Hybrid Cross-Linked Lipase Aggregates with Magnetic

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Hybrid cross-linked lipase aggregates with magnetic nanoparticles: a robust and recyclable biocatalysis for epoxidation of oleic acid Jiandong Cui, Lili Cui, Shiru Jia, Zhiguo Su, and Songping Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01939 • Publication Date (Web): 06 Sep 2016 Downloaded from http://pubs.acs.org on September 6, 2016

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Journal of Agricultural and Food Chemistry

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Hybrid cross-linked lipase aggregates with magnetic nanoparticles:

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a robust and recyclable biocatalysis for epoxidation of oleic acid

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Jiandong Cui 1, 3* Lili Cui3 Shiru Jia1

Zhiguo Su2 Songping Zhang2*

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of Science and Technology, No 29, 13th, Avenue, Tianjin Economic and Technological

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Area (TEDA), Tianjin 300457, P R China

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Academy of Sciences, Beijing, P R China

Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin University Development

National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese

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Hebei University of Science and Technology, Shijiazhang, P R China

Research Center for Fermentation Engineering of Hebei, College of Bioscience and Bioengineering,

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* Corresponding author:

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Jiandong Cui, E-mail:[email protected], Tel: +86-311-81668486

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Address: Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin

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University of Science and Technology, No 29, 13th, Avenue, Tianjin Economic and Technological

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Development Area (TEDA), Tianjin 300457, P R China

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Songping Zhang, E-mail: [email protected], Tel: +86-10-82544958

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Address: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering,

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Chinese Academy of Sciences, Beijing, 100190, P R China

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ACS Paragon Plus Environment


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Abstract

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The highly stable and easy recyclable hybrid magnetic crosslinked lipase aggregates

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(HM-CSL-CLEAs) were prepared by co-aggregation of lipase aggregates with nonfunctionalized

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magnetic nanoparticles and subsequent chemical cross-linking with glutaraldehyde. Analysis by SEM

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and CLSM indicated that the CLEAs were embedded in nanoparticle aggregates instead of covalent

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immobilization. The resulting HM-CSL-CLEAs exhibited higher thermostability, storage stability,

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and reusability than standard CLEAs. For example, HM-CSL-CLEAs maintained more than 60% of

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their initial activity after 40 min incubation time at 60 oC, whereas standard CLEAs lost most of their

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activities. The HM-CSL-CLEAs can be easily recovered from the reaction mixture by an external

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magnetic field. Moreover, the H2O2-tolerance of the lipase in HM-CSL-CLEAs was also enhanced,

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which could relieve the inhibitory effect on lipase activity. High conversion yield (55%) for the

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epoxidation of oleic acid using H2O2 as oxidizing agent was achieved by HM-CSL-CLEAs.

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Keywords: Magnetic nanoparticles; Cross-linked enzyme aggregates; Lipase; Epoxidation; Enzyme

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Immobilization

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1. Introduction

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Epoxides are used widely in pharmaceuticals, cosmetics, and food additives1. Furthermore,

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epoxidized vegetable oils and their derivatives have been used as additives for production of poly

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(vinyl chloride) (PVC) 2. Furthermore, as a higher quantity of additive, PVC has been considered to

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be used in food packaging 3. Over the past twenty years, the enzymatic epoxidation has shown to be

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effective for the epoxidation of vegetable oils with aqueous hydrogen peroxide. Lipases (E.C.3.1.1.3)

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are the most broadly employed biocatalysts due to their variety of reactions such as hydrolysis 4, lipid

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synthesis

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epoxidation of alkenes with hydrogen peroxide (H2O2) 9. Generally, it is necessary to consume H2O2

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during epoxidation of alkenes. However, H2O2 is able to modify the peptide core of the enzymes,

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which results in the oxidation of some cofactors and prostetic groups 8, 9. As a result, the inactivation

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of the lipases occur 10. Moreover, most free lipases display low stability and difficulties in recovery

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and reuse, leading to high cost and the contamination of the products

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enzymes is a promising technology that can overcome these limitations

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dissociation of enzymes subunit can be prevented by multisubunit immobilization 15, and the enzyme

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structure can be rigidified by multipoint covalent attachment 16. Among the various methodologies of

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enzyme immobilization, cross-linked enzyme aggregates (CLEAs) may be considered to be a

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promising method for enzyme immobilization

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volumetric productivities can be simply prepared by cross-linking the physical enzyme aggregates 19,

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20

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treatments, which would cause internal mass-transfer limitations. Especially, it is difficult to handle

5, 6

, transesterification

7

and epoxidation 8. Especially, lipases could be used for the direct

17, 18

11, 12

. Immobilization of

13, 14

. For example,

. CLEAs with superior stability and high

. Unfortunately, the small and fragile CLEAs can form clumps during centrifugation and filtration

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and fully recover the CLEA particle 21-23. Therefore, development of a feasible and efficient approach

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for improving the catalytic properties of traditional CLEAs is highly desirable. In recent years,

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supported CLEAs strategies were developed to overcome these disadvantages. For example, CLEAs

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of papain were prepared in commercial macroporous silica gel. The resulting CLEAs showed an

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impressive stability

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Rhodotorula glutinis into macroporous silica gel exhibited good reusability

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noncatalytic mass of the carrier causes decrease of the volumetric activity and production of the

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biocatalyst. In order to improve these drawbacks, recently, magnetic nanoparticles have attracted

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more and more attention due to their high specific surface area magnetic properties, which can

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efficiently improve enzyme loading and be easily separated using a magnetic field

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was immobilized as CLEAs on amino functionalized magnetite nanoparticles. The magnetic CLEAs

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exhibited excellent thermostability, storage stability and reusability

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were bonded on amino functionalized magnetite nanoparticles. The magnetic CLEAs were recycled

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without appreciable loss of activity

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CLEAs was obtained by cross linking aggregated glucoamylase using dialdehyde pectin with amino

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functionalized magnetite nanoparticles. The magnetic macromolecular CLEAs retained 84% activity

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after 10th cycle without leaching of enzyme which was 22% higher than traditional CLEAs 31. Laccase

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was immobilized as CLEAs (MAC-CLEAs) on amine functionalized magnetic nanoparticles using

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chitosan/1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride as the cross-linking system.

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The MAC-CLEAs exhibited enhanced storage stability over one year at 4℃ and showed better

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temperature resistance compared to its soluble counterpart 32.

24

. The formation of CLEAs of phenylalanine ammonia lyase (PAL) from

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29

25

. However, the

26-28

. α-amylase

. Magnetic CLEAs of laccase

. More recently, the magnetic macromolecular glucoamylase

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Moreover, CLEAs of Candida antarctica lipase B (CALB) were immobilized on magnetic

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nanoparticles by covalently bound. The magnetic CLEAs can be easily recovered and reused for 10

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cycles of 24 h without apparent loss of activity

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magnetic CLEAs could contribute to prepare stable CLEAs. Moreover, the preparation strategy of the

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magnetic CLEAs in these reports is standard. Namely, the iron oxide nanoparticles were first reacted

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with activation agent 3-aminopropyl triethoxysilane (APTES) to synthesize stable amino

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functionalized particles. Afterwords, enzymes were precipitated onto amino functionalized particles.

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Lastly, a strong chemical bond was formed by cross-linker (glutaraldehyde) between primary amine

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group which reacted with the carboxyl terminals (–COOH) on the surface of the precipitated enzymes

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on the magnetic support

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magnetite nanoparticles results in conformational changes in the enzyme. Moreover, this standard

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process is time-consuming and high cost. Considering these drawbacks to the industrial application of

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CLEAs, an innovative approach is required. In this study, a novel strategy was developed to prepare a

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hybrid magnetic CLEA (HM-CSL-CLEAs) by co-aggregated lipase with unfunctionalized magnetite

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nanoparticles and subsequently cross-linked with glutaraldehyde. The differences in this strategy with

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standard magnetic CLEAs are that magnetite nanoparticles are not modified by amino

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functionalization. Moreover, the CLEAs were embedded in nanoparticle aggregates instead of

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covalent immobilization. Compared to covalent immobilization of CLEAs on functionalized

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magnetite nanoparticles, the natural structure of lipase in this strategy can be maintained due to

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prevent the formation of covalent bonds between lipase and magnetite nanoparticles. Generally, the

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activities of the CLEAs are affected by many factors such as the precipitan, the cross linker

33-35

33

. The above reports indicated that the strategy of

. Unfortunately, the formation of covalent bonds between enzymes and

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(glutaraldehyde) and the cross-linking time, etc. Therefore, it is necessary to optimize different

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parameters for the prepararion of CLEAs. Response surface methodology (RSM) is a powerful

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statistical and mathematical tool. This method has been successfully applied in many areas of

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biotechnology. Effect of main factors and their possible interactions on biological process can be

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determined accurately by RSM

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preparation of CLEAs, such as CLEA- hydroxynitrile lyase 36, CLEAs-lipase

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activity of the HM-CSL-CLEAs were first investigated by RSM. Secondly, the catalytic properties of

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HM-CSL-CLEAs were investigated, including temperature, pH, and operational stability. Lastly, the

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catalytic efficiency of HM-CSL-CLEAs and standard lipase-CLEAs for the epoxidation of oleic acid

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using H2O2 as oxidizing agent were evaluated.

34, 35

. Recently, RSM was successfully applied to optimize the 37

and CLEA-protease

. Therefore, in this study, the main immobilization parameters which affect the final volumetric

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2 Materials and Methods

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2.1 Materials

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Candida sp. lipase (30 U/mg) was purchased from Sigma (St. Louis, USA). Olive oil was purchased

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from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Bis (2-ethylhexyl) sulfosuccinate

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sodium (AOT), FeCl2·4H2O, and FeCl3·6H2O were purchased from Xilong Chemical Co., Ltd.

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(Shantou, China). Other reagents used were of analytical grade .The water used in this work was

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deionized water.

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2.2 Preparation of magnetite nanoparticles (MNPs), standard lipase-CLEAs (CSL-CLEAs), and

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HM-CSL-CLEAs

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MNPs were prepared by the modified method of Ranjbakhsh et al 39. Briefly, 1.25 g of FeCl2·4H2O

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and 3.40 g of FeCl3·6H2O were dissolved in 100 mL deionized water under nitrogen gas; then, 6 mL

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of 25% NH3·H2O were added and vigorously stirred for 30–40 min at room temperature. The MNPs

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were recovered and washed several times with the deionized water.

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The CSL-CLEAs were prepared as previously reported

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phosphate buffer (pH 7.5) were precipitated using 80% ammonium sulfate saturation under gentle

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stirring at 4 °C for 1 h. A certain amount of glutaraldehyde (10% v/v in water) were slowly added in

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the mixture for the cross-linking of the aggregates, and stirred at 4 °C for a specified time. Then the

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suspension was centrifuged at 10000×g for 10 min at 4 °C. The precipitate was washed three times by

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50 mM sodium phosphate buffer (pH 7.5) and finally stored in 50 mM sodium phosphate buffer (pH

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7.5) at 4 °C prior to use. For HM-CSL-CLEAs, MNPs (20 mg/mL) and commercial lipase solution

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(1.75 mg/mL) were mixed, and ammonium sulfate was added to a final 80% ammonium sulfate

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saturation under gentle stirring at 4 °C for 1 h. Afterthat, glutaraldehyde (25 % v/v) was added to

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cross-link the precipitate for 2 h under shaking at 200 rpm. Then the immobilized lipases were

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separated by magnet. The biocatalysts were washed three times with distilled water, and finally the

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prepared HM-CSL-CLEAs were stored in 50 mM sodium phosphate buffer (pH 7.5) at 4 oC.

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2.3 The optimization of preparing HM-CSL-CLEAs

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The optimization of preparing HM-CSL-CLEAs

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one-factor-at-a-time experiments and RSM experiments based experimental design. First, the

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influence of main immobilization parameters on the preparation of HM-CSL-CLEAs was evaluated

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by traditional non-statistical technology; they are enzyme concentration, (NH4)2SO4 concentration

40

was

. Lipases (0.1 g) in 50 mM sodium

carried out by combination of

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and glutaraldehyde (GA) concentration. Central composite design (CCD) was used to find the

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optimal concentrations of these three factors and to understand the relationship between the factors

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and lipase activity. The used CCD was generated by “Design-Expert” software (version 7.0, Stat-Ease,

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Inc., Minneapolis, USA)

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glutaraldehyde concentration (X3) were optimized by the experimental plan. Each factor in the design

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was studied at five different levels (Table 1).

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2.4 Protein and activity assay

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The quantities of protein were measured by the method of ultraviolet absorption method at 280 nm 41.

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Lipase activity was determined by measuring the fatty acids produced in reversed micellar solution.

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Fatty acids produced were determined by modified copper soap colorimetry

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used to prepare cupric acetate reagent

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reversed micellar solution was pre-incubated at 37 oC with magnetic stirring (500 rpm). Olive oil

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(10%, v/v) was added to the reversed micellar solution for the hydrolysis. The reactant was collected

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at certain time and diluted with benzene. afterthat, 1 mL of the cupric acetate reagent was added and

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mixed vigorously at 180 rpm for 10 min at 37 oC. After centrifugation, the rate of formation of free

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acids was determined by measuring the absorbance of isooctane solution at 710 nm against the

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control which contains no free fatty acids. One unit of lipase activity was defined as the amount of

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lipase releasing 1 mmol fatty acids from olive oil per minute. The activity recovery in

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HM-CSL-CLEAs and CSL-CLEAs were calculated as given in Eq.1:

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Activity recovery (%)

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2.5 Morphology of HM-CSL-CLEAs

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. The enzyme concentration (X1), (NH4)2SO4 concentration (X2) and

43

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. Lowry method was

. The bis (2-ethylhexyl) sulfosuccinate sodium/isooctane

activity of CLEAs (U) = Total free lipaseTotal activity used for CLEAs production (U)

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1)

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Morphology of the MNPs, CSL-CLEAs and HM-CSL-CLEAs was examined by scanning electron

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micrographs (SEMs) (JEOL JSM6700, Japan). The distribution of lipase within CLEAs was observed

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by Confocal Laser Scanning Microscopy (CLSM) (Leica Camera AG, Germany). Before observation,

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modified FITC labeled lipase was first prepared by adding 50 mg/mL FITC solution (FITC in acetone)

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into lipase solution for 3 min, and then was immobilized 44. The samples were excited at 390 nm and

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the emitted fluorescent light was detected between 460 and 480 nm. Magnetisation measurements

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were carried out using a Magnetic Property Measure System (MPMS, Quantum design) under

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magnetic fields at 300 K.

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2.6 Measurement of apparent kinetic parameters

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The apparent kinetic parameters for free lipase and CLEA were obtained by the Lineweaver-Burk

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double-reciprocal plot method of Michaelis-Menten Equation between 0.12 mM and 4.9 mM olive oil

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concentrations at a constant enzyme concentration (0.5 mg/mL) under assay conditions.

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2.7 Optimal temperature and pH for CLEA activity

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The optimum temperature of free lipase, CSL-CLEAs, and HM-CSL-CLEAs on the activity were

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determined by measuring activity at different temperature (35-55 oC). Meanwhile, the optimal pH

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free lipase, CSL-CLEAs, and HM-CSL-CLEAs on the activity was determined by measuring activity

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at various pHs (3-8). The activity of the free lipase, CSL-CLEAs and HM-CSL-CLEAs was

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determined under assay conditions.

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2.8 The stability of free lipase, CSL-CLEAs and HM-CSL-CLEAs

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The thermal stability of the free lipase, CSL-CLEAs and HM-CSL-CLEAs was determined by

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incubating them in 50 mM sodium phosphate buffer (pH 7.5) without substrate at 60 oC for 10-40

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min. At different incubation time, the residual lipase activity was determined. The storage stability of

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free lipase, CSL-CLEAs and HM-CSL-CLEAs were determined by incubating enzyme samples in 50

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mM sodium phosphate buffer (pH 7.5) at 4 oC. At different storage time, the residual activities of free

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lipase and immobilized lipase were determined. To analyze the reusability of the CSL-CLEAs and

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HM-CSL-CLEAs, olive oil was hydrolyzed as described above. Upon completion of one cycle, the

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HM-CSL-CLEAs were then separated by a magnet (CAL-CLEAs were recovered by centrifugation).

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The recovered CLEAs were washed three times with 50 mM sodium phosphate buffer (pH 7.5) and

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then suspended again in a fresh reaction mixture.The residual lipase activity at each cycle was

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calculated by taking the enzyme activity of the first cycle as 100%.

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2.9 The epoxidation of oleic acid

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The formation of epoxystearic acid from the epoxidation of oleic acid using H2O2 as oxidizing agent

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was evaluated by HM-CSL-CLEAs and CSL-CLEAs. The epoxidation reaction was carried out in a

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50 mL serum bottle. The reaction mixture containing 20 mg/mL oleic acid, 25 mmol/L phosphate

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buffer (pH 7.0), 200 µL H2O2 (30 wt%), and 20 mg immobilized enzymes (10 U/mg) was incubated

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at 40 °C for 96 h at 120 rpm. Every 48 h, the formation of epoxystearic acid was analyzed by

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HPLC-ELSD. HPLC conditions were as follows: injection volume of 10 µL; column temperature of

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40 oC; flow rate of 1 mL/min; gradient elution was applied in chromatographic separation, acetonitrile

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concentration was increasing from 85% to 100%. Substrate and product were detected by ELSD at 40

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o

C, the gas flow rate was 1.5 mL/min, Gain was 1.

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3 Result and discussion

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3.1 Structural characterization and magnetic response of HM-CSL-CLEAs

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General scheme of preparation of HM-CSL-CLEAs was illustrated in Fig. 1. The synthesis of

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HM-CSL-CLEAs involved three main steps. First, some nanoparticles were encapsulated into lipase

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aggregates when lipases and non-functionalized magnetic nanoparticles were mixed and then

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precipitated with ammonium sulfate. Second, enzyme aggregates with nanoparticles were

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cross-linked by glutaraldehyde to form HM-CSL-CLEAs. Lastly, HM-CSL-CLEAs were further

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embeded into the nanoparticles due to aggregation of magnetic nanoparticles. Here, we firstly

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analyzed the size and shape of HM-CSL-CLEAs. SEM images (Fig. 2a) of standard CSL-CLEAs

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revealed no defined morphologies and large size particles. Moreover, the surface of standard

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CSL-CLEAs was relatively smooth, except for a few tiny pores. However, it is obviously observed

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that lipase CLEAs were inlayed within nanoparticles (The CLEAs embedded in the nanoparticles was

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indicated by the arrow). Furthermore, the porous and loose structures of HM-CSL-CLEAs were

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found (Fig. 2b). We estimated that HM-CSL-CLEAs with porous structures decreased mass transfer

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resistance compared to standard CSL-CLEAs, leading to superior catalytic efficiency. To further

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confirm the embedding of CLEAs within the HM-CSL-CLEAs aggregates, we used a confocal laser

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scanning microscopy (LCSM) to observe the distribution of CLEAs in nanoparticles. As shown in Fig.

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2(c) and (d), no fluorescence was observed for nanoparticles without CLEAs (Fig. 2(c)), whereas

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nanoparticles with CLEAs had an apparent fluorescence (Fig. 2(d)), indicating that the CLEAs were

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embedded within the nanoparticles. In addition, the field-dependent magnetisation curve of

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HM-CSL-CLEAs was displayed in Fig. 3. There was no hysteresis observed in low fields, indicating

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that the superparamagnetic behavior of HM-CSL-CLEAs is intact. The HM-CSL-CLEAs with

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superparamagnetic characteristics could be easily recovered from reaction mixture by an external

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magnetic field (Fig. 3b), and re-dispersed easily by a slight shake while removing the magnetic field

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(Fig. 3a), indicating efficient recyclability in industrial application.

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3.2 The optimization of preparing HM-CSL-CLEAs

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In the CLEA formation, the amount of ammonium sulfate, glutaraldehyde concentration, enzyme

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concentration, and cross-linking time play a significant role in CLEA activity 45, 46. For example, too

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little cross-linker is not enough to form the cross-linked aggregates that causes leaching of enzyme

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molecules, while high glutaraldehyde concentration can result in decrease of enzyme activity due to

244

loss of the minimum flexibility 47, 48. In our study, to find the critical factors which have affected on

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activity recovery, critical factors for preparation of HM-CSL-CLEA were first chosen based on the

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one-factor-at-a-time method. The results showed that enzyme concentration, (NH4)2SO4 concentration

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and glutaraldehyde concentration have significant effects on the activity recovery of HM-CSL-CLEA.

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To further find the optimal concentrations of these three factors and relationship between the factors

249

and activity recovery, the CCD experiments for studying the effects of enzyme concentration,

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(NH4)2SO4 concentration, and glutaraldehyde concentration on activity recovery was carried out. The

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results were presented in the Table 2 and Table 3. The model F-value of 7.78 implied that the model

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was significant. The regression equation obtained from analysis of variance (ANOVA) indicated that

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the multiple correlation coefficient of R2 is 0.8752. The value of the determination coefficent

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indicates that the model can explain 87.52% variation in the response. The high R2 value indicated a

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high significance of the model 49. The above results showed that the models were adequate to predict

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the activity recovery within the range of variables studied.

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The analysis of variance indicated that the model terms of X1, X12, X32, and X1X3 were significant

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(“probe>F” less than 0.05), and the interactive effect of X1X3 was significant (“probe>F” less than

259

0.05). However, the interactive effects of X1X2, and X2X3 were not significant. It indicates that

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glutaraldehyde concentration and enzyme concentration play a key role on activity recovery. A

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second-order polynomial equation was obtained by multiple regression analysis of the experimental

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data:

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Y=41.6 + 4.26X1 + 2.53X2 +1.48X1X3 – 6.9X12 – 0.56X1X2 + 6.6X1X3 – 2.51X22 – 1.34X2X3 – 7.7X32.

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where Y is the response, that is activity recovery (%), and X1, X2 and X3 are the coded values of the

265

test variables lipase, (NH4)2SO4 and glutaraldehyde, respectively.

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The 2D contour plots and 3D response surface are generally the graphical representations of the

267

regression equation, and 2D contour plots and 3D response surface are presented in Fig. 4. As

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mentioned by Muralidhar et al 50, the interactions are negligible between the corresponding variables

269

when the contour plots of response surfaces is circular. In contrast, the interactions between the

270

corresponding variables are significance while the contour plots exhibit an elliptical shape. The

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contour plots in Fig. 4 (c, d) showed that there was significant interaction between enzyme

272

concentration and glutaraldehyde concentration. The optimal conditions were extracted by Design

273

Expert Software. They were enzyme concentration at 1.95 mg/mL, (NH4)2SO4 concentration at 2.56

274

M, and glutaraldehyde concentration at 0.23%, respectively. Moreover, the model predicted that the

275

maximum activity recovery that can be obtained under the above optimum conditions was 38..67%.

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The maximum activity recovery obtained experimentally was found to be 40.86%. This is obviously

277

in close agreement with the model prediction, which thus proved the validity. In a word, an 19.32%

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increase in activity recovery of HM-CSL-CLEAs was acquired under optimized conditions.

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3.3 Measurement of apparent kinetic parameters

280

Apparent kinetic properties of free lipase, CSL-CLEAs, and HM-CSL-CLEAs showed that Km was

281

increased when lipase was immobilized (Table 4). The increase of Km value of CLEA could be due to

282

the mass transfer limit for the substrate as well as the rigid conformation of lipase caused by

283

cross-linking 48, 51 . However, Km of HM-CSL-CLEAs was lower than that of standard CSL-CLEAs.

284

Moreover, the catalytic efficiency (Kcat/Km) of HM-CSL-CLEA was higher compared to standard

285

CSL-CLEAs, indicating that HM-CSL-CLEAs were an overall better catalyst. The porous and loose

286

structures of HM-CSL-CLEAs are critical for ensuring high catalytic efficiency, due to an increase in

287

mass substrate transfer for interaction with the interior enzymes of CLEAs. This phenomenon was

288

also observed in other reports 52, 53.

289

3.4 Optimal pH and temperature on enzyme activity

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Fig. 5 showed the effect of pH on the activity of free lipase, CSL-CLEAs, and HM-CSL-CLEAs. The

291

optimum pH of the free lipase was at pH 7.0, which shifted to pH 6.0 for CSL-CLEAs and

292

HM-CSL-CLEAs. Moreover, compared to free lipase and CSL-CLEAs, HM-CSL-CLEAs appeared

293

a much broader pH range of 4 to 8 with higher activity. The shift in pH could be due to the change in

294

acidic and basic amino acid side chain ionization in the microenvironment around the active site, as

295

well as the interaction between the cross-linking agent and the enzyme

296

temperature on the activity of free lipase and all immobilized lipase samples was compared. As

297

shown in Fig. 6, the optimum reaction temperature for free lipase was at 45 oC. However, the optimal

298

temperature on the activity of both CSL-CLEAs and HM-CSL-CLEAs were at 50 oC. It indicates that

14


54

. In addition, optimal

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immobilization improves the thermostability of the enzyme. The high thermostability could be due to

300

the increased rigidity of enzymes by the cross-linking among enzyme molecules 30.

301

3.5 Stability of HM-CSL-CLEAs

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The thermal stability of free lipase, CSL-CLEAs and HM-CSL-CLEAs at 60 oC was shown in Fig. 7.

303

All CLEAs exhibited the higher thermal stability than that of free lipase. Moreover, HM-CSL-CLEAs

304

showed the highest stability. For example, free lipase and CSL-CLEAs lost most of their activities

305

after 40 min incubation time at 60 oC, whereas HM-CSL-CLEAs retained more than 60% of their

306

initial activity in the same conditions. These results indicated that the thermal stability of

307

HM-CSL-CLEAs was significantly improved. This enhancement of HM-CSL-CLEAs in thermal

308

stability could be due to the following reasons: (1) magnetic nanoparticles provide a suitable

309

protection to retard heat transfer and collapse of the CLEAs structure at high temperatures, (2) the

310

rigidity of the active conformation was improved by cross-linking among enzyme aggregates. In

311

addition, storage stability of the free lipase, CSL-CLEAs and HM-CSL-CLEAs was presented in Fig.

312

8. Most activity of both CLEAs forms was retained after 28 days storage. However, free lipase

313

retained only 28% of its initial activity at the same storage time. HM-CSL-CLEAs still retained more

314

than 75% of its initial activity after 38 d. However, the CSL-CLEAs only retained 54% of its initial

315

activity. The reusability of CSL-CLEAs and HM-CSL-CLEAs was shown in Fig. 9, although the

316

activities of both CSL-CLEAs and HM-CSL-CLEAs decrease with increasing number of recycles,

317

the HM-CSL-CLEAs retained more than 65% of its initial activity until 10 cycles. However, the

318

CSL-CLEAs almost lost activity. The result showed that the HM-CSL-CLEAs presented good

319

reusability. This reusability property of HM-CSL-CLEAs is necessary for cost-effective use of the

15



Page 16 of 37

320

enzyme in industrial applications. In the past few decades, lipases have been immobilized in a great

321

variety of supports, such as, mesoporous silica

322

calcium carbonate microcapsule

323

activity and reusability of the biocatalysts are undesirability.

324

3.6 The epoxidation of oleic acid

325

In this section, the epoxidation of oleic acid with H2O2 was tested using the HM-CSL-CLEAs and

326

CSL-CLEAs. The results showed that the final conversion yield was 55% after 96 h when

327

HM-CSL-CLEAs were used. However, only 36% of conversion yield was obtained at the same

328

reaction time when standard CSL-CLEAs were used (Fig.10). The results indicated that

329

HM-CSL-CLEAs had excellent stability of the HM-CSL-CLEAs against high concentrations of H2O2.

330

Therefore, the study not only further improves the catalytic properties of traditional CLEAs, but also

331

provides a robust and recyclable biocatalysis for the epoxidation of alkenes with low cost.

57

55

, Poly (lactic acid)/chitosan fiber mats

56

, and

. However, compared with magnetic CLEAs, the volumetric

332 333

Acknowledgments

334

The authors thank support from the National Natural Science Foundation of China (Grant Nos.

335

21676069, 21376249, 21336010), 973 Program (2013CB733604). National Major Scientific

336

Equipment Development Project (2013YQ14040508), Natural Science Foundation of Hebei Province,

337

China (project no. B2014208054), and the Key Foundation of Hebei University of Science and

338

Technology (project no. SW5).

339 340

16


Page 17 of 37


341

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494 495 496 497 498 499 500 501 502 503 504 505 506 507

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Page 25 of 37


508

Caption of Figures

509

Figure 1 General scheme for hybrid magnetic CLEAs.

510

Figure 2 SEM images of (a) CSL-CLEAs magnified 30000×; (b) HM-CSL-CLEAs

magnified

511

30000×; as well as confocal laser scanning microscopy (LCSM) images of (c) magnetite

512

nanoparticles with

513

treatment.

fluorescamine treatment and (d) HM-CSL-CLEAs with fluorescamine

514

Figure 3 Magnetisation curves of immobilized lipase and the separation process of immobilized

515

lipase from solution by magnet. (a) Immobilized lipase was dispersed in solution; (b)

516

immobilized lipase was collected by an external magnet.

517

Figure 4 2D and 3D contour plots at center point. 2D and 3D plot of (NH4)2SO4 concentration and

518

glutaraldehyde concentration (A, B); 2D and 3D plot of (NH4)2SO4 concentration and

519

glutaraldehyde concentration (C, D); 2D and 3D plot of glutaraldehyde concentration and

520

enzyme concentration (E, F).

521

Figure 5 The effect of pH on the activity of free enzyme, CSL-CLEAs, and HM-CSL-CLEAs.

522

Figure 6 Influence of temperature on activity of free enzyme, CSL-CLEAs, and HM-CSL-CLEAs.

523

Figure 7 The thermal stability of free enzyme, CSL-CLEAs, and HM-CSL-CLEAs at 60 oC.

524

Figure 8 Storage stability of the free enzyme, CSL-CLEAs, and HM-CSL-CLEAs.

525

Figure 9 Reusability of the CSL-CLEAs and HM-CSL-CLEAs.

526

Figure 10 The conversion rates of CSL-CLEAs and HM-CSL-CLEAs for the epoxidation of oleic

527

acid with H2O2

528

25



Page 26 of 37

529 530 531

Table 1 Experimental range levels of enzyme concentration, (NH4)2SO4 concentration,

532

and glutaraldehyde concentration in terms of actual factors

Variable

Symbols

Coded-variable levels -1.68

-1

0

1

1.68

Enzyme concentration（mg/mL）

X1

1.33

1.50

1.75

2.00

2.17

(NH4)2SO4 concentration (%)

X2

1.16

1.50

2.00

2.50

2.84

Glutaraldehyde concentration (%)

X3

0.12

0.15

0.20

0.25

0.28

533 534 535 536 537 538 539 540

26


Page 27 of 37


+ Fe3O4

Aggregation Cross-linking

Enzyme

Cross-linker

Figure 1



Figure 2


Page 28 of 37

Page 29 of 37


Figure 3



Figure 4


Page 30 of 37

Page 31 of 37


Figure 5



Figure 6


Page 32 of 37

Page 33 of 37


Figure 7



120

Free enzyme CSL-CLEAs HM-CSL-CLEAs

100

Residual activity(%)

Page 34 of 37

80 60 40 20 -5

0

5

10

15

20

25

30

35

Time(d) Figure 8


40

45

Page 35 of 37


Figure 9



Figure 10


Page 36 of 37

Page 37 of 37


Graphic Abstract


Hybrid Cross-Linked Lipase Aggregates with Magnetic

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