Immobilization of Lipase from Pseudomonas fluorescens on Porous

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Immobilization of Lipase from Pseudomonas fluorescens on Porous Polyurea and Its Application in Kinetic Resolution of Racemic 1-Phenylethanol Hui Han, Yamei Zhou, Shusheng Li, Yinping Wang, and Xiang Zheng Kong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b07979 • Publication Date (Web): 12 Sep 2016 Downloaded from http://pubs.acs.org on September 14, 2016

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ACS Applied Materials & Interfaces

Immobilization of Lipase from Pseudomonas fluorescens on Porous Polyurea and Its Application in Kinetic Resolution of Racemic 1-Phenylethanol Hui Han1,2, Yamei Zhou1, Shusheng Li1,3, Yinping Wang1 and Xiang Zheng Kong1,* 1

College of Chemistry and Chemical Engineering, University of Jinan, 250022, China

2

University of Chinese Academy of Sciences, Beijing 100049, China

3

College of Chemistry and Chemical Engineering, Shandong University, 250100, China

ABSTRACT: A porous polyurea (PPU) was prepared through a simple protocol by reacting toluene diisocyanate with water in binary solvent of water-acetone. Its amine group was determined through spectrophotometric absorbance based on its iminization with pnitrobenzaldehyde amines. PPU was then used as a novel polymer support for enzyme immobilization, through activation by glutaraldehyde followed by immobilization of an enzyme, lipase from Pseudomonas fluorescens (PFL), via covalent bonding with the amine groups of lipase molecules. Influences of glutaraldehyde and enzyme concentration and pH in the process were studied. The results revealed that the activity of the immobilized PFL reached a maximum at GA concentration of 0.17 mol/L and at pH 8. Immobilization rate of 60% or higher for PFL was obtained under optimized condition with an enzyme activity of 283 U/mg. The porous

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structure of PPU, prior to and after GA activation and PFL immobilization, was characterized. The activity of the immobilized PFL at different temperature and pH, and its stability at 40 °C as well as its reusability were tested. The immobilized enzyme was finally used as enantioselective catalyst in kinetic resolution of racemic 1-phenylethanol (1-PEOH), and its performance compared with the free PFL. The results demonstrate that the enzyme activity and stability were greatly improved for the immobilized PFL, and highly pure enantiomers from racemic 1-PEOH were effectively achieved using the immobilized PFL. Noticeable deactivation of PFL in the resolution was observed by acetaldehyde in-situ formed. In addition, the immobilized PFL was readily recovered from the reaction system for reuse. 73% of the initial activity was retained after 5 repeated reuse cycles. This work provides a novel route to preparation of a polyurea porous material and its enzyme immobilization, leading to a novel type of immobilized enzyme for efficient kinetic resolution of racemic molecules.

KEYWORDS: Porous polyurea, lipase from Pseudomonas fluorescens, enzyme immobilization, 1-phenylethanol, kinetic resolution

INTRODUCTION Practical use of lipase has been realized in various industrial processes, and is being expanded in new fields, such as enantiomer synthesis,1-4 biosensors,5,6 and biofuel cells.6-8 Relative to chemical catalysts, the most significant advantages by using enzymes are the extraordinary rate enhancements and the high degree of selectivity. Enzymes have become powerful tools in the synthesis of value-added chemicals from low-costing and widely available starting materials.2 However, the free or native enzymes themselves are usually not usable for commercial


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application because they are prone to denature under environment conditions and difficult to be recovered. Immobilization of free enzyme onto a solid material as support6,9-12 has become a promising protocol to overcome these limitations, which renders a number of advantages over the free enzyme, including for instance, the enhanced stability, facility in separation, and the easiness in recovery and reuse.4,11-13 Up to date, a variety of processes have been developed for enzyme immobilization, including, for instance adsorption, entrapment, covalent coupling, crosslinking, reversed micelle and so on.11-14 Among these methods, covalent coupling is the most commonly used one.6,10,13,14 In addition, with porous materials as the support, high enzyme loading can be achieved with chemical and mechanical stability and ordered mesostructure because of the high specific surface area in these materials.4,15 Nowadays, to find new support has been considered as one of the promising choice to maintain high catalytic activity for an enzyme while achieving the advantages of immobilization.12 Our studies have been focused on the preparation and characterization of uniform polymer microspheres.16-21 Through precipitation polymerization of isophorone diisocyanate (IPDI) in water or its mixture with a solvent, polyurea microspheres of different size with varied morphology were also prepared.18-21 When using toluene diisocyanate (TDI) instead of IPDI, a porous polyurea (PPU) was also prepared,22-24 which was demonstrated to be an attractive adsorbent for removal of anionic dyes from wastewaters.22,23 This PPU is of high specific surface area, and abundant amine groups are present on its surface. In this study, PPU as prepared was evaluated as a new support for enzyme immobilization. Amount of amine groups on PPU surface was first determined, followed by immobilization of an enzyme, lipase from Pseudomonas fluorescens (PFL), via a treatment of PPU with glutaraldehyde (GA) as the linkage between PPU and PFL. The immobilized lipase was used in


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kinetic resolution of racemic 1-phenylethanol (1-PEOH) based on the selective transformation for one of the enantiomers of the racemate. The activity, stability and enantioselectivity of the immobilized lipase were studied under different experimental conditions. To the best of our knowledge, this work is the first to use PFL immobilization on a porous polyurea, which was prepared through a simple one-step precipitation polymerization and bearing amine groups on their surface without specific surface amination required. EXPERIMENTAL Chemicals and Reagents. The relative information is described in Part 1 in Supporting Information. Preparation of Porous Polyurea. Porous polyurea, PPU, was prepared by reacting TDI with water to yield diamine first, followed by step polymerization of TDI with the in-situ formed amine, a precipitation polymerization proceeding in binary solvent of water and acetone.23 Briefly, 90.0 g of H2O-acetone mixture (mass ratio 3/7) was first put into a round bottom flask of 250 mL capacity, and the flask was then immersed in a water bath at 30 °C. Under stirring at 300 r/min, TDI (10.0 g) was added at a rate of 20 mL/h. The polymerization was continued for 2 h after completion of TDI addition. The solid product was separated through centrifugation and filtration, rinsed with H2O-acetone mixture and vacuum dried. Determination of Amine Group Density on PPU. The density of amine groups on PPU was determined through spectrophotometery based on absorbance of p-nitrobenzaldehyde (NBA).25,26 The process includes two steps (Details given in Part 2 in Supporting Information): Formation of imine by reacting PPU with NBA as depicted by Figure S1 in Supporting Information, and full hydrolysis of the imine by redispersing the iminized powder PPU into a mixture of anhydrous


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acetonitrile and water, in order for the PPU-imine to hydrolyze back to its original PPU and NBA, as shown by the reversing reaction in Figure S1 in Supporting Information. Reaction of Amine PPU with Glutaraldehyde and PFL Immobilization. Enzyme PFL can not be directly immobilized onto amine-bearing PPU through covalent bonding. In order to accomplish the immobilization, PPU was first activated by reacting with GA so that to attach an aldehyde functional group onto its surface, a well known practice.11,27-28 The details of the process and that of lipase immobilization are given in Part 3 in Supporting Information. Determination of PFL Activity. To determine the activity of the PFL enzyme, the immobilized PFL was used as the catalyst in the hydrolysis of NPP (p-nitrophenyl palmitate) to yield NP (p-nitrophenol). The activity of the enzyme is expressed by enzyme unit (U), which is defined as the amount of PFL protein required to yield 1.0 µmol of NP per minute in the hydrolysis of NPP under specified conditions. This definition for enzyme unit was used to express the activity for both the immobilized and the free enzyme. Detailed information for activity measurements is given in Part 4 in Supporting Information. Kinetic Resolution of (R,S)-1-PEOH. The immobilized enzyme on PPU was used as enantioselective catalyst in kinetic resolution of racemic 1-PEOH, through its esterification with vinyl acetate at 40 °C using isooctane or octane as solvent, and its performance compared with the free PFL enzyme. The detailed process is given in Part 5 in Supporting Information. Instrumentation and Supplementary Tests. Other tests or characterization with regard to PPU and its derivatives (iminized, the hydrolysate and PPU with PFL immobilized) were also done. These included, for instance, surface morphology by SEM and porous properties of PPU and its derivatives by Hg intrusion, thermal stabilities and reusability of the immobilized PFL,


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and effect of pH on its activity was also tested. The instrumentation and the process of the tests are described in details in Part 6 and Part 7 in Supporting Information. RESULTS AND DISCUSSION Reaction Time and NBA Amount Required for Amine Determination. One of the primary advantage of the process for the PPU preparation, through precipitation polymerization of TDI with in-situ formed amines, is the synthesis of a porous polyurea (PPU) with rich amine groups on the surface without need for any additives in the polymerization and without any chemical modification for the outcome product.23,24 The presence of the amine groups is an important feature, which is essential for enzyme immobilization. The density of the amine groups on PPU was therefore determined through spectrophotometry based on absorbance of NBA.25 A detailed description of the method is available in Supported Information, Part 2. The results showed that 0.153 mmol of NBA was reacted per gram of PPU (0.023 mmol/150 mg. See Part 2 in Supporting Information, Figure S2). Taking into account of the specific surface area of PPU (161.7 m2/g, see Table 2 hereafter), the density of amine groups on this PPU was readily obtained to be 0.57 NH2/nm2. Compared with reported values, which varied from 3 NH2/nm2 to 23 NH2/nm,2, 26,29,30 the present value (0.57 NH2/nm2) was lower. However, when this was expressed in number of amines per gram of support materials, the density of amine for PPU was 0.153 mmol/g, which was significantly higher than those reported (5 to 71 µmol NH2/g support).29-31 The higher specific surface area of PPU led to the lower amine density expressed by surface area. Since the iminization of PPU amine by aldehyde on NBA is a reversible reaction, an appropriate reaction time must be met to have all amine groups reacted with aldehyde groups of


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NBA. The test showed that 4 h was the reaction time needed in order to get the amine groups full reacted with NBA amount in excess. Details for the test and the result are also given in Part 2 in Supporting Information. Effect of pH in PPU Activation by Glutaraldehyde. GA activation is one of the most popular techniques to covalently immobilize enzymes on supports, since it is simple, efficient and inexpensive.10,28,32,33 PPU was activated using GA to attach an aldehyde group on its surface by covalent bonding, followed by enzyme immobilization through conjugation with the amine groups of lipase molecules. Influences of different experimental conditions, including GA concentration, enzyme concentration and pH in the process were studied. PPU activation by GA imposes significant influence on the usability of the end enzyme. This activation has been reportedly done in buffer solutions of different pH 32,34-36 or in water 37-38 under divers circumstances. Knowing that PPU was a novel material in this application, GA activation was conducted in buffer solutions of different pH and in water, followed by subsequent PFL immobilization. Different properties of the immobilized PFL were tested and given in Table 1. The data show that the immobilized PFL amount and its immobilized ratio (amount of immobilized PFL relative to that added in each run) were kept practically constant with regard to different pH in PPU activation done in buffer solution, whereas a lower PFL immobilization was detected with PPU activation conducted in water. As to the enzyme activity after immobilization, PPU activated at pH 8 showed a higher activity in comparison with the rest of the samples regardless of the medium used in PPU activation. It is well known that support activation by GA through its reaction with amine group on the support was easily affected by a numerous factors,


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including ionic strength, temperature, GA and enzyme concentration etc.13,15,32,35,39 It has been previously reported that, depending on the pH and ionic strength in GA reaction with the supports, GA molecules were presented with a vast number of different forms (i.e. monomer, dimmer or polymer of different degree with different chemical structures).13,15,27,32,35,39 Table 1. Effect of pH in PPU activation by GA on PFL immobilization and its activity pH in PPU activation by GA H2O 7.0 8.0 9.0

a

Immobilized enzyme (mg/g PPU)

80

94

92

93

Immobilized PFL ratio (%)

52

63

60

61

Activity (U/mg)

167.5

161.3

271.9

159.4

Activity retention (%)a

45

43

73

43

Value obtained with the activity of the free enzyme as 100%.

Several reports32,40 have suggested also that each amino group on the support was activated by chemical bonding with mainly one single monomeric GA under low pH and with low GA concentration; With pH increase up to 7 or slightly higher (pH8, polymeric GA was abundantly present, and their formation was hard to be controlled. By consequence, largely more than two units of GA were chemically linked to each amine group on the support. Sensitively depending on the ways of the linkage between amino and GA molecules (including the dimers and polymers), the conformation of enzyme


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molecules is prone to dramatic changes.39,41-44 And this conformational change has often great impact on its catalytic activity. Therefore, use of different immobilization strategies may yield lipase derivatives with different activity and/or enantioselectivity. In their research, both Migneault et al. 40 and Tashima et al.45 have reported that GA dimer was detected with GA treated by aqueous alkaline solution (pH 8.5), and suggested that this GA dimer was responsible for the higher activity of certain immobilized enzymes prepared using alkali-treated GA solution as a coupling agent. Based on these reports, the higher activity for PFL immobilized at pH 8 (Table 1) may be most likely caused by the reactivity of the PPU amine groups at this pH. That is to say that the reaction between the aldehyde and the amino groups is favored at alkaline pH. However, the aldehyde is a very reactive reagent and at more alkaline pH (pH>9, for instance) it reacts with itself to form polymers, and hampers the reaction with the amine groups on PPU surface. Therefore, it is very likely that the reaction of the aldehyde with amino groups will be better at pH values around 8, depending on the pKa of the amino groups and GA concentration. More aldehyde groups on the material surface, particularly more GA dimmers formed, more effective will be the immobilization procedure and higher will be the activity of the immobilized PFL. Effect of GA Concentration in PPU Activation on PFL Immobilization. In PPU activation by GA, GA concentration is also of great importance on PFL immobilization, because it imposes important influence on its structure and also on the activity of the immobilized enzyme in the subsequent step, as mentioned in the previous section. To this end, PPU activation with GA concentration varied from 0.33 wt% to 5.00 wt% (about 0.03 mol/L to 0.50 mol/L), followed immediately by PFL immobilization as afore-described. Amount of immobilized PFL (mg/g PPU)


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and the ratio of PFL immobilized (%, PFL immobilized relative to PFL added in the process) were determined. The results are presented in Figure 1A. Figure 1A shows that an increase was observed for the immobilized PFL with increasing GA concentrations up to 0.10 mol/L, followed by a plateau region with GA concentration from 0.10 mol/L to 0.33 mol/L. PFL immobilization was significantly decreased with further increase in

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Activity of immobilized PFL (U/mg)

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Concentration of GA (mol/L)

Activity retention of immobilized PFL (%)

GA concentration. This observation was in good agreement with previously reported works.32,46

Immobilized PFL (mg/g PPU)

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Figure 1 Effect of GA concentration on immobilized PFL amount (A, ●), its immobilization rate (A, ○), activity (B, ●) and activity retention of the immobilized PFL (B, ○) In Figure 1B is shown also the effect of GA concentration on the activity of immobilized PFL and activity retention relative to the free PFL. The first observation was that, not only the immobilized PFL amount was largely affected by GA concentration (Figure 1A), so was the specific activity (U/mg PFL) of the immobilized PFL (Figure 1B, solid line) and the activity retention (Figure 1B, dash line). Figure 1B indicated that the activity of the immobilized PFL was in sharp increase at low GA concentration, reaching a maximum at GA concentration of 0.17 mol/L, and followed by an abrupt decline thereafter. Influence of GA concentration in the activation on the immobilized enzyme has been largely studied.27,32,40,45,47 A well accepted conception is that an adequate GA concentration in the


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activation of enzyme support was needed to have maximal activity for the immobilized enzyme, considerable inactivation occurred when a higher GA concentration was used, while poor performance was often observed at a lower GA concentration.39 Similar to the effect of pH in PPU activation, a single monomeric GA was believed to be linked through the reaction between its aldehyde group and the amine on the support; polymeric GA was bonded the same way at high GA concentration; and only within an appropriate range of GA concentration, GA was attached to PPU surface under dimer form. With monomeric GA as the spacer, enzyme was closely attached on the surface of PPU support, the inactive closedstructure was mostly adopted for the enzyme immobilized this way.32,33,40 With polymeric GA as the spacer between PPU support and PFL, PFL was also likely to adopt the closed-structure on one hand;43 and on the other hand, the presence of abundant GA polymer around one single PFL molecule would also obstruct the active site on the enzyme, leading to a reduced activity. It was this very GA dimer link between the polymer support and the enzyme molecule which endowed the immobilized enzyme with a higher activity than the enzymes immobilized either by monomeric GA or by its polymer as repeatedly reported.32,46,40, 43,45,48 Figure 1B indicates that the highest activity retention of 76% for the immobilized PFL was obtained. Although the increase in enzyme activity was reported after immobilization,49 the decrease in activity for immobilized enzyme has been a common observation.39,41,43,48-51 With PFL immobilized on different agarose support, about 70% of the enzyme activity in the free enzyme was retained;41 with laccase immobilized on polyacrylonitrile nanoﬁbrous membranes, the immobilized laccase exhibited 72% of the free enzyme activity.43 With PFL immobilized on woolen cloth using polyimine with glutaraldehyde cross-linking under optimized conditions, the immobilized enzyme was found to have an expressed activity of 178.3 U with retained activity of


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30.2%.10 Only about 2% of the activity of the free enzyme was kept with PFL immobilized on polysiloxanes based polymer with GA as the coupling agent;51 With a lipase immobilization on poly(vinylalcohol-co-ethylene) membrane,50 the activity retention of the immobilized enzyme was about 65% with the membrane activated with GA solution of 25 wt% concentration, whereas this value was reduced to about 10% when the activation was done with GA solution of 0.3 wt%. In comparison, 76% of activity retention for immobilized PFL was relatively high in the present study. It is to point out that this activity retention of 76% for immobilized PFL, obtained with GA concentration of 0.167 mol/L, was slightly higher than that given in Table 1 (73%), which was obtained with GA concentration at 1.0 wt% (0.10 mol/L) in GA activation step, shown also in Figure 1B. Effect of PFL Concentration on Its Immobilization. The effects of enzyme concentration, on PFL immobilization (Figure 2A) and on the activity (Figure 2B) of the immobilized PFL, were studied with the enzyme concentration varied from 0.64 to 3.36 mg/mL. It was found that, similar to the case of Candida rugosa lipase immobilization on a support based on poly(ethylene-vinyl alcohol),46 the amount of immobilized PFL increased with PFL concentration, and reached a maximal immobilization (95.2 mg/g) at PFL concentration of 2.56 mg/mL. This value was kept practically constant with further increase in PFL concentration. Figure 2A showed that PFL immobilization ratio was in linear decline with PFL concentration. At very low PFL concentration (0.64 mg/mL), nearly all PFL was immobilized onto PPU, which gave an immobilization rate of 100%. This reveals that, with increase in PFL concentration, unimmobilized PFL was in constant accumulation, albeit the immobilized amount was also increasing.


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PFL concentration (mg/mL)

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Activity retention of immobilized PFL (%)

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PFL immobilization rate (%)

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PFL immobilized (mg/g PPU)

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Activity of immobilized PFL (U/mg)

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Figure 2. Effect of lipase concentration on the immobilized amount (A, ●), immobilized rate (A, ○) of PFL, activity (B, ●) and activity retention of the immobilized PFL (B, ○)

The molecular mass of PFL is of about 50 kDa.46,52 From the maximal immobilization of PFL (95.2 mg/g PPU), one was able to conclude that the number of PFL molecule immobilized on PPU was 1.147×1018/g PPU. Taking into account of the specific surface area of PPU (161.7 m2/g), PFL amount immobilized on PPU support was obtained to be 7.09×10-3/nm2. Knowing the density of the amine on PPU surface (0.57 NH2/nm2, or 0.153 mmol/g PPU), the number of PFL molecules per amine group (-NH2) on PPU surface was 1.24×10-2. In other words, there were about 80 amine groups for each PFL molecule. It is reported that the size of globular PFL molecule is of 3 nm × 4 nm × 5 nm with an average molecular diameter of about 5 nm,53 much larger compared to a single amine group. And in addition, PFL are known to aggregate often by hydrophobic interactions, forming further larger aggregates.53,54 In this way, one PFL aggregate will cover several amine groups, making a portion of amine groups on PPU inaccessible to the PFL. At the same time, the covalent bonding of PFL molecules to the surface amine groups in pores of small size (pore size99.5 >99.5 >99.5 94.1 93.6 0.8 C (%) 6.36 14.5 14.5 25.1 28.1 E >200 >200 >200 44.6 43.4 eep (%) >99.5 >99.5 >99.5 94.8 94.0 C (%) 4.22 8.38 12.6 15.1 17.6 0.4 E >200 >200 >200 44.6 39.2 94.2 93.4 91.6 91.1 88.9 eep (%) 0.8 C (%) 32.7 39.3 50.8 52.9 54.7 E ----84.0 ---eep (%) >99.5 >99.5 98.7 96.7 96.2 C (%) 13.7 24.9 39.0 46.3 49.7 0.4 E >200 >200 195.4 188 196 eep (%) >99.5 >99.5 98.5 98.3 97.5 0.2 C (%) 9.94 19.0 33.3 38.0 43.3 E >200 >200 >200 >200 181 eep (%) >99.5 >99.5 >99.5 >99.5 95.7 0.1 C (%) 6.34 9.86 14.6 15.7 17.3 E >200 >200 >200 >200 195.2 Immobilized PFL Enzyme

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a

Enzyme (mg) used in 4 mL of the reaction system containing 0.5 mmol of 1-PEOH and 1.0 mmol of vinyl acetate, prepared by mixing 2.0 mL of isooctane solution of 1-PEOH (0.25 mol/L) with 2.0 mL of isooctane solution of vinyl acetate (0.50 mol/L).

As to the reaction catalyzed with the immobilized PFL (Table 3), the relationship of the substrate conversion with the enantioselectivity was quite similar to that observed with the free PFL. With 0.8 mg of the immobilized PFL, the conversion was 50.8% at 8 h of the reaction, the corresponding E was 84 and the eep was 91.6%. With the enzyme amount decreased to 0.4 mg, the E value and the eep, at 8 h of the reaction, were largely increased to 195.4 and eep>98.7, respectively. The substrate conversion was reduced from 50.8% to 39.0% in accordance. These results demonstrate that, the catalytic enantioselectivity was improved with a lowered enzyme loading with the immobilized PFL, as observed for the free enzyme. However, when the results from the two types of the enzymes were compared, it is easy to conclude that the enantioselectivity was significantly improved with the immobilized PFL. Using 0.4 mg of the


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immobilized PFL and with the reaction time extended to 12 h, quite high enantioselectivity (E=196) was detected with a substrate conversion of 49.7%, which indicated that R-1-PEOH was practically fully reacted with vinyl acetate, and extreme-highly pure enantiomers were effectively obtained. Since the enantioselectivity was significantly improved with reduced enzyme amount, experiments were carried out using further lowered amount of immobilized PFL (0.1 mg and 0.2 mg) in the resolution. The results given in Table 3 (bottom section) revealed that, the enantioselectivity of the kinetic resolution was indeed enhanced at given reaction time. Nevertheless, this enhancement of enantioselectivity was obtained with a severe sacrifice of substrate conversion. For instance, with 0.2 mg of immobilized PFL, E>200 was maintained at 8 h of reaction time while E=195.4 was detected with 0.4 mg of immobilized PFL at the same reaction time; the substrate conversion decreased from 39% (with 0.4 mg of immobilized PFL) to 33.3% with 0.2 mg of immobilized PFL. With extended reaction time to 10 h, E value with 0.2 mg of the immobilized PFL was still kept above 200, the substrate conversion increased to 38%, still lower than that at 8 h of reaction with 0.4 mg of immobilized PFL. At 12 h of the reaction with 0.2 mg of immobilized PFL, the substrate conversion finally increased to 43.3, with the E value decreased now to 181. These comparisons demonstrate that, to get highly pure optical enantiomers, the amount of enzyme relative to the substrate is an important factor. In the present case, 0.4 mg of the immobilized PFL was the optional amount taking into account of the enantioselectivity and the reaction time. This suggests that an optimal amount for the immobilized PFL exists for an effective kinetic resolution, the resolution is less effective with either a higher or lower immobilized enzyme amount used.


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It is to point out that, with this novel PPU as the support, the immobilized PFL not only outperformed its counterpart of free enzyme, it demonstrated as well a higher enantioselectivity along with a higher substrate conversion in comparison with a number of reports on immobilized lipases or other enzymes as catalyst in the resolution of different racemates.3,50,53,60,62 With lipase from Burkgolderia cepacia immobilized inside zirconia particles as the catalyst for kinetic resolution of 1-PEOH in different solvents,60 the highest conversion of 18.7% was reported with the resolution carried out in octane after 48 h of reaction at 37 °C. Suan et al. tested 6 immobilized lipases of different microorganisms in kinetic resolution of 1-PEOH in isooctane at 35 °C,63 the one with the best performance was the lipase from Pseudomonas cepacia, which gave a conversion of 45%. PFL was also immobilized onto magnetic nanoparticles and used for resolution of 2-octanol with vinyl acetate as acyl donor in 6 organic solvents.64 Under optimized conditions, the highest enantioselectivity with E=71.5 was obtained. A starch film and its blend with polyethylene oxide at different compositions were also used for immobilization of lipases from different microorganisms.65 The immobilized lipases were used as biocatalysts in the resolution of 1-PEOH with vinyl acetate, which showed that all immobilized lipases were stereopreferential towards the (R)-enantiomer, with high enantiomeric ratio (E>200) and high eep (>99). However, extreme low conversion was obtained with all four types of lipases tested after 72 h of reaction at 35 °C. The highest conversion was 10% with immobilized lipases from Aspergillus niger. All these demonstrate that the present PPU is an effective enzyme support; highly pure enantiomers were actually achieved in 1-PEOH resolution using this immobilized PFL as the biocatalyst. In kinetic resolution by enantioselective catalysis, it is commonly accepted that efficient resolutions is possible even with moderate enantiomeric ratios (E>10); Reactions with E≥100


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allow, in general, the formation of both product and residual substrate in high optical purity.62 In principle, E should be independent of conversion, and good kinetic resolution should results in and lead to pure enantiomeric products at 50% conversion when E is higher than 200. However, the results in Table 3 seemingly indicated that the substrate conversion leveled off at early conversions; whereas the enantiomeric ratio E was decreased with time, particularly in the runs with high dosage of the catalyst. This unexpected decrease in E value has been observed and was rarely fully interpreted.3,43,66,67 It was stated that exceedingly high E values, E>200 for instance, were prone to even minor experimental errors.3,62 Small variations on enantiomeric excess may cause significant change in E value. This is the reason why expression of E>200, rather than an accurate value, is often used as in Table 3. However, the observed E variation in Table 3 should not be attributed simply to experimental errors, because it occurred in all the runs with repeated tests. In fact, the stereoselectivity of enzyme is known to be susceptible to deactivation by a number of factors, such as the medium, impurities, diverse agents present or in-situ formed in the reaction system.3,43, 66,67 The hydrolysis of the product enantiomer to compete with the acylation of the substrate was also possible reason, especially with immobilized enzyme. However, the fact that a decrease in E value with the reaction time was observed for both the free and the immobilized enzyme, suggested strongly that the immobilization was not the cause of the E value decrease. We noticed that acetaldehyde was reported to inhibit enzyme catalytic activity and enantioselectivity.68-70 Aldehyde reacts with certain amine groups to form a Schiff-base; several follow-up reactions lead to an irreversible modification for enzyme. Depending on the nature of the enzyme and the reaction concerned, different effects on various enzymatic properties, including the activity and enantioselectivity, can be imposed.68 Knowing that acetaldehyde is


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produced (Figure S3, Supporting Information), this may help to understand the decrease in E value seen in Table 3. To this end, two parallel runs to that with 0.2 mg of immobilized PFL in Table 3 were carried out in hexane, with known amount of acetaldehyde deliberately added. Conversion of racemic 1-PEOH and enantioselectivity were determined and collected in Table 4. Table 4. Influence of acetaldehyde on the kinetic resolution of 1-PEOH using immobilized PFL as the biocatalyst Acetaldehyde to Reaction time (h) the resolution Properties 4 8 12 16 24 systema eep (%) >99.5 >99.5 >99.5 99.1 98.6 0.00

0.025 mol/L

0.056 mol/L

C (%)

16.8

29.6

37.9

42.7

47.6

E

>200

>200

>200

>200

>200

eep (%)

>99.5 >99.5

98.5

97.5

96.2

C (%)

15.6

27.3

35.6

41.3

47.3

E

>200

>200

>200

160.5 145.8

eep (%)

>99.5

98.1

97.5

96.9

93.5

C (%)

14.4

25.9

33.8

40.1

45.1

E

>200

144.0

130.5 125.4

69.6

a

Enzyme (mg) used in 4 mL of the reaction system, containing 0.5 mmol of 1-PEOH (0.125 mol/L) and 1.0 mmol of vinyl acetate (0.250 mol/L), prepared by mixing 2.0 mL of hexane solution of 1-PEOH (0.25 mol/L) with 2.0 mL of hexane solution of vinyl acetate (0.50 mol/L).

Comparing the results in Table 4, it was seen that both the activity and the enantioselectivity of the catalyst were deteriorated in the presence of acetaldehyde. While the conversion was slightly reduced, more significant decreases were observed for the enantiomeric ratio E, indicating a deactivation of the enantioselectivity of the biocatalyst. With the concentration of the added acetaldehyde at 0.025 mol/L, which corresponded to 25% of the conversion of the Renantiomer, E value fell from >200 to 160 at 16 h of the reaction, and it was further decreased to 125.4 with the concentration of the acetaldehyde at 0.056 mol/L, the concentration


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corresponding to 45% of the conversion of the R-enantiomer. Based on these results, the data in Table 3 can be well interpreted taking into account that acetaldehyde was in constant increase in the process of racemic 1-PEOH resolution. CONCLUSIONS Through spectrophotometric absorbance of p-nitrobenzaldehyde, surface amine group density of a porous polyurea (PPU), prepared through a simple and one step precipitation polymerization of toluene diisocyanate with water in binary solvent of water-acetone, was first determined to be 0.57 NH2/nm2. This PPU was then activated by the reaction of its surface amine groups with GA, and followed by immobilization of an enzyme, PFL, through covalent bonding with the amine groups of lipase molecules. The immobilization was carried out under different experimental conditions, including GA concentration prior to immobilization, enzyme concentration and pH in the process. The results demonstrate high immobilization rate of PFL with high activity, determined through its catalysis in hydrolysis of p-nitrophenyl palmitate, was achieved at pH 8.0. GA concentration has an important effect on PFL immobilization and the activity of the immobilized enzyme. While high PFL immobilization was obtained with GA concentration between 0.10 mol/L and 0.33 mol/L, the highest activity of 283 U/mg for the immobilized PFL was observed at GA concentration of 0.167 mol/L (1.67 wt%), which represented an activity retention of 76% relative to that of the free PFL. The porous structure of PPU was also characterized after its activation by glutaraldehyde and PFL immobilization, which indicated that the specific surface area in initial PPU was decreased after GA activation, and further reduced after PFL immobilization. The activity of the immobilized PFL at different temperature and pH and the stability at given temperature were all enhanced by immobilization. The immobilized enzyme was finally used as enantioselective catalyst in kinetic resolution of racemic 1-PEOH,


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and its performance compared with the free PFL enzyme. Highly pure enantiomers from racemic 1-PEOH were effectively achieved using the immobilized PFL. The activity and enantioselectivity of PFL were deactivated at certain extent, owing to the in-situ formed acetaldehyde, confirmed by parallel tests with added acetaldehyde in the resolution process. In addition, the immobilized PFL was readily recovered from the reaction system and reusable. 73% of the initial activity was retained after 5 repeated reuse cycles. This PPU is therefore a promising support for enzyme immobilization and for subsequent application as biocatalyst. ASSOCIATED CONTENT Supporting Information Supporting Information, which contains the detailed information on chemicals and reagents used in this work, processes for determination of amine amount and enzyme activity, process for GA activation and enzyme immobilization on the PPU material and the relative method for the characterization of the PPU, testing experiments for thermostability and reusability of the immobilized PFL etc, is provided. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. ACKNOWLEDGMENT This work is supported by grants from National Natural Science Foundation of China (NSFC 21274054 and 51473066).


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Immobilization of Lipase from Pseudomonas fluorescens on Porous

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