Site-specific, covalent immobilization of an engineered enterokinase onto magnetic nanoparticles through transglutaminase-catalyzed bioconjugation

Accepted Manuscript Title: Site-specific, covalent immobilization of an engineered enterokinase onto magnetic nanoparticles through transglutaminase-catalyzed bioconjugation Authors: Jing-Hong Wang, Ming-Ze Tang, Xiao-Tian Yu, Chong-Mei Xu, Hong-Ming Yang, Jin-Bao Tang PII: DOI: Reference:

S0927-7765(19)30092-X https://doi.org/10.1016/j.colsurfb.2019.02.018 COLSUB 10009

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

13 October 2018 2 February 2019 8 February 2019

Please cite this article as: Wang J-Hong, Tang M-Ze, Yu X-Tian, Xu CMei, Yang H-Ming, Tang J-Bao, Site-specific, covalent immobilization of an engineered enterokinase onto magnetic nanoparticles through transglutaminasecatalyzed bioconjugation, Colloids and Surfaces B: Biointerfaces (2019), https://doi.org/10.1016/j.colsurfb.2019.02.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Site-specific, covalent immobilization of an engineered enterokinase onto magnetic nanoparticles through transglutaminase-catalyzed bioconjugation Jing-Hong Wanga, Ming-Ze Tangb, Xiao-Tian Yua, Chong-Mei Xu a, Hong-Ming Yanga,*, Jin-Bao Tanga,* School of Pharmacy, Weifang Medical University, Weifang 261053, Shandong

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a

b

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Province, China

School of Science, China Pharmaceutical University, Nanjing 211198, Jiangsu

Province, China

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* Corresponding author. Tel.: +86 536 8462497; fax: +86 536 2602083. E-mail address:

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[email protected] (H.-M. Yang); [email protected] (J.-B. Tang).

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Graphical abstract

Highlights •

An engineered enterokinase light chain (EKLC) was ingeniously constructed.

•

EKLC was orientedly immobilized on amine-modified magnetic nanoparticles.

•

Site-specific immobilization of EKLC relies on MTG-catalyzed bioconjugation.

•

Immobilized EKLC exhibits high intrinsic activity and stability. -1-

•

Reusability of immobilized EKLC was proved in fusion partner cleavage over 10 cycles.

ABSTRACT Enterokinase (EK) is one of the most popular enzymes for the in vitro cleavage of

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fusion proteins due to its high degree of specificity for the amino-acid sequence (Asp)4-Lys. Enzyme reusability is desirable for reducing operating costs and facilitating

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the industrial application of EK. In this work, we report the controlled, site-specific and

covalent cross-linking of an engineered EKLC on amine-modified magnetic nanoparticles (NH2-MNPs) via microbial transglutaminase-catalyzed bioconjugation for

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the development of the oriented-immobilized enzyme, namely, EKLC@NH2-MNP

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biocatalyst. Upon the site-specific immobilization, approximately 90 % EKLC enzymatic

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activity was retained, and the biocatalyst exhibited more than 85 % of initial enzymatic activity regardless of storage or reusable stability over a month. The EKLC@NH2-MNP

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biocatalyst was further applied to remove the His tag-(Asp)4-Lys fusion partner from

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the His tag-(Asp)4-Lys-(GLP-1)3 substrate fusion protein, result suggested the EKLC@NH2-MNP possessed remarkable reusability, without a significant decrease of enzymatic activity over 10 cycles (P > 0.05). Supported by the unique properties of

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MNPs, the proposed EKLC@NH2-MNP biocatalyst is expected to promote the

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economical utilization of enterokinase in fusion protein cleavage. Keywords:

Engineered

enterokinase;

Site-specific

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transglutaminase; Enzymatic cleavage; Reusability

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immobilization;

Microbial

1. Introduction Enterokinase (EK, EC 3.4.21.9) is a serine proteinase of the intestinal brush border found in the duodenum, and holoenzyme is a disulfide linked two-chain polypeptide that is derived from a single-chain precursor: an N-terminal heavy chain that anchors EK in the intestinal membrane and a C-terminal light chain, which is the catalytic

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subunit [1, 2]. EK exhibits a high degree of specificity for the amino-acid sequence (Asp)4-Lys, and thus useful for the in vitro cleavage of fusion proteins [3–5]. Specifically, with any fusion partner downstream of the (Asp)4-Lys sequence, EK can

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generate a digestion product with a native N-terminus without leaving any unwanted

amino acid residues on their amino termini [3]. Consequently, EK exhibits a more prominent advantage than the other proteases (e.g., tobacco etch virus protease, SUMO

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protease, thrombin [6, 7]) for the in vitro cleavage of fusion protein at defined cleavage

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sites. Thus far, the catalytic subunit, that is, EK light chain (EKLC) with full activity, has

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been expressed in several cell lines, such as COS cell, Pichia pastoris, Aspergillus niger,

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Saccharomyces cerevisiae, and Escherichia coli [8–11]. Despite these achievements, the high manufacturing cost of EK remains a challenge with the growing demands of

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commercial EK in practical industrial applications. Reusability of EK is an alternative approach to reducing the costs associated with this

industrial

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process, and enzyme immobilization provides a convenient and recyclable design for biocatalyst

applications.

Nowadays,

various

strategies

of

protein

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immobilization, such as adsorption, covalent binding, encapsulation, entrapment and cross-linking are readily available [12–16]. Owing to the irreversible and stable attachment, covalent conjugation appears to be the most attractive approach [17, 18].

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Based on the active side groups (amine, carboxyl, thiol, and so on) within proteins, enzymes can be immobilized onto functional support materials via covalent bonds [13, 14]. However, proteins usually contain several residues with similar reactivity, thereby complicating the immobilization; this method is efficient but suffers from uncontrolled and random conjugation, and enzymes often suffer from decreased in activity upon immobilization due to the alteration of the protein’s functional structure surrounding the -3-

active site [19–21]. Consequently, the development of site-specific and covalent immobilization methodologies for retaining the intrinsic functions of enzymes is in high demand. Enzyme-catalyzed bioconjugation offers the advantage of improved selectivity and compatibility with sensitive biological systems relative to traditional chemical methodologies, and so far, the flexibility and customizability of enzyme-catalyzed

including

protein–protein

conjugates,

protein–small

molecule

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bioconjugation have promoted its applications in site-specific protein modification conjugates

and

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protein–DNA conjugates [22–24]. For instance, microbial transglutaminase (MTG, EC 2.3.2.13) is a unique enzyme that catalyzes the formation of a covalent bond between the γ-carboxyamide group of a peptide-bound glutaminyl residue (acyl-donors) and

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various primary amines (acyl-acceptors), including the amino group of lysine [25, 26].

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To data, MTG has been continuously applied for successful conjugation in applications,

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such as food processing, site-specific labeling of proteins, and fabrication of unique biomaterials [26–28]. Furthermore, MTG-catalyzed conjugations for covalent protein

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immobilization have also been fairly demonstrated via facile monitored molecules (such

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as bacterial alkaline phosphatase and fluorescent protein) in several studies [29–32]. In addition, this bioconjugation approach was applied for immobilized E. coli biotin ligase (BirA) in our previous study [33]. Despite these successes, the tool enzymes for

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removing fusion tag in recombinant proteins, whose covalent immobilization onto solid support with a defined structure and uniform-specific conjugation by employing

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MTG-catalyzed bioconjugation, are still underway. This study aims to develop the controlled, site-specific and covalent immobilization of EK via MTG-catalyzed bioconjugation to improve its stability and reusability. In this

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approach, a glutamyl donor substrate peptide (Met-Leu-Ala-Gln-Gly-Ser, Gln-donor tag [29]) was incorporated the C-terminus of EKLC at locations distant from the N-terminal active site of the enzyme. To be specific, a ZZ peptide was fused at the N-terminus of EKLC, as the molecular chaperone which promotes the proper folding of its C-terminal fusion protein. As well as, the EK recognition sequence was inserted between ZZ and EKLC. In doing so, we found that nearly all of the inclusion bodies were properly -4-

refolded after the renaturation process. Thus, a final EKLC-Gln construct, with its native N-terminus and high specific activity, was successfully fabricated after the autocatalytic cleavage reaction. Subsequently, the engineered EKLC-Gln was attached onto amine-modified

magnetic

nanoparticles

(NH2-MNPs)

via

MTG-catalyzed

bioconjugation (Fig. 1). Then, the efficacy of enzyme-catalyzed bioconjugation, the activity and stability of EKLC after immobilization, were characterized in detail, and the

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reusability of immobilized EKLC was also evaluated by removing the His tag from a substrate fusion protein.

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2. Materials and methods 2.1. Preparation of Gln-donor tagged EKLC

DNA sequence of the synthetic construct bovine enterokinase catalytic subunit gene

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(sBEKLC; GenBank Accession No.DQ265741) containing (Asp)4-Lys DNA at its 5' end

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and Gln-donor tag DNA at its 3' end, was synthesized using a chemical method

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(consigned to Sangon Biotechnology Co., China), and then sub-cloned into the BamH

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I/Hind III site of pGreen-S plasmid [34], resulting in an interim vector. Subsequently, the DNA sequence of ZZ peptide with 5' end 6×His tag was obtained by colony PCR

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using pEZZ 18 (Amersham, USA) as the template and then inserted into the EcoR I/BamH I site of the above interim vector, yielding the expression vector for producing

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recombinant protein. E. coli BL21 (DE3) cells harboring the expression vector were cultivated in Luria-Bertani medium (100 μg/mL ampicillin) at 37 °C overnight. The

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cells were harvested by centrifugation, and then resuspended in 50 mM Tris-HCl and 150 mM NaCl at pH 7.5 (buffer A). Cells were lyzed on ice by ultrasonication, and then inclusion bodies were harvested by centrifugation. After washing with buffer A, the

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inclusion bodies were dissolved in buffer A containing 8 M urea. For in vitro folding process, the solution was successively dialyzed against refolding solutions (50 mM Tris-HCl, 150 mM NaCl, 3 mM GSH, 0.6 mM GSSH, 5% (v/v) glycerol at pH 7.5, containing 6, 4, 2, and 1 M urea, respectively) at 4 °C for 12h. Finally, the solution was dialyzed against binding buffer (50 mM Tris-HCl, 500 mM NaCl and 50 mM imidazole, pH 7.5) at 4 °C for 12 h. -5-

The renatured supernatant was loaded onto a HisTrap FF crude column (5 mL; GE Healthcare, USA) and eluted with a gradient of 50–500 mM imidazole elution buffer according to the instructions of the manufacturer. The pooled fraction was desalted and concentrated using an Amicon® Ultra-15 centrifugal filter (Ultracel-10; Millipore, USA) and analyzed by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

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For releasing the native N-terminus of EKLC-Gln, 1 U commercial EK (Bio Basic Inc.,

China) was added to 10 mL recombinant protein solution (2 mM CaCl2, 50 mM NaCl

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and 25 mM Tris-HCl, pH 7.6; protein concentration at 1 mg/mL), and the mixture was stirred at 25 °C for 6 h. Then the digested production was passed through a HisTrap FF crude column (5mL) and the flow-through fraction was collected and analyzed by 15%

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SDS-PAGE.

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2.2. Immobilization of engineered EKLC-Gln onto NH2-MNPs

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Immobilization experiments were performed by mixing 0.5 mL of engineered EKLC-Gln (1 mg/mL) with 1 mL of amine-modified magnetic nanoparticles (NH2-MNPs, Fe3O4

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core particles, diameter: 100–200 nm; BaseLine, China) in 5 mL PBS (20 mM, pH 8.0)

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in the presence or absence of MTG (Yiming Biological, China; 5 U/mL) with gentle shaking at 27 °C for 6 h. Subsequently, the MNPs were separated from the reaction solution via magnetic separation, followed by washing five times with PBST (PBS

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containing 0.01% Tween-20) under ultrasonic condition. The amount of immobilized enzyme was determined using bicinchoninic acid (BCA) protein assay with bovine

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serum albumin (BSA) as the standard according to the manufacturer's instruction. Briefly, 100 μL series of dilutions of BSA protein standard and 100 μL MNPs were mixed with 2 mL of BCA working reagent, respectively. And those mixtures were

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incubated for 2 h at 25 °C. Then the sample of MNPs mixed BCA working reagent solution was firstly separated using magnetic separation, and the supernatant was simultaneously determined the 560 nm value with BSA protein standard. To evaluate the specific of MTG-catalyzed bioconjugation, a commercial EK was used in a control-experiment. 2.3. Enzyme activity assay -6-

The enzymatic activity was determined by utilizing a synthetic substrate containing the EK recognition sequence, Gly-(Asp)4-Lys-β-naphthylamide (Sigma-Aldrich, USA) as described in previous study [35]. The reaction mixture contained 25 μL substrate (1 mM) and 5 μL analyte (free or immobilized EKLC, or commercial EK; 50 ng protein/mL), with a supplemental 70 μL standard reaction buffer (2 mM CaCl2, 50 mM NaCl and 25 mM Tris-HCl, pH 7.6), and the mixture was incubated for 1 h at 37 °C. Then the MNPs

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were firstly removed from reaction solution, and the supernatant was compared with the reaction mixtures of the free enzymes for enzyme activity assay. The released

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β-naphtylamine was reacted with 100 μL 0.05% N-(1-naphthyl)-ethylenediamine (0.2%

sodium nitrite and 0.5% ammonium sulfamate buffer) for color generation, and the absorbance was measured at 580 nm. A standard solution of β-naphtylamine was used

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for calibration. One unit of EK was defined as 1 nM of β-naphtylamine released after 1

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h incubation.

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2.4. Characterization on stability of immobilized enzyme The storage stabilities of the free and the immobilized enzymes were investigated by

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measuring enzymatic activity after storage at 4 °C every 5 days for a one month. As for

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the reusable stability of immobilized EKLC, the enzymatic activity of 100 μL EKLC coated MNPs was repetitiously examined at 5-day intervals for 7 cycles. No examination, the immobilized enzymes were stored at 4 °C in 20 mM PBS buffer

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(0.01% sodium azide, pH 7.0) for the next experiment. At each of the respective time points, the enzymatic activities were assessed as described earlier and compared with

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the corresponding initial activity, respectively. 2.5. Application of immobilized enzyme The reusability of immobilized EKLC for cleaving fusion partner in practical application

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was assessed over several cycles by removing the His tag from the triplet of glucagon-like

peptide-1

(GLP-1)

recombinant

protein,

which

namely

His

tag-(Asp)4-Lys-(GLP-1)3 (produced in our Lab, not published). In brief, 50 μL EKLC coated MNPs and 50 μL protein substrate were mixed with 100 μL two-folded reaction buffer (4 mM CaCl2, 200 mM NaCl and 40 mM Tris-HCl, pH 7.6) at 25 °C for 6 h. After one reaction, the MNPs were collected via magnetic separation after washing five -7-

times with PBST under ultrasonic condition, and then applied to the next catalytic reaction. The digested productions were analyzed by 15% tricine-SDS-PAGE.

3. Results and discussion 3.1. Production of Gln-donor tagged EKLC protein Given that the crystal structure of EKLC shows that the C-terminal portion is exposed at

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the outer surface and the N-terminal residue is positioned inside the protein for salt bridge, N-terminal surplus may be deleterious to the enzyme activity [36, 37]. To take advantage of MTG-catalyzed bioconjugation for the controlled and site-specific

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immobilization of EKLC onto amine-modified solid supports, an obligatory handle should be utilized. Accordingly, a glutamyl donor substrate peptide (Gln-donor tag) was genetically incorporated into the C-terminus of EKLC, at locations distant from the

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N-terminal active site of the enzyme. For generating the native N-terminus of EKLC, the

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EK cleavage sequence was installed at its N-terminal. We fully understand the fact that

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EKLC contains four disulfide bonds that make the correct folding of expressed protein in

promote

the

proper

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cytoplasm practically difficult. So, a hydrophobic peptide, ZZ peptide, which can folding

of

its

C-terminal

fusion

protein

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during the process of protein translation in vivo [38], with a N-terminal 6×His tag, was introduced at front of EK recognition sequence (Fig. 1).

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After overnight cultivation at 37 °C, the designed target protein was successfully produced in gene engineering bacteria (Fig. 2 A). However, the construct was expressed

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as inclusion body and hardly soluble, thus, we emphasized the inclusion body renaturation procedure to obtain the soluble target protein. Interestingly, the results showed that nearly all inclusion bodies were converted into soluble form at high

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concentration (approximately 5 mg/mL) after the renaturation process (Fig. 2 B). Next, the successful refold of inclusion bodies was witnessed by the fact of autocatalytic cleavage reaction. Stirring by commercial EK, the initial released EKLC-Gln could exert its intrinsic bioactivity, and thereby producing and assigning more EKLC-Gln moieties to the cleavage reaction (Fig. 2 C). The refolding results suggested that ZZ peptide can promote the proper folding of its downstream fusion partner not only in vivo [38], but -8-

also in vitro. Thus, we presented the ZZ peptide-assisted refolding approach is significant as the refolding process for proteins with intramolecular disulfides at high concentration are known to be problematic. Subsequently, the released EKLC-Gln moieties were separated from the autocatalytic cleavage reaction solution by nickel chelating affinity chromatography with approximately 95 % purity (Fig. 2 C). 3.2. Immobilization of engineered EKLC-Gln onto NH2-MNPs

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Currently, various materials of different origin, from inorganic through organic to

hybrids and composite supports, can be used as supports for enzyme immobilization [12,

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13]. Moreover, magnetite is often combined with inorganic moieties and can also be mixed with organic materials [13, 39–40]. Due to the unique size and physical

properties, magnetic nanoparticles (MNPs) are important carrier materials and the

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enzymes attached on the carrier can be easily recovered using an external magnetic field

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and recycled for iterative uses to save costs [41–45]. Here, a commercially available

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amine-modified MNPs (NH2-MNPs) was used. Catalyzed by MTG, a new amide linkage (R1-HN-CO-R2) was formed between the glutamyl group of Gln tag and the

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primary amines of NH2-MNPs, thus, EKLC-Gln was immobilized onto NH2-MNPs with

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site-specific and covalent manner. As shown in Fig. 3, the amount of EKLC-Gln immobilized onto the surface of MNPs was approximately 0.2 mg protein per mg of MNPs. The other three control-experiments, EKLC-Gln without MTG, and commercial

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EK with or without MTG, were scarcely conjugated to MNPs. These results indicated that EKLC-Gln with a specific Gln residue, was recognized by MTG and the reaction

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proceeded actively on the amine-modified MNPs. The resulting immobilized enzyme, with a site-specific and oriented manner, named as EKLC@NH2-MNP biocatalyst. To determine the enzymatic activity, the free EKLC-Gln was first compared to the

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activity of commercial EK, and presented approximately the same activity as the commercial one based on the equimolar amounts of protein. Considering that the enzyme activity of commercial EK is 4000 U/mg, the activity of free EKLC-Gln was estimated at approximately 4000 U/mg. And the activity of the immobilized EKLC-Gln, was approximately 3700 U per mg protein which was conjugated to the surface of MNPs, that suggesting that more than 90 % enzymatic activity was detained after -9-

MTG-catalyzed immobilization. Generally, coupled with the carboxylic and sulfhydryl groups of the enzyme, EK can be immobilized onto the functionalized carriers via chemical covalent conjugation methodology. However, the enzymatic activity retentions between 20% and 60% reported in previous studies [46–48], the decrease in activities might be due to the uncontrolled and site-unspecific conjugation that resulted in steric hindrance effect, structural distortion and active site blockage [19, 20, 49]. Here, the

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immobilized EKLC via the bioconjugation showed the activity retention of more than 90 % could be mainly due to the fact that the site-specific conjugation occurring along

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the engineered handle of its C-terminus was far from the active site, thereby presenting a significant improvement on the activity retention of the immobilized enzyme.

Confirming the stability of the immobilized EKLC is crucial for consecutive applications.

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Next, the storage and reusable stability of the EKLC@NH2-MNP was assessed. As

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shown in Fig. 4, the free enzyme remained over 75 % of the initial activity after 30 days

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of storage at 4 °C, and the EKLC@NH2-MNP exhibited a high stability, which remained approximately 90 % of the initial activity over time. Therefore, the reusable stability of

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the EKLC was at least 85 % of the initial activity with no significant difference with

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respect to the storage stability, even after seven discontinuous cycles. After determination on the reusable enzymatic activity of the immobilized EKLC, the apparent zeta potential of EKLC@NH2-MNP was further investigated, and exhibited the similar

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value with NH2-MNP (Fig. 5). Remarkably, after enduring 7 cycles for cleaving Gly-(Asp)4-Lys-β-naphthylamide, the EKLC@NH2-MNP biocatalyst still presented the

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similar apparent zeta potential. Those results indicated that the EKLC immobilized on the surface of NH2-MNP has a little impact on the physical characteristic of MNP, which similar with previously reported that the BSA-coated gold nanoparticles (AuNPs)

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fully maintained the colloidal properties of the parental AuNPs [50]. Taken together, the favorable stability of the EKLC@NH2-MNP biocatalyst suggested its significant potential for removing fusion partner application. 3.3. Application of EKLC@NH2-MNP biocatalyst The reusability of immobilized enzyme is an economically important goal for industrial enzymatic process; next, a preliminary study with a fusion protein containing the EK - 10 -

recognition sequence was used to evaluate the EKLC@NH2-MNP biocatalyst in 10 continuous operation cycles. The substrate protein with a total molecular weight of 11 kDa which originates 6×His affinity tag and triplex GLP-1 (9.6 kDa), is interrupted by the (Asp)4-Lys sequence. Before the clinical experiment on the treatment of type 2 diabetes, the His tag must be removed and released the therapeutic (GLP-1)3. For the proper estimation of the cutting efficiency, surplus fusion protein substrate was supplied

15%

tricine-SDS-PAGE, (excluding

the

released invisible

(GLP-1)3 His

fragment accounted

tag-(Asp)4-Lys

band)

for

was

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the total protein

and the

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to EKLC@NH2-MNP. The digested productions by EKLC@NH2-MNP were separated by

calculated by gray scanning. As shown in Fig. 6, the EKLC@NH2-MNP exhibited approximately 90% of the initial activity over 10 cycles of catalytic reaction, suggesting

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that the activity of the EKLC@NH2-MNP remained relatively constant, without a

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significant decrease over 10 cycles (P > 0.05). Furthermore, the EKLC@NH2-MNP also

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simplified the separation of the enzyme from the reaction mixture, significantly, thereby avoiding protease contamination and the instability of the final mature protein (i.e.,

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tagless).

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In summary, an engineered catalytic subunit of EK fused with Gln-donor tag, was ingeniously constructed and site-specifically, covalently immobilized onto NH2-MNPs via MTG-catalyzed bioconjugation, resulting in an EKLC@NH2-MNP biocatalyst. The

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immobilized enzyme effectively retained its intrinsic activity and without a significant loss in enzymatic activity after several cycles of operation. Supported by the unique

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properties of magnetic nanoparticles, the proposed EKLC@NH2-MNP biocatalyst is expected to promote the economical utilization of enterokinase in fusion protein cleavage. In addition, the proposed methodology here for the site-specific and covalent

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immobilization of EK also supplies a candidate strategy for the reusability of other instrumental enzymes. Conflict of interests None

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Acknowledgements This work was supported by the National Natural Scientific Foundation of China (81201346, 31600386) and the Natural Scientific Foundation of Shandong Province (ZR2018MC009, ZR2017LC002). References

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Figure captions Fig. 1. Illustration of site-specific and covalent immobilization of the engineered EKLC-Gln on NH2-MNPs by employing MTG-catalyzed bioconjugation for construction of the reusable EKLC@NH2-MNP biocatalyst.

the

gene engineering E.

coli

BL21

(DE3)

cells

for

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Fig. 2. SDS-PAGE assays. Lane 1 and 2 show the control E. coli BL21 (DE3) cells and production

of

His

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tag-ZZ-(Asp)4-Lys-EKLC-Gln, respectively. Lane 3 and 4 show the supernatant and

insoluble of the gene engineering E. coli cells after ultrasonication, respectively (A). Lane 5 shows the supernatant after renaturation procedure and lane 6 shows the purified

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His tag-ZZ-(Asp)4-Lys-EKLC-Gln by HisTrap Chelating HP column (B). And C shows the autocatalytic cleavage reaction process, lanes 1–7 show the reaction for removing

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His tag-ZZ-(Asp)4-Lys at 0, 1, 2, 3, 4, 5, 6 h, respectively. After autocatalytic cleavage

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A

reaction, the purified EKLC-Gln by HisTrap Chelating HP column is shown in lane 8.

Fig. 3. Site-specific immobilization of engineered EKLC onto NH2-MNPs via

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MTG-catalyzed bioconjugation. The amount of immobilized enzyme was examined by BCA protein assay (n = 3). After chromogenic reaction, NH2-MNPs were firstly

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separated and the supernatants were for the determination of 560 nm value (the protocol is shown in the insert). The signs of + and – indicate those reagents are present or absent

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in the bioconjugation system, respectively.

Fig. 4. The stabilities of EKLC@NH2-MNP biocatalyst were investigated by monitoring

A

enzymatic activity. The storage stability of EKLC@NH2-MNP was evaluated using 7 samples of the equal fresh EKLC@NH2-MNP at 5-days interval for one month. For the reusable stability of EKLC@NH2-MNP, the EKLC@NH2-MNP was examined and stored at 4 °C for the next evaluation within 5-days interval for 7 cycles. The free EKLC-Gln was used as a control. Those activities at different times were compared with the corresponding initial activity, respectively (n = 3). *p < 0.05, two-sided Student’s t-test. - 18 -

Fig. 5.

The apparent zeta potentials of NH2-MNP and EKLC@NH2-MNP. The zeta

potentials of NH2-MNP (A) and EKLC@NH2-MNP (B) were firstly investigated, and those of EKLC@NH2-MNP endured 3 cycles (C) and 7 cycles (D) of cleaving Gly-(Asp)4-Lys-β-naphthylamide were also investigated.

Fig. 6. The reusability of EKLC@NH2-MNP biocatalyst was assessed by removing His

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tag of the His tag-(Asp)4-Lys-(GLP-1)3 fusion protein. Slight superfluous substrate protein was supplied to the EKLC@NH2-MNP for continuous 10 cycles. Lanes a, b show

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the intact fusion protein (11 kDa) and digested by free EKLC-Gln, respectively. The digested productions were separated by 15 % tricine-SDS-PAGE, the uncut fusion protein and released (GLP-1)3 fragment (9.6 kDa) were calculated by gray scanning (the

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small Met-His tag-(Asp)4-Lys fragment was invisible in the gel). The cleavage activity

A

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A

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of the first cycle is set to 100 %.

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