Biocatalyst Immobilization by Anchor Peptides on an Additively

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Biocatalyst Immobilization by Anchor Peptides on an Additively Manufacturable Material Niclas Buescher, Giovanni Sayoga, Kristin Rübsam , Felix Jakob, Ulrich Schwaneberg, Selin Kara, and Andreas Liese Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.9b00152 • Publication Date (Web): 12 Jul 2019 Downloaded from pubs.acs.org on July 19, 2019

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Organic Process Research & Development

Biocatalyst Immobilization by Anchor Peptides on an Additively Manufacturable Material Niclas Büscher1, Giovanni V. Sayoga1, Kristin Rübsam3,4, Felix Jakob3,4, Ulrich Schwaneberg3*, Selin Kara1,2*, Andreas Liese1* 1Institute

of Technical Biocatalysis, Hamburg University of

Technology, Denickestr. 15, D-21073 Hamburg, Germany 2Department

of Engineering, Biocatalysis and Bioprocessing,

Aarhus University, Gustav Wieds Vej 10, DK-8000 Aarhus, Denmark 3DWI

-

Leibniz-Institute

for

Interactive

Materials,

Forckenbeckstrasse 50, D-52074 Aachen, Germany 4RWTH

Aachen University,Lehrstuhl für Biotechnologie, Worringerweg

3, D-52074 Aachen, Germany

*Corresponding authors E-mail: [email protected]; [email protected]; [email protected]

ACS Paragon Plus Environment

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TABLE OF CONTENTS


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ABSTRACT Additive manufacturing refers to manufacturing methods, which are being used to build up three-dimensional (3D) structures by adding a certain material stepwise onto a support. Nowadays, these

manufacturing

efficient

compared

defined production

methods to

can

be

conventional

of a

more

material-

methods

wide variety

and

of 3D

and

cost

allowing

the

structures using

computer aided design (CAD). A broad range of materials can be additively manufactured (AM) resulting in specific properties and highly diverse structures making them promising matrices for the utilization as enzyme carriers. The variety of materials offers the possibility to select materials with properties needed for particular biocatalytic processes. This is especially true for hybrid

reactor

concepts

where

multiple

operations,

including

catalytic reactions and downstream processes, are combined into a single apparatus. For the enzymatic decarboxylation of ferulic acid, polyethylene terephthalate (PET) has been chosen as an additively manufacturable carrier material for the immobilization of

phenolic

acid

decarboxylase

(PAD)

from

Mycobacterium

columbiense. The genetic fusion of PAD with anchor peptides enabled the

adsorptive

immobilization

on

PET.

Starting

from

an

immobilizate activity of 0.39 ± 0.19 U m–2 and a conversion of 19.2 ± 3.7 % after two hours the optimization of the peptide and spacer

sequence

immobilizates

between

with

anchor

activities

peptide up

to

and

PAD

resulted

1.80 ± 0.41 U m–2

in and


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conversions of 59.9 ± 3.9 % after two hours. Moreover, within this study integrating an in situ product removal, enabled by an extraction with n-heptane, the altering of surface hydrophobicity of PET and a conversion of 88.0 ± 3.8 % after two hours could be observed.

KEYWORDS: additive manufacturing, anchor peptides, protein engineering, adsorptive immobilization, biocatalysis

INTRODUCTION The optimization of the environment in which a chemical or biochemical reaction takes place is the research focus within the design of smart reactors. The geometrically forced guidance of reaction components in macro- and micro scale can increase yields and process efficiencies. One method for the intensification of biocatalytic processes and their increase in efficiency is the synergy of reaction and downstream operation.1,2 An example of such a synergy is the in situ extraction of the formed product. Regarding this, the liquidliquid extraction is the most frequently used extraction method3 and represents an advantageous downstream operation especially for temperature sensitive products.4 With an intelligent choice of the extraction agent the reaction product can selectively be


4

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removed from the reaction phase.3 An in situ product removal (ISPR) from the reaction medium has not only the advantage of minimizing the number of apparatus needed for the overall process of producing purified products, additionally it can also affect thermodynamics and kinetics of biocatalytic reactions in a positive way.5,6 One approach to combine reaction and extraction is the implementation of those in flow reactors. Since the extraction efficiency depends on the interfacial area between reaction and extraction phase as well as the concentration gradient between the phases, a sufficient mixing and dispersion needs to be realized e.g. by the help of static mixers as reactor packings. In this respect, additive manufacturing gives the opportunity to create packing structures in a way that they are specifically adjusted to the needs of in situ extractions. The immobilization of enzymes often leads to higher operational stabilities and easier enzyme recovery for re-use.7–9 In industrial applications enzymes are mostly immobilized on solid, porous and spherical carriers e.g. glucose isomerase on controlled-pore alumina10 or lipases on polymers like Lewatit VP OC 160011 or Accurell MP 1000.12,8,13 While porous carriers often provide large specific surface areas (e.g. 100 m2 g–1)10, the biocatalytic reaction might become diffusion limited.9 When it


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comes to the use of immobilized enzyme preparations in continuously operated packed bed reactors, high pressure drops14, non-homogeneous flows15 and wall effects16,17 influence the utilization of the catalyst and the efficiency of the process. The use of additively manufactured (AM) packings can overcome these limitations and further simplify the design of hybrid reactors for which reaction and extraction take place in one apparatus. High precision additively manufactured structures can guide the reaction components in optimal flow regimes, designed on basis of multiphase simulations, overcoming mass transfer limitations in reaction and extraction. One limitation in the application of AM structures in the field of biocatalysis is a generally applicable immobilization technology for different materials being used in additive manufacturing. The existing immobilization methods are mostly not transferable to every combination of enzyme and carrier.18,19 Basically, immobilizations on carrier materials are distinguished in adsorptive and covalent attachments. The reversible adsorptive immobilization allows a fast enzyme replacement subsequent to deactivation. Additionally, adsorptive immobilization can be applicable for a wide range of supports and enzymes.20 The major shortcoming is loss of biocatalysts by leaching (desorption)


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especially in aqueous media since, in principle, adsorptive immobilization is an exothermic equilibrium reaction. The oriented non-covalent immobilization of proteins and enzymes to surfaces by anchor peptides (also known as material binding peptides) at room temperature was reported for surfaces such as polypropylene21,22, carbon23, gold24,25 , and

chlorine-doped

polypyrrole.26,27 Recently, a cutinase degrading polyesterpolyurethane particles was genetically fused to a polyurethane binding anchor peptide: The generated fusion enzyme accelerated the degradation of polyester-polyurethane microplastics up to 6.6-fold due to the high affinity of the anchor peptide towards the polymer surface.28 The active immobilization of the monooxygenase cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium on plane gold surface was achieved by the genetic fusion of gold binding peptides (BP) namely HighSP-BP and CysBP.29 A 47-residue cationic antimicrobial peptide (AMP) found in Bacillus subtilis called LCI30 was reported to serve as an adhesion promotor to polypropylene and enabled the immobilization of the laccase CueO in 96-well microtiter plates.31 Furthermore, several successful directed evolution campaigns to increase the binding strength of anchor peptides in the presence of surfactants such as alkylbenzene sulfonates (LAS) and Triton X-100 were reported for the anchor peptide


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Tachystatin A2 (TA2) and LCI.22,32 Within such approaches, surface-specific anchor peptides are covalently attached and/or genetically fused to the target protein. In addition, the freedom for the use of spacers between the target enzyme and anchor peptide allows enhancing mobility as well as spatial arrangement of the enzyme and provides diverse possibilities for enzyme-spacer-anchor peptide combinations (Figure 1). As a model, the biocatalyst phenolic acid decarboxylase (PAD)33 was chosen, catalyzing decarboxylation of ferulic acid (FA) to form the 25- to 30-fold34 valuable 2-methoxy-4-vinylphenol (MVP), which is a severe inhibitor of PAD. As a consequence,

in situ

product removal by extraction with n-heptane is required.35,33 In this contribution, we report the systematic assessment of an additively manufactured material as a solid support for PAD with surface-specific anchor peptides. The model PAD construct is anchored on polyethylene terephthalate (PET) powder with a particle size below 300 µm and a specific surface area of 0.344 m2 g–1 the subsequent conversion of ferulic acid is presented.35 PET material was chosen as a carrier as a result of initial screening of AM material that were compatible with the reaction conditions in a two-liquid phase system (Figure 1).


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Figure 1. Principle of the adhesive immobilization strategy: The target protein phenolic acid decarboxylase (PAD) is covalently bound to a spacer, which is attached to a surface-specific anchor peptide. The in situ product removal (ISPR) with nheptane allows a minimization of the severe product inhibition. Reaction conditions: T = 37 °C, 0.5 M KPi buffer. FA = ferulic acid; MVP = 2-methoxy-4-vinyl-phenol. RESULTS AND DISCUSSION Material selection The selection of an appropriate additively manufacturable material was based on the requirements for the decarboxylation of ferulic acid (Figure 1) and the in situ product extraction with n-heptane. One appropriate material, which can be additively manufactured, is chemically resistant against n-


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heptane, the buffer system (0.5 M KPi buffer, pH7), and temperature stable up to at least 40 °C, is PET. Its vast availability, the possibility to be manufactured with the most common printing process fused deposition modeling (FDM), and its stability were reasons to show the principle of PAD immobilization on PET as a first example material in this contribution. Preliminary screening step Binding of anchor peptides to the surface of additively manufactured PET (cube with dimensions 10x10x5 mm – printed with Ultimaker 2 pro extend, Ultimaker B.V., The Netherlands) was investigated by the enhanced green fluorescent reporter protein (EGFP). The enhanced green fluorescent protein was genetically fused to three selected anchor peptides: SI (DSI), EGFP-LCI32, and EGFP-Tachystatin A2 (TA2).32 Anchor peptides LCI and TA2 were selected due to their reported strong binding to hydrophobic polymers such as polypropylene (PP).21 This selection was complemented with the potential anchor peptide DSI that was selected due to its high number of hydrophobic residues (50 %).36 PET wafers were coated with EGFP (as control) and EGFP-DSI, EGFP-TA2, and EGFP-LCI. In a subsequent washing procedure unspecifically adsorbed proteins were removed and the surface was analyzed by fluorescence microscopy. For the EGFP control a


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weak fluorescent signal was detected on the coated PET wafers (Figure 2). In contrast, PET surfaces incubated with EGFP-DSI, EGFP-LCI and EGFP-TA2 showed significantly higher fluorescence signal after the washing steps, indicating that the selected anchor peptides DSI, LCI and TA2 are solely responsible for PETbinding.

250 µ m

Figure 2. Visualization of EGFP-anchor peptide adhesion on PET surface by fluorescence microscopy: PET surfaces were incubated with EGFP, EGFP-Dermaseptin (DSI), EGFP-LCI, and EGFPTachystatin A2 (TA2), followed by two washing steps. Successful binding of the anchor peptide to PET is visualized by the green fluorescence of EGFP (excitation 488 nm, emission 500–600 nm) Adhesive binding of the designed constructs on PET The preliminary anchor peptide binding studies showed that the three selected anchor peptides LCI, TA2, and DSI are able to bind on additively manufactured PET. After fusion of the three selected anchor peptides to the catalytically active PAD domain


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(separated by a stiff 17X spacer sequence consisting of 17 amino acids: AEAAAKEAAAKEAAAKA)37, the activity of the free and immobilized fusion enzymes (immobilized on PET powder; d

Biocatalyst Immobilization by Anchor Peptides on an Additively

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