect of PEI or glutaraldehyde modification produced similar stabilization. The double modification was even more stabilizing. The inactivation course was again multiphasic, and the last fraction was even much more stable. The inactivation of the different TLL preparations in dioxane offered a similar picture to LU in acetonitrile; highly loaded preparations were more stable than the low 15

loaded ones, PEI coating presented the most positive effects and gave the most stable preparations, and glutaraldehyde modification is only positive when using the highly loaded preparations. The main difference is that modification of OC-TLL-Glu with PEI gave even slightly less stable preparations than just PEI modification. This may be explained considering that PEI should generate a hydrophilic enzyme environment [50, 78, 79], mainly using highly loaded preparations, while glutaraldehyde may have an effect mainly if intramolecular or/and intermolecular crosslinkings are introduced, being the latter easier in the highly loaded preparations. The higher stabilization of LU high loaded preparation compared to the TLL can be related to the differences in the degree of intermolecular crosslinkings introduced for each enzyme and the effect of the intramolecular crosslinking and one point modifications on the TLL and LU properties.

The activities of the different preparations versus pNPB were analyzed in the presence of growing concentrations of solvent. The low-load LU preparations decreased their activities quite rapidly, being the PEI treated preparations the most resistant to the activity reduction promoted by this solvent. The results were qualitatively similar using highly loaded preparations, but with higher activity retention in all cases. The only exception was the glutaraldehyde treatment. This treatment had a clear positive effect, while glutaraldehyde treated LU coated with PEI did not reach the positive effects of just PEI-treated enzymes. Using TLL, the highly loaded preparations were more resistant to inhibition using low solvent concentrations than the lowly loaded ones, although results were similar using 16

higher concentrations of solvent and TLL-OC-PEI offered the highest resistance to the solvent presence. This PEI effect was reduced using the enzyme modified with glutaraldehyde, although the modification with glutaraldehyde alone was also positive. Thus, highly loaded enzyme biocatalyst retained more activityin both cases in the presence of organic solvents, perhaps because the hydrophilicity generated by the protein molecules crowding that in certain way generated a hydrophilic nano-environment in the enzyme surroundings due to the near enzyme molecules proximity [63-64], decreasing the concentration of solvent near the active center. The PEI effect should be also related to this partition of the organic solvent [78-79]. 3.4.- SDS-PAGE analysis of the different preparations. This study was performed to check the promotion of covalent intramolecular enzyme crosslinkings [54] and how this may explain some of the results reported above. The 8 preparations (2 loads and 3 modifications, plus the unmodified biocatalysts) were used in this study, the glutaraldehyde preparations were reduced to ensure the stability of the glutaraldehyde-enzyme or glutaraldehyde-PEI bonds during the boiling step. Starting with lowly loaded OC-LU, Figure 6a shows that the treatment with glutaraldehyde reduced the intensity of the LU band, while the treatment with PEI gave a wider band with an apparent slightly higher molecular weight but more diffuse. As PEI was released from the support under boiling SDS, this was attributed to the presence of the PEI during the electrophoresis. PEI may somehow alter the enzyme electrophoretic mobility of the enzyme, perhaps by interacting with the enzyme. PEI treatment of the glutaraldehyde treated enzyme produced a reduction of the LU band, but this band still is very significant. 17

Dimers or trimers of the enzyme were not visualized. Using the highly loaded preparation, the effect of PEI is similar to the one described above (there are not any irreversible bonds). Modification with glutaraldehyde produced a significant decrease in the intensity of the LU band, and dimers, trimers and other larger aggregates are visible. That meant that some intermolecular crosslinking had been achieved. However, many LU molecules are in monomeric state, even using this crowding preparation. This indicates the difficulties in getting a full intermolecular crosslinking using small bifunctional reagents. The incubation of this LU-OC-Glu preparation with PEI produced a further reduction of the LU band, but a significant amount of monomeric enzyme (around 10-15%) may be visualized. This may explain the multiphasic shape of some of the inactivation courses of highly loaded LU treated with PEI and glutaraldehyde, although many molecules are forming very large aggregates that are not released to the medium, there is a significant percentage of monomers, dimers and trimers that may have different stabilities (e.g., may be released from the support at high temperatures) [27-30]. Using TLL results were fairly similar, except that aggregates can be visualized after glutaraldehyde treatment even using the low load preparation and treatment of glutaraldehyde TLL with PEI using the high load preparation gave a very small band of TLL, not accounting for more than 5% of initial protein. Apparently, TLL crosslinking is easier than LU crosslinking, both with PEI or glutaraldehyde, suggesting that the enzyme molecules could be near each other with this enzyme. Thus, the use of low load preparations caused the intermolecular crosslinking with either glutaraldehyde or glutaraldehyde/PEI treatments to be only moderate. Thus, the main effect of the treatment of lowly loaded preparations will be the intramolecular chemical

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modifications. This includes glutaraldehyde one point modification, that may have positive or negative effects on enzyme stability, and crosslinking, that should have a positive effect on enzyme stability but may be very difficult to achieve [54, 74]. The treatment with PEI of the enzyme preparations activated with glutaraldehyde will prevent protein intramolecular chemical crosslinkings by reacting with amino-glutaraldehyde moieties. This may decrease the stabilizing effect of the glutaraldehyde treatment if the PEI effects were e nor clearly positive. A larger percentage of enzyme molecules crosslinked via glutaraldehyde (there is less protein in the supernatant) may be obtained using derivatives with high load. However, even using PEI and highly loaded enzyme preparations to increase the possibilities of a massive intermolecular crosslinking, a certain percentage of enzyme molecules remained not crosslinked even with just other enzyme molecule. These results may explain the biphasic behavior that highly loaded preparations treated with glutaraldehyde has in many of the inactivations presented in this paper. The failure in getting a full intermolecular crosslinking combining glutaraldehyde and PEI show the difficulty of achieving a massive intermolecular crosslink, 4.- Conclusions This paper is a new example of how enzyme crowding may affect the enzyme stability; in this case LU stability is improved in all inactivations assayed, while the results obtained with TLL results confirm previous reports [77]. The modifications assayed produced significant and always more relevant stabilizations using biocatalysts with a high loading. The use of covalent bonds confirmed 19

that the main difference between high and low load biocatalysts is the percentage of molecules involved in intermolecular crosslinking, previously used as a hypothesis but not clearly shown by the SDS-PAGE. The stabilizing effects are more relevant under certain conditions that under other conditions. Although glutaraldehyde is considered a very adequate reagent for enzyme stabilization, PEI tends to be the most stabilizing modifier. This occurs even if this is just a physical modification and PEI is a very flexible polymer that may hardly increase enzyme rigidity. This could be mainly based in the prevention of enzyme desorption by physical intermolecular crosslinking. This paper also shows for first time that the stabilizing effects of glutaraldehyde modification depends on enzyme loading. They become maximized using highly loaded preparations, where intermolecular crosslinking affects a large percentage of the enzyme molecules (as shown by SDS-PAGE). This suggests that the main stabilizing effect of this reagent is the promotion of intermolecular crosslinkings. Coating of glutaraldehyde treated molecules with PEI in many cases even reduces the stability compared to the enzyme treated just with PEI, even though glutaraldehyde treatment alone is also positive. This may be related to the decrease of intramolecular crosslinking, being the main effect of glutaraldehyde in this case the one point chemical modification. In any case, the double modification seems to be positive for enzyme stability with scarce effect on enzyme activity, and therefore it may be a proper option to be used in the strategy of enzyme coimmobilization described in [59], one of the objectives of this paper. 20

A similar strategy may be valid for other immobilization where the enzyme is only physically immobilized on the support, like in the case of enzyme nanoflowers [80-83]. Acknowledgments We gratefully recognize the support from the MINECO from Spanish Government, (project numbers CTQ2013-41507-R). Hadjer Zaak thanks the Algerian Ministry of higher education and scientific research for her fellowship. The authors wish to thank Mr. Ramiro Martínez (Novozymes, Spain) for kindly supplying some of the enzymes used in this research. The help and suggestions of Dr. Ángel Berenguer (Instituto de Materiales, Universidad de Alicante) during the writing of this paper are gratefully recognized

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29

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Figure Legends Figure 1. Thermal inactivation courses at pH 5 of OC-LU at 52ºC (A) and OC-TLL at 73ºC (B) after different treatments. Other specifications are described in the Methods section. Solid lines, solid symbols: lowly loaded preparations, Dashed lines, empty symbols: highly loaded preparations. Circles: OC-enzyme preparations; Triangles: OCenzyme-Glu preparations, Squares: OC-enzyme-PEI preparations, Rhombus, OC-enzymeGlu-PEI preparations. Figure 2. Thermal inactivation courses at pH 7 of OC-LU at 50ºC (A) and OC-TLL at 70.5ºC (B) after different treatments. Other specifications are described in the Methods section. Solid lines, solid symbols: lowly loaded preparations; Dashed lines, empty symbols: highly loaded preparations. Circles: OC-enzyme preparations, Triangles: OCenzyme-Glu preparations, Squares: OC-enzyme-PEI preparations, Rhombus, OC-enzymeGlu-PEI preparations. Figure 3. Thermal inactivation courses at pH 9 of OC-LU at 50ºC (A) and OC-TLL at 65ºC (B) after different treatments. Other specifications are described in Methods section. Solid lines, solid symbols: lowly loaded preparations; Dashed lines, empty symbols: highly loaded preparations. Circles: OC-enzyme preparations, Triangles: OCenzyme-Glu preparations, Squares: OC-enzyme-PEI preparations, Rhombus, OC-enzymeGlu-PEI preparations. Figure 4. Inactivation courses of OC-LU in 30% acetonitrile at pH 7 and 25ºC (A) and OC-TLL in 60% dioxane at pH 7 and 40ºC (B) after different treatments. Other specification are described in the Methods section. Solid lines, solid symbols: lowly loaded 35

preparations; Dashed lines, empty symbols: highly loaded preparations. Circles: OCenzyme preparations, Triangles: OC-enzyme-Glu preparations, Squares: OC-enzyme-PEI preparations, Rhombus, OC-enzyme-Glu-PEI preparations. Figure 5. Effect of the presence of organic solvents in the pNPB activity of differently modified OC-LU (A, acetonitrile) and OC-TLL (B, dioxane). Other specifications are described in the Methods section. Solid lines, solid symbols: lowly loaded preparations; Dashed lines, empty symbols: highly loaded preparations. Circles: OC-enzyme preparations, Triangles: OC-enzyme-Glu preparations, Squares: OC-enzyme-PEI preparations, Rhombus, OC-enzyme-Glu-PEI preparations. Figure 6. SDS-PAGE analysis of differently modified OC-LU. Samples obtained after boiling in SDS the lowly loaded derivatives (A) or the the highly loaded derivatives (B). The immobilized enzymes were submitted to the processes described in the Methods section. Lane 1: OC-LU preparations. Lane 2: OC-LU-Glu preparations. Lane 3: OC-LUPEI preparations. Lane 4: OC-LU-Glu-PEI preparations. Figure 7. SDS-PAGE analysis of differently modified OC-TLL. Samples obtained after boiling in SDS the lowly loaded derivatives (A) or the the highly loaded derivatives (B). The immobilized enzymes were submitted to the processes described in the Methods section. Lane 1: OC-TLL preparations. Lane 2: OC-TLL-Glu preparations. Lane 3: OCTLL-PEI preparations. Lane 4: OC-TLL-Glu-PEI preparations.

36

Figure(s)

A

B 100

Residual activity (%)

Residual activity (%)

100 80 60 40 20 0

80 60 40 20 0

0

2

4 Time (h)

6

0

1.5

3

4.5

Time (h)

Figure 1

6

7.5

A

B

100

Residual activity (%)

Residual activity (%)

100 80 60 40 20 0

80 60 40 20 0

0

20

40

60

80 100 120 140 160

Time (h)

0

1

2 Time (h)

Figure 2

3

B

A 100

Residual activity (%)

Residual activity (%)

100 80 60 40 20 0

80 60 40 20 0

0

15

30 Time (minutes)

45

60

0

1.5

3 Time (h)

Figure 3

4.5

6

B

A

100

Residual activity (%)

Residual activity (%)

100 80 60 40 20 0

80 60 40 20 0

0

2

4 Time (h)

6

0

2

4 Time (h)

Figure 4

6

B

A

100

Residual activity (%)

Residual activity (%)

100 80 60 40 20

80 60 40 20 0

0 0

5

10

15

Percentage of acetonitrile, (%)

20

0

5

10

15

20

Percentage of dioxane, (%)

Figure 5

25

30

A

kDa 97

B

1

2

3

4

kDa 97

66 45

66 45

30

30 20.1 20.1 14.4

Figura 6

1

2

3

4

A

B

kDa

1

2

3

4

kDa

45

97 66 45

30

30

20.1

20.1

14.4

14.4

66

Figura 7

1

2

3

4

Table 1. Effect of the different treatments on the different biocatalysts activity. Experiments were performed as described in Methods section. Values are given as relative activity considering the unmodified biocatalyst.

Treatment Enzyme

Load

None

PEI

Glutaraldehyde Glutaraldehyde plus PEI

LU

Low

100±3

110±3

63±2

91±3

LU

High

100±4

102±2

80±3

90±4

TLL

Low

100±4

104±3

97±3

110±4

TLL

High

100±3

100±5

80±4

100±3

37