Scholarly article on topic 'Carboxylesterase from Spodoptera Litura: Immobilization and use for the Degradation of Pesticides'

Carboxylesterase from Spodoptera Litura: Immobilization and use for the Degradation of Pesticides Academic research paper on "Chemical sciences"

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Abstract of research paper on Chemical sciences, author of scientific article — Jianxiong Diao, Guangyu Zhao, Yuanqing Li, Jinli Huang, Ying Sun

Abstract The immobilization of carboxylesterase from Spodoptera Litura in mesoporus molecular sieves was studied in this paper. Two different types of mesoporous sieves (MCM-41, SBA-15) were initially used as enzyme support to compare the efficiency of immobilization. The outcome showed the maximum enzyme loadings, twice as many in MCM-41, are 0.0267 L/g in SBA-15 support. The most efficient pH value for immobilization was at 6.5 for both types of the support and the optimal immobilizing time was 2h for SBA-15 and 4h for MCM-41. Hence, the SBA-15 mesoporous support was determined as the enzyme immobilization matrix to continue the investigation because of its superior properties for enzyme immobilization. Then a series of characters of the free and immobilized Carboxylesterase were compared. As a result, the immobilized enzyme was substantiated to be obviously more stable than the free enzyme under the variant conditions. The experiments of pesticides degradation show that, relative to degradation in natural environment, immobilization enzyme had a higher ability to reduce the organic compounds with ester bonds.

Academic research paper on topic "Carboxylesterase from Spodoptera Litura: Immobilization and use for the Degradation of Pesticides"

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Environmental Sciences

Procedia Environmental Sciences 18 (2013) 610-619

2013 International Symposium on Environmental Science and Technology (2013 ISEST)

Carboxylesterase from Spodoptera Litura: Immobilization and use for the degradation of pesticides

Jianxiong Diaoa, Guangyu Zhaob, Yuanqing Lia, Jinli Huangb, Ying Suna*

aCollege of Resources and Environmental Sciences, China Agricultural University, Beijing 100193, China bDepartment of Chemistry, China Agricultural University, Beijing 100193, China

Abstract

The immobilization of carboxylesterase from Spodoptera Litura in mesoporus molecular sieves was studied in this paper. Two different types of mesoporous sieves (MCM-41, SBA-15) were initially used as enzyme support to compare the efficiency of immobilization. The outcome showed the maximum enzyme loadings, twice as many in MCM-41, are 0.0267 L/g in SBA-15 support. The most efficient pH value for immobilization was at 6.5 for both types of the support and the optimal immobilizing time was 2 h for SBA-15 and 4 h for MCM-41. Hence, the SBA-15 mesoporous support was determined as the enzyme immobilization matrix to continue the investigation because of its superior properties for enzyme immobilization. Then a series of characters of the free and immobilized Carboxylesterase were compared. As a result, the immobilized enzyme was substantiated to be obviously more stable than the free enzyme under the variant conditions. The experiments of pesticides degradation show that, relative to degradation in natural environment, immobilization enzyme had a higher ability to reduce the organic compounds with ester bonds.

© 2013 The Authors. Published by Elsevier B.V.

Selection and peer-review under responsibility of Beijine Institute of Techno logy. Keywords: Casboxylrsfrsasr; Immobilization; Mesoposous moleculas sieves; pesticide degradation

1. Introduction

Pesticides, the most cost-effective means of pest and weed control, ase dramatically incseased due to the incasing agsicultuse practices. The sepeated pesticide usage cassies away from Seated fields to aiT, othes land and wates bodies which causes de^^rnta! effect on the environment and biota[1,2]. Most pesticides sequin mose effectively practical methods on account of theis toxicity and high chemical stability [3]. Nowadays, sevesal novel methods ase applied to the degsadation of pesticides, such as the

* Corresponding authors. Tel./fax: +86 10 62734676. E-mail address: sunying@cau.edu.cn

1878-0296 © 2013 The Authors. Published by Elsevier B.V.

Selection and peer-review under responsibility of Beijing Institute of Technology.

doi: 10.1016/j.proenv.2013.04.084

photocatalytic and the enzymatic degradation [4-7]. Among these methods, the enzymatic degradation and hydrolysis exhibits promising application by virtue of its high bio-catalytic and environmental-friendly characteristics. Carboxylesterase (CbE), equipped with hydrolases group, have the capacity of hydrolyzing carboxyl esters. In insects, esterases play an important role in the degradation of organophosphates, carbamates and synthetic pyrethroids insecticides. The enzyme thusly could be applied referring to dispose those relevant pesticides which possess carboxyl esters. The ability of CbE to hydrolyze these insecticides brings expansive prospect of using it to deal with insecticides pollution. In order to develop feasible application of CbE, this work was focused on the issue how to use the enzyme so efficient that it can be turned to practical account.

So much is certain: even though enzymes are highly active biocatalysts, they cannot be always used for biotechnological applications due to their low stability. This deficiency can be improved via the immobilization on solid supports. The approach permits highly selective catalysis to be performed using materials that are chemically and mechanically stout and readily separated from reaction mixtures. Enzyme stability may be enhanced due to reduced autolysis in the case of protease enzymes, and more generally reduced protein aggregation, as a result of separation of enzyme molecules adsorbed on the surface[8]. In principle, the ordered mesoporous materials are one of the potential candidates. Such materials were firstly synthesized via sol-gel route[9,10]. They were well-known to apply to many fields such as catalysts[11,12]; recovery of metal ions[13,14]; Adsorption of Formaldehyde vapor[15] and also to immobilization of enzymes or protein[16,17]. The comparatively large pore dimensions of these mesoporous materials offer the possibility of accommodating small enzymes within the channels, Taking advantages of their large pore sizes and high surface areas, enzymes can be directly immobilized through the interaction with surface silanol groups by hydrogen-bonding[8,18]. As a result, the enzyme immobilized on mesoporous supports may show higher thermal and pH stability than free enzyme.

In the present work, the conditions of CbE's immobilization in mesoporous MCM-41 and SBA-15[19] was studied, then the SBA-15 was determined to serve as the better support to continue the investigation that focus on the stability of the immobilized enzyme.

2. Materials and methods

2.1. Preparation of Carboxylesterase

Carboxylesterase (CbE), from Spodoptera Litura, was obtained as solution in pH7.0 buffer (50 mmol/L phosphate) from Prof. Qiao Chuanling, Institute of zoology in Chinese Academy of Sciences. The initial active unit of CbE was in the cell powder, determined with the (3-Naphthyl acetate as substrate, was about 0.8771U/g. Before immobilization, the enzymatic protein from the cell powder was released and dissolved in buffer.

2.2. Preparation of pure silica MCM-41 and SBA-15 mesoporous molecular sieves

MCM-41 was prepared starting with a reaction mixture containing CTAB (Cetyltrimethylammonium bromide, as template): TEOS (Tetraethylorthosilicate): H2O:NH3«H2O in molar ratios of 0.23:1.55:100:12.22. After stirring for 2 h, the mixture was heated for 60 h at 373 K in an autoclave under static conditions. The white solid was filtered before being washed by distilled water and dried at 50 °C for 24 h. The dry precipitate was calcined at 550°C for 9 h under air.

Pluronic P123 triblock copolymer (EO20-PO70-EO20) was used as surfactant template to synthesize SBA-15. The mixture was stirred at 40 °C for 4 h after dissolving a mass of 2 g surfactant in 0.06 L of 2 mol/L HCl and 0.015 L distilled water. Then 0.00456 L of TEOS was added and the solution was

continued to stir at the same temperature for 5 h. Finally, the mixture was transferred into a sealed Teflonlined autoclave at 100 °C for 72 h. The white precipitate was filtered, washed with distilled water and air dried. Then the white solid was calcined at 550 °C for 6 h under air.

2.3. Characterization of mesoporous molecular sieves

The calcined MCM-41 and SBA-15 products were analyzed on a Philips X'Pert System diffractometer using CuK radiation. The scans were taken from 29 = 0.5° to 10°.The BET surface area and pore volume of calcined MCM-41 and SBA-15 were measured by nitrogen adsorption-desorption at -196 °C by using a Quantachrome AS-6 appliance.

2.4. Activity assay

The CbE activity was measured via the hydrolysis reaction of P-Naphthyl acetate. Each assay sample contains 10 nL of enzyme protein solution or 0.05 g immobilized enzyme, 200 nL of 0.03 mol/L p-Naphthyl acetate which dissolved in ethanol and 5ml of 0.02 mmol/L phosphate buffer (pH 7.0). The reaction was terminated by SDS solution and the produce of hydrolysis was dyed with fast blue B salt before checking up by measuring the absorbance at 555 nm under a temperature of 298K using a PE UV-Vis spectrophotometer. All reactions were performed in triplicate.

2.5. Immobilization of CbE to mesoporous materials

The immobilization of enzyme in Mesoporous materials was carried out according to the following procedures. 0.15 g mesoporous supports in 5 ml pH 7.0 phosphate buffers were added to 2ml Carboxylesterase solution in the flask. The mixture was rotated at 180 r/min for several hours in icy-water environment. The immobilized enzyme was separated from solution by filtering and washed with phosphate buffer until the residual liquid didn't display any enzymatic activity. The immobilized CbE had to be kept moist in tube, since drying causes the inactivation of the enzyme[20].

2.6. Determination of immobilization conditions

The method involved a feasible orthogonal design with 25 runs, 3 factors (enzyme volumes, pH value and immobilizing span of time) and 5 levels (Table 1). The general process involved following step: Certain volumes of enzyme solutions were added to a suspension which contained 0.15 g mesoporous supports and 5 ml phosphate buffer of which values ranged from 6 to 8 at every 0.5 intervals in flasks. The mixture was rotated at speed 180 r/min for several hours in ice water environment. After being filtered and washed by phosphate buffer, the immobilized CbE enzymes were kept in tubes so as to measure activity assay.

Table 1. factor-levels table of orthogonal experiment.

Levels enzyme volumes(ml) pH value immobilizing span of time(h)

1 0.5 6.0 0.5

2 1.0 6.5 1.0

3 2.0 7.0 2.0

4 4.0 7.5 4.0

5 8.0 8.0 8.0

2.7. Investigation of the characters of immobilized CbE

In osdes to investigate the characteristics of immobilization enzyme, its activities in diffesent conditions was studied, compared with the fsee enzyme. The expesimental psogsams compsised following psoceduses.

Psoceduse 1: Consesving the free enzyme and immobilized enzyme at upwasd tempesatuses (277 K, 298 K, 308 K, 323 K, 343 K) in wates bath fos 30 minutes, then the activity of fsee enzyme and immobilized enzyme wese detesmined at 298 K.

Psoceduse 2: Aftes 5 minutes catalytic seaction in wates bath at series of tempesatuses at 301 K, 310 K, 323 K, 328 K, 333 K and 338 K, the activities of the fsee and immobilized enzymes wese measused.

Psoceduse 3: the enzymes wese added in phosphate buffes with variant pH values, the selative activity was detesmined aftes the hydsolysis seaction fos 5 min in 310 K wates bath.

Psoceduse 4: three diffesent osganic solvents wese added (methanol, ethanol and acetone) into pH 7.0 phosphate buffes (V/V = 1:5) and consesved the enzymes in the osganic solutions sespectively fos 30 min. Then the activities of the enzymes wese detected.

Psoceduse 5: mixed in diffesent sosts and concentrations of the inosganic salt solutions fos 30 minutes in 298 K, the activities of fsee enzymes and supposted enzymes then wese detesmined sespectively.

Psoceduse 6: in osdes to compare the stabilities between fsee and supposted enzyme in denatusant, 3 mol/L usea solution was psepared as enzymic denatusant, then consesved both sosts of enzymes in the solution fos diffesent span of time. Finally, the activities of enzymes wese detesmined.

Psoceduse 7: how long the enzyme could consesve is anothes sticking factos to the stability. Sevesal amounts of fsee and supposted enzymes wese stosed fos 3 months and monitosed the seduction of then activities on time, so as to compare the stability within them.

2.8. Degradation of pesticides

Likewise, in osdes to evaluate the enzymatic (CbE) activity of the biocatalyst on the degsadation of pesticides, the degsadation of malathion and die^iT^a^ fsom an aqueous solution was studied. In this case, catalytic tests wese cassied out by adding the immobilized enzyme to 10 mg/L malathion and diethofencarb aqueous solution containing 0.2 mol/L phosphate buffes pH 7.0 (seposted pH fos maximum activity of CbE).

Pesticides (malathion and diethofencasb) monitoring was sealized using a Agilent 1200 HPLC equipped with a reverse phase column (CI8 Platisil ODS 5|im, 250*4.6mm) with methanol/wates (70/30, v/v%, malathion) and (90/10 v/v%, diethofencarb) mobile phase at a flow rate of 1.0 mL min-1. UV detectos of HPLC is set 254 nm fos malathion and 244 nm fos diethofencasb samples. Samples collected at regular intervals were filtered through 0.2 lm micro syringe filters (Millipore millex PVDF, 13 mm).

3. Results and discussion

3.1. Characterization of pure siliceous MCM-41 and SBA-15

The XRD pattern of calcined MCM-41 and SBA-15 are depicted in Fig.1.a and Fig.1.b, as well as the N2 isothesm sosption/desosption cusve of the both supports ase shown in Fig. 2.a and Fig. 2.b, in which a type IV isothesm with a H hystesesis of typical mesoposous matesials ase well postrayed. Meanwhile, the BET surface aseas, BJH pose diametess and mesopose volumes of two moleculas sieve samples wese calculated and shown in Table 2. TEM images of the mesoposous samples in Fig. 3.a and Fig. 3.b. The typical hexagonal pattesn and cylindsical parallel channels can be cleasly discesned.

Table 2. Physical properties of mesoporous molecular sieves MCM-41 and SBA-15.

Sample Pore diameter Pore volume

(nm) (m2/g) (cm3/g)

MCM-41 2.74 807 1.10

SBA-15 32.9 730 1.17

Fig. 1. XRD of the MCM-41(a) and SBA-15^) from 20 = 0.5-10°.

Fig. 2. N2 adsorption/desorption isotherms for calcined MCM-41(a) and SBA-15(b).

Fig. 3. TEM images of the mesoporous samples. The typical hexagonal pattern and cylindrical parallel channels can be clearly discerned.

3.2. Comparison of immobilization conditions

After orthogonal test, Table 3 displayed the optimal generalization in enzyme-loading volumes, pH conditions and immobilizing span of time. The figures indicated that comparing to MCM-41, SBA-15 was capable of loading more volumes of enzyme (0.0267 L/g versus 0.0133 L/g) as well as attaining a loading-unloading equilibrium in a shorter span of time (2 h versus 4 h) when other latent factors which could probably affect the relative activity of immobilized enzyme were consistent. These advantages might be attributed that SBA-15 support possesses a larger pore diameter than MCM-41 support, that means, a further increase in the channel size of the support renders the enzymes were entrapped in SBA-15 channels more readily. In addition, the optimal pH value amidst the course of immobilization was 6.5 for both mesoporous supports, on the inner surface of which was conjectured that due to the weak acidic hydroxyl, the enzyme could be tied tightly with the support via hydrogen bonds.

To sum up the results above, a conclusion that SBA-15 mesoporous sieves possess more superior characteristics than MCM-41 supports in immobilization of Carboxylesterase can be drawn. Considering these advantages, SBA-15 mesoporous sieves was selected as enzyme supports to continue the following study so that it may give rise to more evident results relatively.

Table 3. Optimal immobilizing conditions in mesoporous molecular sieves MCM-41 and SBA-15.

Optimal enzyme-loading Optimal immobilizing Optimal immobilizing span

volumes(L/g) pH values of time(h)

MCM-41 0.0267 6.5 2

SBA-15 0.0133 6.5 4

3.3. Investigation of the characters of SBA-15 immobilized CbE

In order to investigate the superiority of immobilized enzyme, its activities in different conditions were studied, comparing with the free enzyme.

It shows that the relative activity of immobilized enzyme declined more slowly than free enzyme before 308 K in Fig. 4. , which denoted the thermal stability of the immobilized enzyme was better than emancipated enzyme within a range of temperature. The channel wall's providing protection could account for this stability. The enzymes entrapped in the channel were isolated from heat outside relatively. Nevertheless, After 320 K, the two sorts of enzyme merely have few activities.

Fig. 4. Thermal stability of immobilization enzyme and free enzyme at different temperatures

Fig.5. shows the optimal reaction temperature of the free enzyme was ^303 K,while that of the immobilized enzyme was about 310 K, which showed that the heat-resistance of the immobilized enzyme

obtained a manifestly enhancement. With the increase of the temperature, the activity of the free enzyme decreased after 301 K, while that of the immobilized enzyme had an ascendant trend from 301 K to 310 K and then declined slowly. Heat could activate the enzyme but increase the loss of activity.

Fig. 5. Stability of immobilization enzyme and free enzyme at different reaction temperatures

Solution condition such as pH values as one of evident factors could alter the activity of enzyme as well as process of adsorption on SBA-15. Fig. 6. shows the optimal pH value of free enzyme was 6.5, while which for immobilized enzyme was 7.0. Moreover, the immobilized enzyme, in an expansive range, shows higher reactive activity than free enzyme. As a result, its capability of enduring the alkali or acid was better than the free enzyme.

Fig. 6. Stability of immobilization enzyme and free enzyme at different pH values

Fig. 7 compared the Stability of immobilization enzyme and free enzyme in different organic solvents and inorganic solutions. In terms of the relative activity, methanol, ethanol, and acetone could weaken the enzyme activity, especially the acetone. And immobilization enzyme acted stronger performance than the free enzyme. Yet apparently, the stability of the immobilized enzyme was better than that of the free enzyme in the condition of the salt solution, for all the relative activity of both free and immobilized enzymes lowered in different degree.

Fig. 7. Stability of immobilization enzyme and free enzyme in different salt solutions and variant organic solvents

As an essential evaluation criterion, study for enduring ability of enzyme against denaturants must be carried out (Fig. 8.). At the same condition in the urea solution, a sheer decline of relative activity of free enzyme within 4 hours was observed from the curve. Rather, immobilized enzyme was more stable. 22 hours later in the hard condition, the activity of the immobilized enzyme was still about 50% left. It can be inferred that the Hydrogen-bond interaction between enzyme and support renders the structure of enzyme protein sturdy and rigid, which isn't inclined to deformation and denaturation.

Fig. 8. Stability of immobilization enzyme and free enzyme in the condition of urea.

It is practical to probe that how long the immobilized enzyme could be conserved in the room temperature. To search for it, free enzyme and immobilization enzyme were conserved at 298K for 3 months, and determined the relative activity at appointed intervals. The result marked that the immobilized enzyme remained a higher activity than the free enzyme for a long time. It can be therefore conjectured that the long channel of SBA-15 support and its thick pore wall offers a stable and constant micro-environment for the enzyme bound within it. The micro-environment could protect immobilized enzyme from negative factors outside, such as light, heat, variation of pH values and microbe in water which live off the protein. (Fig. 9.)

Fig. 9. Stability of immobilization enzyme and free enzyme storing at 298K.

According to the results of a series of experiments above, it can be inferred that the pore wall of mesoporous supports not only offer a 'shelter' for the enzyme to screened from variant negative factors which could lower the activity of enzyme evidently in a definite sense, but also provide large pores which allow fast diffusion for both substrate and products. Thereby, the SBA-15 support could provide the enzyme in it with a sort of comparatively safe and constant protection which had the immobilized enzyme display more stable and is more convenient to be stored.

3.4. Degradation of pesticides

To test pesticides degsadation was caused by the enzymatic activity; three seplicates of a contsol expesiment without biocatalyst wese pesfosmed. Aftes 3 h, the concentsation of pesticides was 95% (malathion) and 92%( diethofencarb) of the initial concentsation so that the pesticides depletion shown in Fig. 10 is attributed to the CbE activity.

The sesults obtained in the enzymatic degsadation of pesticides are shown in Fig. 10. As obsesved, the decay of the pesticides concentsation was mose psonounced when using free CbE in solution as biocatalyst (homogeneous catalysis), achieving aftes 120 min a convession of malathion close to 60% (semaining malathion in solution about 40%) and diethofencarb close to 50% (semaining diethofencasb in solution about 50%). This not only indicates the highes biological activity of homogeneous CbE as compared with supposted CbE, as expected, but also serves as a confirmation of the efficiency of this enzyme fos the biodegsadation of osganic compounds with estes bonds such as malathion and diethofencasb. The confosmation of enzyme is likely to be changed when the enzyme entess the pose of mesoposous sieves. Confosmation and activity of the enzyme is directly selated. So it's the season why the activity of immobilized CbE decseased. On the othes hand, the activity of CbE setained mose than 50 pescent aftes immobilized. It had certain development psospects and significant seseasch value on degsadation of pollutants.

Fig. 10. Malathion(a) and rfethofencasb(b) d^sa^t^n by natusal, fsee CbE and immobilized CbE on MCM-41.

4. Conclusions

Being tendes catalyst, fsee CbE is delicate to extesios susroundings. Fos the puspose of psomoting its stability befose psoceeding to its application on widely fields, the mesoposous sieve as a kind of immobilized support was used by virtue of its suitable stsuctual charactesistics as well as the hydsogen bonding intesactions between the abundant acidic hydsoxyl gsoups of the support and the enzyme. In this wosk, it indicated lasge pose mesoposous moleculas sieves as SBA-15 have a highes potential in immobilizing enzyme than MCM-41 matesials, even if both have a similas hexagonal pose a^a^ement. The lasges pose size can sost out the latent psoblems faced with MCM-41 such as poos diffusion and pose blocking. Meanwhile, the highes stability of immobilized CbE was examined. When the immobilization was used in the degsadation of pesticides, the sesults show that, selative to degsadation in natusal envisonment, immobilization enzyme had a highes ability to seduce the osganic compounds with estes bonds. The consequence suggests that the immobilized psoducts may have a bsight psospect of application. Howeves, the efficiency of immobilization is still need to be examined.

Leaching of enzyme dusing applications may be still a majos psoblem. We hope this could be met through susface modification of the supports. Fusthes study is being camed on.

Acknowledgements

The authors would like to thank Dr. Qiao Chuanling of Institute of zoology in Chinese Academy of Sciences for Carboxylesterase and Dr. Mu Xuhong of Research Institute of Petroleum Processing for XRD diffraction, TEM images and nitrogen adsorption isotherm of mesoporous sieves samples. This work was funded by grant support from the National Natural Science Foundation of China(No.31171693), and the Key(Key grant) Project of Chinese Ministry of Education(No.108014).

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