Stoichiometrically Controlled Immobilization of Multiple Enzymes on

Nov 16, 2015 - Stoichiometrically Controlled Immobilization of Multiple Enzymes on Magnetic Nanoparticles by the Magnetosome Display System for Effici...
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Stoichiometrically Controlled Immobilization of Multiple Enzymes on Magnetic Nanoparticles by the Magnetosome Display System for Efficient Cellulose Hydrolysis Toru Honda, Tsuyoshi Tanaka, and Tomoko Yoshino Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01174 • Publication Date (Web): 16 Nov 2015 Downloaded from http://pubs.acs.org on November 21, 2015

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Stoichiometrically Controlled Immobilization of Multiple Enzymes on Magnetic

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Nanoparticles by the Magnetosome Display System for Efficient Cellulose Hydrolysis

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Toru Honda, Tsuyoshi Tanaka, and Tomoko Yoshino*

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Division of Biotechnology and Life Science, Institute of Engineering, Tokyo University of

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Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan

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*Corresponding author: Tomoko Yoshino; Division of Biotechnology and Life Science,

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Institute of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho,

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Koganei, Tokyo 184-8588, Japan

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Telephone: +81-42-388-7021

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Fax: +81-42-385-7713

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E-mail: [email protected]

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Keywords: magnetosome display system; cellulosome; protein complex; magnetic nanoparticle

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Abstract

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The immobilization of multiple cellulase complexes receiving attention for use in the efficient

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hydrolysis of celluloses. In this study, the magnetosome display system was employed for the

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preparation of systems mimicking natural multiple cellulase complexes (cellulosomes) on

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magnetic nanoparticles (MNPs). Initially, two fluorescent proteins, namely green fluorescent

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protein and mCherry, were immobilized on MNPs. Fluorescence analysis revealed the close

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proximity of two different proteins on the MNPs. Enzyme-linked immunosorbent assay analysis

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showed that stoichiometrically equivalent amounts of the proteins were immobilized on the

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MNPs. Next, endoglucanase (EG) and β-glucosidase (BG) were immobilized on MNPs to give

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EG/BG-MNPs. The resulting MNPs were applied for the hydrolysis of celluloses, with rapid

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hydrolysis of carboxymethyl cellulose being observed. Furthermore, the fusion of the cellulose-

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binding domain to EG/BG-MNPs promoted improved hydrolysis activity against the insoluble

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cellulose. We could therefore conclude that the magnetosome display system can expand the

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possibilities of mimicking natural cellulosome organization on MNPs.

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1. Introduction

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Cellulose is the most abundant and promising biomass for bioethanol production. Its hydrolysis

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by cellulase is key for this process, however, the large consumption of cellulase makes it costly,

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resulting in a bottleneck in cellulose-based bioethanol production 1. An alternative approach is

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the utilization of immobilized cellulase on solid materials, as immobilization provides

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advantages in terms of handling, reusability, and cellulase stability2-6.

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Although a number of studies have been reported for the preparation of immobilized cellulase,

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the immobilization of multi-enzyme complexes, known as cellulosomes7, has also attracted

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considerable attention. Cellulosomes has been discovered in several cellulolytic bacteria, such as

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Clostridium cellulolyticum, Clostridium thermocellum, and Ruminococcus flavefaciens8. It is

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composed of a scaffold protein containing a cohesin domain, a cellulose-binding domain (CBD),

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and several cellulases with the dockerin domain (i.e., endoglucanase, exoglucanase, and β-

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glucosidase). The dockerin domain can specifically bind to the cohesin domain on the scaffold

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protein, allowing the construction of highly organized multi-enzyme complexes in close

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proximity to one another. This close proximity leads to synergistic effects in enzymatic activity,

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resulting in efficient cellulose hydrolysis9. Recently, to mimic the cellulosome on solid materials,

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the immobilization of multiple cellulases onto magnetic particles has been attempted10,

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However, reproduction of the highly organized multiple enzyme assembly was difficult, as the

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enzymes were randomly immobilized on the magnetic particle surfaces through chemical cross-

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linking or Au-S bonding.

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We previously proposed a novel process for the immobilization of recombinant proteins onto

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magnetic nanoparticles (MNPs) using the magnetosome display system with a magnetotactic

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.

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bacterium, Magnetospirillum magneticum strain AMB-1

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in magnetotactic bacteria, consisting of a magnetite core covered by a lipid bilayer membrane,

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and an average diameter of 75 nm 12 (see also Figure 1(A)). In the magnetosome display system,

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foreign proteins can be immobilized on magnetosomes using the mms13 gene as a fusion partner

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by genetic engineering13. Mms13, a protein kwon to double-pass transmembrane protein and

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tightly bind to the magnetite core. Therefore, M. magneticum AMB-1 was transformed by the

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fusion gene of mms13 and target protein gene, and then desired target protein can be efficiently

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expressed (or immobilized) on magnetosomes via Mms13 in M. magneticum. Until now, various

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proteins, including single-chain variable fragments

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complex

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have been successfully immobilized on MNPs using the magnetosome display system. The

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genetically engineered MNPs can be easily extracted and purified from the M. magneticum

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AMB-1 transformant cell lysate by magnetic separation. The complexes of foreign proteins with

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MNPs have been applied to immunoassays, ligand screening assays, and cell separation systems

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as magnetic carriers18-23. The magnetosome display system can therefore enable us to construct

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highly organized multi-enzyme complexes on MNPs.

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We report the preparation of highly organized protein complexes on MNPs using the

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magnetosome display system, for application in the hydrolysis of celluloses. Initially, two

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fluorescent proteins, namely green fluorescent protein and mCherry, will be immobilized on

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MNPs for the proof-of-concept study. Fluorescent microscopy and fluorescence resonance

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energy transfer analysis will be used to determine the proximity of two different proteins on the

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MNPs based on the interaction between the cohesin and dockerin domains. Furthermore, ELISA

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analysis will be used to determine the quantities of proteins immobilized on the MNPs.

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, a tropomyosin receptor kinase A

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. Magnetosome is a unique organelle

, the class I major histocompatibility

, and a thyroid-stimulating hormone receptor

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,

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Subsequently, endoglucanase and β-glucosidase immobilized on MNPs will be prepared using

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the magnetosome display system and tested for cellulose hydrolysis activity. We expect to

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confirm synergistic effects when using two close proximity enzymes and the CBD on MNPs,

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resulting in the efficient degradation of celluloses. We predict that the magnetosome display

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system will expand the possibilities to mimic natural cellulosome organization.

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2. Experimental Section

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2.1 Bacterial Strains and Culture Conditions

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Escherichia coli TOP10 (Life technologies, CA, US) and EPI300 (ARBROWN, Tokyo, Japan)

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were used for gene cloning. E. coli transformant cells were cultured at 37 °C in Luria-Bertani

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(LB) agar plates or in ampicillin-containing media (50 µg mL-1). The Magnetospirillum

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magneticum AMB-1 (ATCC 700264) mms13 gene deletion mutant (strain AMB-1 ∆mms13) was

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used for the production of magnetic nanoparticles (MNPs)24. Strain AMB-1 ∆mms13 was

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microaerobically cultured in magnetic spirillum growth medium (MSGM) containing

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gentamycin (2.5 µg mL-1) at 28 °C as described previously

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constructed by electroporation

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and ampicillin (5 µg mL-1). Furthermore, the expression of target proteins was induced by the

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addition of anhydrotetracycline hydrochloride (500 ng mL-1, Kanto Chemical, Tokyo, Japan) at

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the mid-log phase, and the mixture cultured for a further 12 h.

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. AMB-1 transformants were

and cultured in MSGM containing gentamycin (2.5 µg mL-1)

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2.2 Plasmid Construction

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A schematic diagram representing the co-expression plasmids in Figure S1. Co-expression

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plasmids were comprised of two fusion gene sets, namely “mms13-cohesin” genes and “target-

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dockerin” genes. Cohesin domains from Clostridium thermocellum (CohC: CipA 364-702 amino

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acids) and Ruminococcus flavefaciens (CohR: ScaB 29-186 amino acids) were fused to mms13.

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Dockerin domains from C. thermocellum (DocC: CelS 673-741 amino acids) and R. flavefaciens

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(DocR: ScaA 787-879 amino acids) were fused to target proteins (GFP and mCherry, or

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endoglucanase celCCA (EG) and β-glucosidase Ccel_2454 (BG). EG and BG were removed

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signal sequence and native dockerin. The gene sequences of two cellulases and the cellulose

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binding domain (CBD) were also obtained from C. thermocellum. All genes were generated by

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artificial gene synthesis (Integrated DNA Technologies, Coralville, IA, USA). Fusion genes were

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cloned into pUMtOR which is a tetracycline-inducible vector for M. magneticum AMB-1 27. The

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linker is a hydrophilic polypeptide linker containing repeating amino acid chains of 4 asparagine

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residues and 1 serine residue (linker length: 10 amino acids)28. The thrombin cleavage site was

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designed between the linker and CohC. All prepared transformants are listed in Table S1.

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2.3 Preparation of MNPs

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Cultured cells of M. magneticum AMB-1 transformants using MSGM were collected by

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centrifugation at 9000 g for 10 min at 4 °C. Collected cells were re-suspended in HEPES-Ca

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(40 mL, 10 mM, supplemented with 1 mM CaCl2, pH 7.4), and disrupted by passing through a

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French press at 1500 kg cm-2 (Ohtake Works, Tokyo, Japan). MNPs were collected from the

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disrupted cell suspension using a columnar neodymium-boron magnet. The MNPs were washed

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10 times with HEPES-Ca (20 mL). The concentration of MNPs in suspension was determined by

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measuring the optical density at 660 nm using a UV-2200 spectrophotometer (Shimadzu, Kyoto,

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Japan). A value of 1.0 corresponded to 172 µg MNPs (dry weight) per mL.

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2.4 Enzyme-Linked Immunosorbent Assay (ELISA)

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Sandwich ELISA analysis was performed for the quantification of GFP and mCherry on the

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MNPs. Mouse anti-GFP IgG (Takara Bio, Otsu, Japan) and alkaline phosphatase (ALP)-

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conjugated anti-GFP IgG (Rockland Immunochemicals, PA, USA), or mouse anti-mCherry IgG

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(Takara Bio, Otsu, Japan) and ALP-rat anti-mCherry IgG (Life Technologies, CA, USA) were

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used as primary and secondary antibodies, respectively. ALP conjugation to rat anti-mCherry

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IgG was performed using the Alkaline Phosphatase Labeling Kit–NH2 (Dojindo, Kumamoto,

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Japan). Primary antibodies (2 µg mL-1, 100 µL) in bicarbonate buffer (pH 9.8) were added to a

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96 well palate (Costar 3925, Corning Inc., NY, USA) and incubated at 4 °C overnight to allow

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sufficient time for adsorption to take place. After washing three times with PBST (phosphate-

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buffered saline, PBS, containing 0.1% Tween-20, at pH 7.4), the plates were blocked with 0.2%

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bovine serum albumin (BSA) in PBS for 1.5 h at room temperature. After washing with PBST,

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the protein samples (100 µL) were added, and the plates incubated for 1 h at 37 °C. The protein

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fractions on the MNPs were extracted according to a modified literature procedure

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washing with PBST, secondary antibodies (1 µg mL-1 for GFP, 2 µg mL-1 for mCherry) were

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added and incubated at 37 °C for 1 h. After washing once more with PBST, Lumi-Phos 530

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(100 µL, Wako Pure Chemical Industries) was added as a luminescence substrate. After

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incubation for 5 min, the luminescence intensity was measured using a microplate reader SH-

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9000 (Corona Electric, Ibaraki, Japan). The quantities of GFP and mCherry were estimated using

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. After

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luminescent curves of the GFP and mCherry standards (Cell Biolabs, Inc., CA, USA). Finally,

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the quantities of GFP (27 kDa) and mCherry (29 kDa) on a single MNP were calculated as

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described previously28.

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2.5 FRET Analysis

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Prior to fluorescence measurements, the protein complexes were purified from MNPs by

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thrombin digestion to avoid interference from MNP light scattering30. The MNPs (1 mg) were

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suspended in thrombin solution (100 µL, 100 U mL-1 in PBS) and incubated at 4 °C overnight.

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Following centrifugation at 18000 g for 1 min, the supernatants were collected and used for the

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measurements. Fluorescence emission spectra using an excitation wavelength of 483 nm were

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acquired between 540 and 680 nm to detect the fluorescence resonance energy transfer (FRET)

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between GFP and mCherry using a microplate reader SH-9000 (Corona Electric, Ibaraki, Japan).

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2.6 Hydrolysis of Soluble and Insoluble Celluloses using MNP-Immobilized Cellulases

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MNPs (50 µg) were added to a substrate solution (0.5% carboxymethyl cellulose (CMC) or 1%

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Avicel in 100 mM Tris-HCl containing 1 mM CaCl2, at pH 5.5). Following incubation at 30 °C,

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the MNPs were removed from the suspension by magnetic separation, and the supernatant was

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collected. The supernatant (5 µL) was added to tetrazolium blue chloride assay buffer (195 µL,

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composition = 1 mg mL-1 tetrazolium blue chloride, 0.5 M potassium sodium tartrate, 50 mM

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NaOH)

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absorbance of the solutions at 704 nm was measured using a microplate reader SH-9000

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. After boiling at 100 °C for 3 min, the solutions were cooled using an ice bath. The

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(Corona Electric, Ibaraki, Japan). The concentrations of reducing sugars present were calculated

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from a standard curve using glucose.

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3. Results and Discussion

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3.1 Protein Proximity Analysis of Marker Proteins on MNPs

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Prior to preparation of the cellulase-MNP complexes, two marker proteins, namely GFP and

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mCherry, were immobilized onto MNPs for the evaluation of spatial protein-protein proximity.

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Immobilization was achieved using the magnetosome display system. A schematic diagram of

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the transformants used in this section is given in Figure 1(A). In this study, the dockerin domains

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from C. thermocellum (DocC) and R. flavefaciens (DocR) were fused to GFP and mCherry,

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respectively. Furthermore, the cohesin domains from C. thermocellum (CohC) and R.

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flavefaciens (CohR) were fused to mms13. The CohC and CohR were linked with a peptide

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linker of 10 amino acids. GFP and mCherry were expected to be co-localized on magnetosomes

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arranged in a chain through interactions between cohesin and dockerin in AMB-1/CohCR-GmC

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(details of the M. magneticum AMB-1 transformants are given in Table S1). As expected,

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fluorescence from both GFP and mCherry was observed at the same positions, with

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magnetosome chains present in AMB-1/CohCR-GmC (Figure 1(B), top), although no

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fluorescence was detected in the wild type (Figure 1(B), bottom). Furthermore, fluorescence

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from mCherry was widely distributed in the cytoplasm of CohR-lacking AMB-1/CohC-GmC

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(Figure 1(B), middle), indicating that two different proteins are co-immobilized onto the MNPs

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through cohesin-dockerin interactions.

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The spatial protein-protein proximity was then investigated by FRET analysis. The MNPs were

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extracted from cell lysates of AMB-1 transformants by magnetic separation, followed by

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purification of the MNP protein fractions by enzymatic cleavage, as described in the

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Experimental Section (Section 5). Figure 2 shows the emission spectra of fluorescent proteins

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under an excitation wavelength of 483 nm. No FRET signal was detected from the MNP/CohC-

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GmC protein complexes (Figure 2, dashed line), as these protein complexes include only GFP. A

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slight increase in the signal at ~610 nm was observed, even when free mCherry was added to the

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GFP-containing protein complexes (Figure 2, gray solid line). In contrast, mCherry fluorescence

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was detected in protein complexes purified from MNP/CohCR-GmC (Figure 2, black solid line,

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indicated by an arrow). mCherry may therefore act efficiently as a fluorescence acceptor towards

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GFP, which acts as a fluorescence donor in the FRET system on MNP/CohCR-GmC. FRET

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occurs when GFP and mCherry are co-localized within 1-10 nm of each other, with the highest

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efficiency reached between 4-6 nm

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CohR) were linked with a peptide linker consisting of 10 amino acids. If the DocC and DocR

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fused to fluorescence proteins were respectively bound to each scaffold protein, two fluorescence

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proteins were located within about 4 nm corresponding to a peptide unit’s length of 0.38 nm33.

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Therefore, this closer position led to the generation of FRET.

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The quantities of GFP (1.3 ± 0.2 µg mg-1-MNP) and mCherry (1.4 ± 0.1 µg mg-1-MNP) present

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on the MNPs were estimated using enzyme-linked immunosorbent assay (ELISA) analysis.

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Based on these results, the number of GFP and mCherry molecules per single MNP were

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calculated according to literature methods28, and found to be 33 molecules/MNP for both

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proteins. This value was in agreement with previously reported results, i.e., 22 molecules-Protein

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A/MNP

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. The distance between two scaffold proteins (CohC and

and 20-34 molecules-Protein G/MNP23. These results indicate that stoichiometrically

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equivalent amounts of GFP and mCherry were successfully immobilized on MNPs using the

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magnetosome display system. This demonstrates that if desired, the number of recombinant

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proteins present on MNPs can be altered by genetic engineering.

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3.2 Hydrolysis of Carboxymethyl Cellulose (CMC) using Endoglucanase (EG) and β-

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Glucosidase (BG) Immobilized on MNPs (EG/BG-MNPs)

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According to GFP and mCherry immobilization conditions, endoglucanase (EG) and β-

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glucosidase (BG) were immobilized onto MNPs via cohesin-dockerin interactions (Figure 3(A),

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EG/BG-MNPs) for application in the hydrolysis of the soluble cellulose, carboxymethyl

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cellulose (CMC). A schematic diagram of the transformants used in this section is given in

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Figure 3(A). To evaluate the proximity effect of the two enzymes on CMC hydrolysis, the

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mixture of EG-MNPs and BG-MNPs was used as a control (Figure 3(A), EG-MNPs and BG-

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MNPs). It should be noted that equal quantities of the two enzymes were used for CMC

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hydrolysis. When the CMC solution was treated with EG/BG-MNPs, the quantity of reducing

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sugar increased with time, reaching a plateau at >3 h (Figure 3(B), closed circles). In contrast,

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the hydrolysis rate of the EG-MNPs and BG-MNPs mixture was significantly slower than that of

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EG/BG-MNPs, with the quantity of reducing sugar reaching a plateau beyond 7 h (Figure 3(B),

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open circles). This indicates that the close proximity of EG and BG may contribute to the higher

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hydrolysis rate in EG/BG-MNPs. The previous report revealed the higher activity of

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immobilized three enzymes (EG, BG and exoglucanase) against CMC than that of free

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enzymes9. The resulting concentration of reducing sugars was increased from about 3.2 mM to

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3.9 mM. These values (3.2 to 3.9 mM) are similar level with the obtained value (1.6 mM) in this

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study. Based on these results, we have concluded that the co-immobilization by our proposed

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method has an advantage over using free enzymes. In general, cellulose is degraded to

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oligosaccharides by EG, and the oligosaccharides are subsequently degraded to glucose by BG.

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Thus, the spatial proximity of these two enzymes leads to synergistic enzyme reactions for the

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efficient degradation of cellulose, suggesting that the efficient degradation of CMC by EG/BG-

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MNPs may be caused by the oligosaccharides being unable to diffuse away from the reaction

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center due to the proximity of EG and BG.

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The reusability of the EG/BG-MNPs was then investigated (Figure 3(C)). Following hydrolysis

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of CMC, the MNPs were magnetically collected, and reused in further CMC hydrolysis cycles

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under the same conditions. The hydrolysis activity in the first trial was defined as 100%, and the

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activity of EG/BG-MNPs was maintained at >70% after five trials. Previous reports using

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chemically conjugated cellulases and magnetic particles indicate that enzyme activity dropped to

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20-50% after five trials, even with the use of thermostable cellulases and optimal conditions for

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CMC hydrolysis

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system show good performance in terms of cellulase reusability. The slight decrease in

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enzymatic activity could be explained by two enzymes being released from or inactivated on

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MNPs during the hydrolysis process. It is also possible that the amount of MNPs used gradually

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decreased during the magnetic separation process.

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. This suggests that the MNPs prepared using the magnetosome display

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3.3 Effect of the Cellulose-Binding Domain (CBD) on Hydrolysis of Microcrystalline

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Cellulose

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The EG/BG-MNPs were applied in the hydrolysis of the insoluble cellulose, Avicel, but little

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hydrolysis was detected (Figure 4(B)). In order to improve the hydrolysis activity, the cellulose-

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binding domain (CBD) was fused to the end of the scaffold protein attached to the MNPs (Figure

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4(A), EG/BG-CBD-MNPs). The CBD plays an important role in the binding of cellulosome to

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the crystalline cellulose34-36. When compared to the EG/BG-MNPs, the production of reduced

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sugar was significantly enhanced when EG/BG-CBD-MNPs were used (Figure 4(B)). The

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binding of the CBD to microcrystalline cellulose may promote the hydrolysis of EG and BG on

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MNPs. Although the hydrolysis rate of Avicel was approximately 10 times lower than that of

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CMC (Figure 3(B)), this tendency correlates with previous results.

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4. Conclusions

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We reported the construction of highly organized protein complexes on magnetic nanoparticles

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(MNPs) using the magnetosome display system. A proof-of-concept study using green

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fluorescent protein (GFP) and mCherry revealed the close proximity (