Cell Debris Self-Immobilized Thermophilic Lipase: a ... - Springer Link

Appl Biochem Biotechnol (2013) 170:399–405 DOI 10.1007/s12010-013-0152-z

Cell Debris Self-Immobilized Thermophilic Lipase: a Biocatalyst for Synthesizing Aliphatic Polyesters Yang Sun & Yan Yang & Chenhui Wang & Jiaming Liu & Wei Shi & Xiaobo Zhu & Laijin Lu & Quanshun Li

Received: 21 August 2012 / Accepted: 18 February 2013 / Published online: 28 March 2013 # Springer Science+Business Media New York 2013

Abstract The paper explored the catalytic activity of a cell debris self-immobilized thermophilic lipase for polyester synthesis, using the ring-opening polymerization of ε-caprolactone as model. Effects of biocatalyst concentration, temperature, and reaction medium on monomer conversion and product molecular weight were systematically evaluated. The biocatalyst displayed high catalytic activity at high temperatures (70–90 °C), with 100 % monomer conversion. High monomer conversion values (>90 %) were achieved in both hydrophobic and hydrophilic solvents, and also in solvent-free system, with the exception of dichloromethane. Poly(ε-caprolactone) was obtained in 100 % monomer conversion, with a number-average molecular weight of 1,680 g/mol and a polydispersity index of 1.35 in cyclohexane at 70 °C for 72 h. Furthermore, the biocatalyst exhibited excellent operational stability, with monomer conversion values exceeding 90 % over the course of 15 batch reactions. Keywords Cell debris . Immobilization . Thermophilic lipase . Ring-opening polymerization . ε-Caprolactone

Yang Sun and Yan Yang contributed equally to the work.

Y. Sun : Y. Yang : C. Wang : J. Liu : W. Shi : Q. Li (*) Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, College of Life Science, Jilin University, Changchun 130012, China e-mail: [email protected] Y. Sun : X. Zhu Department of Neurosurgery, the Second Part of First Hospital, Jilin University, Changchun 130023, China L. Lu (*) Department of Hand Surgery, First Hospital, Jilin University, Changchun 130023, China e-mail: [email protected]

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Appl Biochem Biotechnol (2013) 170:399–405

Introduction Aliphatic polyesters are a group of remarkably biocompatible and biodegradable polymers, and are widely used as resorbable implant materials for tissue engineering and as vehicles for drug/gene delivery [1]. Generally, they are synthesized using toxic organometallic catalysts, e.g., aluminum alkoxides and tin carboxylates. To avoid the harmful effects of these catalysts, enzymatic polymerization has been extensively developed over the past two decades [2–7], and it is of potential to be a powerful alternative to the toxic, metal-based catalytic systems. Moreover, it has the advantages of mild reaction conditions, recyclability of enzymes, and high chemo-, enantio- and regioselectivity [3]. To date, many lipases and esterases have been reported to possess the ability to catalyze the synthesis of aliphatic polyesters, e.g., porcine pancreatic lipase, Pseudomonas cepacia lipase, Candida antarctica lipase B (CALB), Humicola insolens cutinase, and Archaeoglobus fulgidus esterase [8–12]. Among them, CALB showed the highest catalytic activity toward a wide range of monomers [3]. Using ε-caprolactone as the monomer, CALB could catalyze the synthesis of poly(ε-caprolactone) with an isolated yield of 85 % and a number-average molecular weight (Mn) of 44,500 g/mol at 70 °C for 4 h [10]. However, enzymatic polymerization is still in the research and development stage, mainly due to the high cost, low catalytic activity, and stability of these enzymes. Thus, developing a commercially viable biocatalyst will be of great significance for the green and sustainable synthesis of polyesters at an industrial scale. One efficient strategy is to construct the immobilized enzymes [13–15] or enzymes displayed on Escherichia coli or yeast [16–20], which provides economic benefits to the biocatalytic process or organophosphorus compound detection through enzyme reuse and improvements in enzyme stability. In the previous study, the thermophilic lipase gene FN1333 from Fervidobacterium nodosum was cloned and over-expressed in E. coli [21], and the recombinant enzyme could efficiently catalyze the ring-opening polymerization of ε-caprolactone [22]. Moreover, the enzyme was probably bound to or associated with the E. coli cell membrane due to its hydrophobicity, and expressed in an active and insoluble form. Thus, the cell debris after cell disintegration might be an efficient self-immobilized biocatalyst for the preparation of aliphatic polyesters. Herein, we first constructed this unique cell debris self-immobilized thermophilic lipase through ultrasonic cell disintegration and then employed it in the synthesis of aliphatic polyesters, using the ring-opening polymerization of ε-caprolactone as model. Effects of reaction conditions on monomer conversion and product molecular weight were systematically evaluated. The operational stability of the biocatalyst was also investigated.

Materials and Methods Materials The recombinant E. coli BL21 strain, which harbors the thermophilic lipase gene FN1333 from F. nodosum, was constructed using the expression vector pET-28a and stored in our laboratory [21]. ε-Caprolactone was purchased from Fluka Chemical Co. in the highest available purity and used as received. Yeast extract and tryptone was purchased from Oxide Ltd. Kanamycin and isopropyl β-D-thiogalactopyranoside (IPTG) were purchased from Sigma. Organic solvents of analytic grade were purchased from Beijing Chemical Co. (Beijing, China) and dried over 4-Å molecular sieves before use.


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Construction of Cell Debris Self-Immobilized Thermophilic Lipase The recombinant E. coli BL21 harboring FN1333 from F. nodosum was cultured in 2YT medium (1 % yeast extract, 1.6 % tryptone, and 0.5 % NaCl) containing kanamycin (50 μg/mL) at 37 °C. When the optical density of culture reached 1.0, the induction was carried out by adding IPTG at a final concentration of 1 mM and shaking for an additional 6 h at 37 °C. The cells were harvested by centrifugation at 8,000 rpm for 15 min at 4 °C. The harvested cells were frozen and thawed three times, and then suspended in 50 mM phosphate buffer (pH8.0) at a ratio of 1:8 (w/v). After ultrasonic cell disintegration and centrifugation at 8,000 rpm for 15 min, the pellet was collected, washed with deionized water, lyophilized, and then used as the catalyst. Ring-opening Polymerization of ε-Caprolactone A quantity of the cell debris self-immobilized enzyme (typically 30–150 mg) was dried in a desiccator overnight and then transferred to a dried screw-capped vial containing 200 μL ε-caprolactone and 600 μL organic solvent (no solvent addition for solvent-free system). Ethylbenzene (50 μL) was added as an internal standard for the quantification of monomer conversion by gas chromatography (GC). The vial was then sealed and placed into a thermostatic reactor, and the reaction mixture was stirred at 180 rpm at the appropriate temperature. Reaction was terminated by adding dichloromethane and filtrating to remove the biocatalyst. The biocatalyst was washed three times with dichloromethane, and the filtrate was collected and evaporated under reduced pressure to remove dichloromethane. Afterward, the viscous sample was precipitated in methanol at −20 °C, and the cloudy solution was centrifuged (8,000 rpm, 15 min, 4 °C). The white precipitate was then collected and dried in a vacuum oven. 1 H NMR (500 MHz, CDCl3) of poly(ε-caprolactone): 1.39 (m, -COCH2CH2CH2-), 1.66 (m, -COCH2CH2CH2CH2CH2O-), 2.31 (t, -COCH2-), 4.06 (t, -CH2O-), 3.66 (t, -CH2OH end group), 2.36 ppm attributed to –COCH2- of cyclic oligomers. Determination of Monomer Conversion Monomer conversion values were determined by GC using a Shimadzu 2014 gas chromatograph equipped with an Rtx-1 capillary column (30 m×0.25 mm×0.25 μm) and a hydrogen flame ionization detector. Nitrogen was used as the carrier gas. The temperatures of injection pool and detector were 200 and 240 °C, respectively. The column temperature was held at 70 °C for 2 min and then programmed to rise at 10 °C/min to a final temperature of 140 °C, which was then maintained for 2 min. The injection volume was 1.0 μL. Determination of Molecular Weight and Polydispersity Index The Mn, weight-average molecular weight (Mw), and polydispersity index (PDI) (Mw/Mn) values of products were determined by gel permeation chromatography (GPC). Analyses were carried out using a Shimadzu HPLC system equipped with a refractive index detector and Shim-pack GPC-804 and GPC-8025 ultrastyragel columns in series. Tetrahydrofuran was used as the eluent with a flow rate of 1.0 mL/min at 40 °C. The sample concentration and injection volume were 0.3 % (w/v) and 20 μL, respectively. The GPC system was calibrated with polystyrene standards of narrow molecular weight distribution.

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Operational Stability Analysis To test the operational stability of the cell debris self-immobilized enzyme, the biocatalyst was recovered by filtration after each batch, and then, the recycled biocatalyst was reused for the next batch reaction under the same reaction conditions.

Results and Discussion Effects of Amount of Cell Debris Self-Immobilized Enzyme The thermophilic lipase from F. nodosum was over-expressed in E. coli in an active and insoluble form, and could be extracted and isolated by a unique thermal solubilization procedure [21]. Thus, the enzyme was probably bound to or associated with the E. coli cell membrane due to its high hydrophobicity. In the present research, we constructed the cell debris selfimmobilized thermophilic lipase through the ultrasonic cell disintegration and lyophilization, and employed the biocatalyst in the ring-opening polymerization of ε-caprolactone. The effects of amount of cell debris self-immobilized enzyme (30–150 mg) were first investigated in toluene at 70 °C for 72 h. As shown in Table 1, compared with the blank reaction, high monomer conversion values (>90 %) could be achieved in the range of 30 to 150 mg of self-immobilized thermophilic lipase. With the increasing of amount of biocatalyst, monomer conversion values exhibited an increasing tendency, and the monomers could be completely converted into the polymer chain when the amounts of biocatalyst were higher than 50 mg. Nevertheless, the amount of biocatalyst had almost no influence on the product Mn values (1,190–1,280 g/mol), and the polymers obtained were of narrow molecular weight distribution with PDI values of ca. 1.30. Thus, 50 mg of self-immobilized enzyme was selected as the optimal amount of biocatalyst in the subsequent research. Effects of Temperature on Monomer Conversion and Mn To examine the thermal stability of the cell debris self-immobilized enzyme, reactions were conducted at different temperatures in toluene for 72 h, and the monomer conversion and Mn values were determined in each case. As shown in Fig. 1, with the increasing of reaction temperature, monomer conversion values exhibited an obvious increasing tendency, and monomers were completely converted when the temperatures were higher than 70 °C. Using 50 mg of free enzyme as catalyst, the monomer conversion only reached 80 % at 70 °C for Table 1 Effects of amount of cell debris self-immobilized enzyme on monomer conversion and product molecular weight. The reactions were conducted in toluene at 70 °C for 72 h Mn (g/mol)

PDI

91

1,190

1.33

100

1,240

1.29

100 150

100 100

1,250 1,280

1.30 1.30

50

No product

Entry

Amount of biocatalyst (mg)

Monomer conversion (%)

1

30

2

50

3 4 Blanka

In the blank reaction, ring-opening polymerization of ε-caprolactone was performed using the E. coli BL21 strain harboring no target gene FN1333

a

Appl Biochem Biotechnol (2013) 170:399–405 1500

Monomer conversion Mn

100

80 1000 60

40

500

Mn (g/mol)


403

20

0

0 50

60

80

70

90

o

Temperature ( C) Fig. 1 Effects of temperature on monomer conversion and product molecular weight Mn. These reactions were conducted in toluene using 50 mg of cell debris self-immobilized enzyme at different temperatures for 72 h

72 h [23]. For the cell debris self-immobilized enzyme, the lipase was associated with the cell membrane and thus caused the beneficial effect of the hydrophobic cell membrane on the stability and catalytic activity of the recombinant enzyme. The Mn values of products were in the range of 930–1,280 g/mol and also showed little dependence on the reaction temperature. From the above results, the synthesized polymers are of low molecular weight and narrow molecular weight distribution, and potential to be widely used as the soft block in a thermoplastic elastomer or carriers for localized drug delivery. Effects of Reaction Medium on Monomer Conversion and Mn When biocatalytic reactions are conducted in non-aqueous medium, the nature of the solvent employed plays a crucial role in determining the stability of the biocatalyst and in the partitioning of substrates and products between the solvent and the biocatalyst [24]. To evaluate the activity and stability of the cell debris self-immobilized enzyme in various organic media, enzymatic polymerization in a series of solvents with different log P values were conducted. The results were summarized in Table 2. High monomer conversion Table 2 Effects of organic solvents on monomer conversion and product molecular weight. The reactions were conducted at 70 °C for 72 h

Solvent

Log P


Mn (g/mol)

PDI

1,4-Dioxane

−1.10

96

830

1.15

Acetone

−0.23

94

770

1.15

Tetrahydrofuran

0.49

100

850

1.14

Dichloromethane

0.93

84

860

1.12

Chloroform

2.00

95

930

1.17

Toluene

2.50

100

1,240

1.29

Cyclohexane n-Hexane

3.09 3.50

100 100

1,680 1,620

1.35 1.35

Solvent-free

–

97

1,540

1.33

404


values (>90 %) were achieved in both hydrophobic (toluene, cyclohexane, and nhexane) and relatively hydrophilic (1,4-dioxane and acetone) solvents, and also in solvent-free system, with an exception of dichloromethane (84 %). Generally, hydrophobic solvents were favorable for enzymatic polyester synthesis with high monomer conversion and product molecular weight [25, 26]. Thus, the cell debris selfimmobilized enzyme could tolerate a broader range of organic solvents than the free enzyme. Relatively higher Mn values were obtained in hydrophobic solvents, with the highest Mn value of 1,680 g/mol in cyclohexane. This phenomenon was usual in enzymatic polymerization and could be explained by the fact that hydrophobic solvents are favorable for the retention of essential water molecules on the enzyme’s surface, which helps it maintain its catalytic conformation and activity. Meanwhile, higher PDI values were also obtained in hydrophobic solvents, and this was probably caused by hydrophobic solvents resulted in improved polyester synthesis activity, but also in increased rates of hydrolysis and transesterification, and, in turn, resulted in a polymer with a broader molecular weight distribution. Operational Stability Analysis To test the operational stability of the cell debris self-immobilized enzyme, a series of consecutive ring-opening polymerization reactions were performed in toluene at 70 °C for 72 h using 50 mg biocatalyst. After each reaction, the self-immobilized enzyme was recovered by filtration, washed with dichloromethane to remove the residual substrate and product, and then reused in the next batch reaction. As shown in Fig. 2, high monomer conversions (>90 %) were obtained over the course of 15 batch reactions, and all the reactions afforded comparable M n values (960– 1,240 g/mol). Thus, this unique self-immobilized enzyme exhibited excellent operational stability than the recombinant E. coli whole-cell biocatalyst [23], and it might offer dramatically improved cost effectiveness relative to conventional enzymatic approaches in industrial settings.

1500

1200

80

60 900

40

Mn (g/mol)


100

600

20

0

0

1

2

3

4

5

6

7

8

9 10 11 12 13 14 15 16 17

300

Batch number

Fig. 2 Monomer conversion and Mn values for a series of consecutive batch reactions conducted using a single 50-mg sample of the cell debris self-immobilized thermophilic lipase; on completion of one reaction, the catalyst was washed and recycled for use in the next. Reactions were conducted at 70 °C for 72 h each, using 200 μL ε-caprolactone and 600 μL toluene


405

Conclusion In conclusion, a unique cell debris self-immobilized thermophilic lipase was successfully constructed and showed a promising activity for the synthesis of aliphatic polyesters. This biocatalyst offered significant advantages over the conventional enzymatic process due to its high catalytic efficiency, low cost of production, and excellent operational stability. Thus, this unique immobilized enzyme is of great potential to be widely used in the mild, metal-free synthesis of polyesters, which are especially used as carriers for drug/gene delivery. Acknowledgments This work was supported by the Natural Science Foundation of China (nos. 21204025, 21074042, and 81102383), the Ministry of Science and Technology of China (International Cooperation and Communication Program 2011DFR51090), Doctoral Fund of Young Scholars of the Ministry of Education (no. 20110061120028), the China Postdoctoral Science Foundation (no. 20110491328), the grant from Jilin Province Science & Technology in China (no. 201101040), and Development and Reform Commission of Jilin Province (no. JF2012C007-3).

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