Origami Cells Research Articles

A Versatile Microfluidic Platform for Extravasation Studies Based on DNA Origami—Cell Interactions

AbstractThe adhesion of circulating tumor cells (CTCs) to the endothelial lumen and their extravasation to surrounding tissues are crucial in the seeding of metastases and remain the most complex events of the metastatic cascade to study. Integrins expressed on CTCs are major regulators of the extravasation process. This knowledge is primarily derived from animal models and biomimetic systems based on artificial endothelial layers, but these methods have ethical or technical limitations. We present a versatile microfluidic device to study cancer cell extravasation that mimics the endothelial barrier by using a porous membrane functionalized with DNA origami nanostructures (DONs) that display nanoscale patterns of adhesion peptides to circulating cancer cells. The device simulates physiological flow conditions and allows direct visualization of cell transmigration through microchannel pores using 3D confocal imaging. Using this system, we studied integrin‐specific adhesion in the absence of other adhesive events. Specifically, we show that the transmigration ability of the metastatic cancer cell line MDA‐MB‐231 is influenced by the type, distance, and density of adhesion peptides present on the DONs. Furthermore, studies with mixed ligand systems indicate that integrins binding to RGD (arginine‐glycine‐aspartic acid) and IDS (isoleucine‐aspartic acid‐serine) did not synergistically enhance the extravasation process of MDA‐MB‐231 cells.

Kresling origami mechanics explained: Experiments and theory

From a kinematics perspective, a Kresling origami cell couples axial displacement (contraction/expansion) with twist, leading to non-rigid origami behavior. From an energy landscape perspective, the selection of the Kresling origami geometry, together with its fabrication process and material, lead to energy envelopes allowing single or multiple stable states. In this context, this paper explores the Kresling origami mechanics through mathematical modeling integrated with experimental testing. On the theoretical mechanics front, we present a comprehensive model incorporating the representative geometrical parameters of the Kresling origami cell into the corresponding energy function in order to capture its essential mechanical behavior. On the experimental mechanics front, we create two fixtures that demonstrate the ability to control axial displacement (contraction/expansion) and twist independently, without imposing any constraints on the chiral arrangement of individual cells within the Kresling origami array (composed of multiple cells). Finally, we show the coexistence of multiple mechanical and morphological configurations within the same Kresling array by programming its loading modes, i.e., compression or twist. The fundamental nature of this work makes it applicable to several fields of engineering, including soft robotics and mechanical computing.

Compression performance evaluation of a novel origami-lattice metamaterial

The thin-walled lattice metamaterials are usually applied into the energy absorption, and the low-weight and high-bearing components. In this paper, a novel kind of open-section origami units with single degree of freedom (DOF) motion from flat foldability to closed tube are firstly proposed and verified based on the symmetry design method. On this base, a novel origami-lattice metamaterial is firstly proposed through stacking multiple symmetry open-section origami cells. Furthermore, the influence of different geometry parameters and compression directions on the energy absorption ability and the deformation mechanism are analyzed in detail. The relative density of the proposed origami-lattice metamaterials will increase with the dihedral angles and wall thickness. Testing results show that the initial peak force, specific energy absorption of the proposed lattice metamaterials in z-direction and y-direction compression could improve by 397.18 % and 138.69 %, 303.84 % and 82.77 % with the increase of the wall thickness from 1 mm to 2 mm. With the increase from 90° to 135°, the initial peak force, EA value, and SEA value in the z-direction could improve by 79.34 %, 55.54 % and 56.93 %. When the z-direction layers increase from the 4 to 6, the initial peak force, EA value, and SEA value in the y-direction could improve 78.10 %, 204.72 % and 101.82 %. Significantly, the proposed lattice metamaterials have the better low-weight and high-bearing and energy absorption capacity, which is promising for the architecture field.

Novel open-section origami tubes with multiple programmable mechanical properties

The diverse folding motion of the origami structures induces a series of extraordinary properties, which enriches their application prospects in engineering. In this paper, we propose a class of open-section origami tubes with multiple extraordinary mechanical properties, and explore their potential applications. First, three types of origami tubes with one degree of freedom are developed. Their motion patterns are studied through the kinematic analysis, which induces several extraordinary mechanical properties. The Poisson’s ratio of the proposed structure undergoes multiple sign-flipping, which can be tuned by the geometric parameters of panels. Also, the origami tubes exhibit multiple mechanical properties under compression such as mono/bistable and zero stiffness mode, which can be tuned by the rotational stiffness assignment of the creases. The self-locking behavior of the tandem origami tubes is also discussed, which shows the remarkable programmability on the number and position of locking. Based on the proposed origami cells, the piecewise-stiffness structure and quasi-zero-stiffness structure are constructed, and their performance is verified through the prototype tests.

Digitized design and mechanical property reprogrammability of multistable origami metamaterials

Origami-based multistable metamaterials have been recognized as a promising platform for diverse applications due to their exceptional mechanical properties. However, the current state of the art in designing origami metamaterials is mostly ad-hoc and narrowly focused on a particular mechanical property. In other words, there is a lack of basic research on deriving generic and systematic methodologies for the design, configuration tuning, and property programming of origami metamaterials, to explore their achievable ranges of mechanical properties comprehensively from the perspective of mechanics. In this work, a mathematically rigorous strategy for synthesizing and reconfiguring multistable origami metamaterials based on two basic modules is put forward, and a systematic approach to analyze and program the material properties is proposed. More specifically, by programming the binary design array of module arrangement and by mathematically expressing the connection constraints, a number of multistable origami cells with exceptional shape reconfigurability are generated. Building upon this, the fundamental mechanics properties, including the multistability, global stress-strain profiles, and tangent elastic moduli, are thoroughly investigated via examining the unit cell, i.e., the “infinitesimal element,” of the metamaterial. Considering the extreme cases of the unit cell architecture, boundaries of the material properties of the synthesized metamaterials at the stress-free configuration are derived, which are of vital importance for material selections and topological optimization. Moreover, due to the topologically different configurations, six categories of metamaterials with qualitatively different material densities and moduli programmability are revealed. In addition to the reprogrammable density and Young's modulus of conventional Miura origami metamaterial, more intriguing properties, such as reprogrammable shearing modulus, locking effect, and inner reconfiguration without modulus change, are discovered for the first time. A prototype made of dual-material 3D printing is created to experimentally evaluate and verify the effectiveness of the analysis method and the characterized material properties. Overall, this work provides a rigorous tool for designing multistable origami metamaterials and an effective strategy for characterizing their fundamental mechanical properties, which will greatly enhance the systematization of creating origami metamaterials.

Multiplexed Detection of Molecular Interactions with DNA Origami Engineered Cells in 3D Collagen Matrices.

The interactions of cells with signaling molecules present in their local microenvironment maintain cell proliferation, differentiation, and spatial organization and mediate progression of diseases such as metabolic disorders and cancer. Real-time monitoring of the interactions between cells and their extracellular ligands in a three-dimensional (3D) microenvironment can inform detection and understanding of cell processes and the development of effective therapeutic agents. DNA origami technology allows for the design and fabrication of biocompatible and 3D functional nanodevices via molecular self-assembly for various applications including molecular sensing. Here, we report a robust method to monitor live cell interactions with molecules in their surrounding environment in a 3D tissue model using a microfluidic device. We used a DNA origami cell sensing platform (CSP) to detect two specific nucleic acid sequences on the membrane of B cells and dendritic cells. We further demonstrated real-time detection of biomolecules with the DNA sensing platform on the surface of dendritic cells in a 3D microfluidic tissue model. Our results establish the integration of live cells with membranes engineered with DNA nanodevices into microfluidic chips as a highly capable biosensor approach to investigate subcellular interactions in physiologically relevant 3D environments under controlled biomolecular transport.

Bio-inspired programmable multi-stable origami

This Letter presents a programmable path to multi-stability of the Kresling origami by introducing bio-inspired nonlinear creases. The origami mathematical model is proposed for the bio-inspired Kresling with the validations by uniaxial compression experiment and finite element analysis. Mono-stability, bi-stability, and tri-stability are demonstrated in one Kresling origami cell. Local energy minimum of multi-stability is found to arise from asymmetric energy barriers. Stable state bifurcation can be tunable under different nonlinearity of the creases and free-stress dihedral angles. Position of stable equilibria can be programmable by varying free-stress dihedral angle of the crease. This work provides a strategy to design programmable multi-stable origami structures.

DNA origami cell sensors for real time probing of membrane interactions with biomolecules in extracellular matrix

Multicellular organisms depend on cell-cell and cell-environment interactions to guide spatiotemporal patterning and organization of tissue. Dysregulation of these interactions underlie disease progression, such as metabolic disorders, autoimmune diseases, and cancer. Thus, the ability to externally monitor cellular interactions is of a great significance. Conventional assays such as ELISA only focus on the end point cell respond and fail to provide real-time information of single cells interaction with its microenvironment.

Graph-theoretic estimation of reconfigurability in origami-based metamaterials

Origami-based mechanical metamaterials have recently received significant scientific interest due to their versatile and reconfigurable architectures. However, it is often challenging to account for all possible geometrical configurations of the origami assembly when each origami cell can take multiple phases. Here, we investigate the reconfigurability of a tessellation of origami-based cellular structures composed of bellows-like unit cells, specifically Tachi-Miura Polyhedron (TMP). One of the unique features of the TMP is that a single cell can take four different phases in a rigid foldable manner. Therefore, the TMP tessellation can achieve various shapes out of one original assembly. To assess the geometrical validity of the astronomical number of origami phase combinations, we build a graph-theoretic framework to describe the connectivity of unit cells and to analyze the reconfigurability of the tessellations. Our approach can pave the way to develop a systematic computational tool to design origami-based mechanical metamaterials with tailored properties.

Origami-Based Bistable Metastructures for Low-Frequency Vibration Control

Abstract In this research, we aim to combine origami units with vibration-filtering metastructures. By employing the bistable origami structure as resonant unit cells, we propose metastructures with low-frequency vibration isolation ability. The geometrical nonlinearity of the origami building block is harnessed for the adjustable stiffness of the metastructure’s resonant unit. The quantitative relationship between the overall stiffness and geometric parameter of the origami unit is revealed through the potential energy analysis. Both static and dynamic experiments are conducted on the bistable origami cell and the constructed beam-like metastructure to verify the adjustable stiffness and the tunable vibration isolation zone, respectively. Finally, a two-dimensional (2D) plate-like metastructure is designed and numerically studied for the control of different vibration modes. The proposed origami-based metastructures can be potentially useful in various engineering applications where structures with vibration isolation abilities are appreciated.

Geometric Mechanics of Origami Patterns Exhibiting Poisson's Ratio Switch by Breaking Mountain and Valley Assignment.

Exploring the configurational space of specific origami patterns [e.g., Miura-ori (flat surface with parallelogram crease patterns), eggbox] has led to notable advances in science and technology. To augment the origami design space, we present a pattern, named "Morph," which combines the features of its parent patterns. We introduce a four-vertex origami cell that morphs continuously between a Miura mode and an eggbox mode, forming an homotopy class of configurations. This is achieved by changing the mountain and valley assignment of one of the creases, leading to a smooth switch through a wide range of negative and positive Poisson's ratios. We present elegant analytical expressions of Poisson's ratios for both in-plane stretching and out-of-plane bending and find that they are equal in magnitude and opposite in sign. Further, we show that by combining compatible unit cells in each of the aforementioned modes through kinematic bifurcation, we can create hybrid origami patterns that display unique properties, such as topological mode locking and tunable switching of Poisson's ratio.

Expression and purification of AtRALF1 from Escherichia coli

Rapid Alakalinization Factors (RALFs) are a family of plant specific peptide growth factors involved in myriad plant processes including growth, development, and response to stresses. These secreted peptides are genetically encoded and processed into an active form from a larger pre-pro-peptide. The active forms are ~5 kDa and have two disulfide bridges. Here we report our attempts to express the active form of AtRALF1 (At1g02900) from the soluble fractions of two different expression strains of E. coli, Origami 2 (DE3) and SHuffleT7 pLysY. Both of these strains are commercially available and genetically altered for expression of proteins with disulfide bonds. Expression of these peptides is challenging because misfolded disulfides or other structural issues result in insoluble peptides. We sought to optimize expression conditions to bulk purify HIS(6x)-AtRALF1 by cobalt column chromatography. Under most conditions the peptide was insoluble. However, at 25oC around 50% of total HIS(6x)-AtRALF1 was soluble in Origami cells. As controls, we expressed mutated versions with alterations in the conserved YISY motif which are physiologically inactive. Surprisingly, these mutant versions were 100% soluble in most conditions tested. All versions of HIS(6x)-AtRALF1 were observed to migrate ~12 kDa in SDS-PAGE gels although their predicted molecular weights is ~8 kDa. Treatment under strong reducing conditions had no effect on this migration pattern. We conclude that AtRALF1’s insolubility issues could be more related to a structure in the YISY motif than to the formation of disulfide bonds.

Short-chain consensus alpha-neurotoxin: a synthetic 60-mer peptide with generic traits and enhanced immunogenic properties.

The three-fingered toxin family and more precisely short-chain α-neurotoxins (also known as Type I α-neurotoxins) are crucial in defining the elapid envenomation process, but paradoxically, they are barely neutralized by current elapid snake antivenoms. This work has been focused on the primary structural identity among Type I neurotoxins in order to create a consensus short-chain α-neurotoxin with conserved characteristics. A multiple sequence alignment considering the twelve most toxic short-chain α-neurotoxins reported from the venoms of the elapid genera Acanthophis, Oxyuranus, Walterinnesia, Naja, Dendroaspis and Micrurus led us to propose a short-chain consensus α-neurotoxin, here named ScNtx. The synthetic ScNtx gene was de novo constructed and cloned into the expression vector pQE30 containing a 6His-Tag and an FXa proteolytic cleavage region. Escherichia coli Origami cells transfected with the pQE30/ScNtx vector expressed the recombinant consensus neurotoxin in a soluble form with a yield of 1.5mg/L of culture medium. The 60-amino acid residue ScNtx contains canonical structural motifs similar to α-neurotoxins from African elapids and its LD50 of 3.8µg/mice is similar to the most toxic short-chain α-neurotoxins reported from elapid venoms. Furthermore, ScNtx was also able to antagonize muscular, but not neuronal, nicotinic acetylcholine receptors (nAChR). Rabbits immunized with ScNtx were able to immune-recognize short-chain α-neurotoxins within whole elapid venoms. Type I neurotoxins are difficult to isolate and purify from natural sources; therefore, the heterologous expression of molecules such ScNtx, bearing crucial motifs and key amino acids, is a step forward to create common immunogens for developing cost-effective antivenoms with a wider spectrum of efficacy, quality and strong therapeutic value.

Origami-based tunable truss structures for non-volatile mechanical memory operation

Origami has recently received significant interest from the scientific community as a method for designing building blocks to construct metamaterials. However, the primary focus has been placed on their kinematic applications by leveraging the compactness and auxeticity of planar origami platforms. Here, we present volumetric origami cells—specifically triangulated cylindrical origami (TCO)—with tunable stability and stiffness, and demonstrate their feasibility as non-volatile mechanical memory storage devices. We show that a pair of TCO cells can develop a double-well potential to store bit information. What makes this origami-based approach more appealing is the realization of two-bit mechanical memory, in which two pairs of TCO cells are interconnected and one pair acts as a control for the other pair. By assembling TCO-based truss structures, we experimentally verify the tunable nature of the TCO units and demonstrate the operation of purely mechanical one- and two-bit memory storage prototypes.

Asymmetric energy barrier and mechanical diode effect from folding multi-stable stacked-origami

Origami, the ancient art of paper folding, has evolved into a design framework of a wide variety of engineering systems such as deployable structures and architected cellular materials. The fundamentally three-dimensional folding is not only a capable tool to generate shape transformation, but also a powerful platform to program sophisticated elastic properties. This study investigates the multi-stability characteristics of a stacked origami cellular solid consisting of multiple Miura-ori sheets, specifically focusing on a dual-cell chain structure. Experiment results indicate that folding imposes a unique kinematic constraint between the two adjacent bistable origami cells, which can significantly increase the energy barrier of the extensional switch from one stable state to another without notably increasing the barrier of the opposite switch, creating an asymmetric barrier. We formulate a geometric mechanics model to examine the underlying physical principles of the observed asymmetric behaviors, and demonstrate the potentials to strengthen this asymmetric energy barrier and to achieve a static mechanical diode effect. That is, the multi-stable origami could be easily compressed via switching between different stable states, but demand a large external force to be extended. Since such diode effect has the potential to rectify reciprocal loads into unidirectional deformation, the results of this study can become the building blocks for origami solids with novel functionalities.

Cloning and Over-expression of xynB Gene of Bacillus subtilis subsp. spizizenii W23 into Escherichia coli Origami Host Cells

<p class="Els-Abstract-text">The <em>xyn</em>B gene of <em>Bacillus</em><em> subtilis</em> subsp. spizizenii W23 is predicted to encode a xylan 1,4-beta-xylosidase. Application of XynB enzymes in industries is wide. Production of this enzyme in its host cells is naturally restricted by repression process. It will give certain beneficial to over-expressed the enzymes in other host-cells under inducing promoter. This study aimed to clone the <em>xyn</em>B gene from <em>Bacillus</em><em> subtilis</em> subsp. spizizenii W23, to pMMB67EH plasmid, and to over-express the <em>xyn</em>B gene in <em>Escherichia coli </em>Origami as host cells. The <em>x</em><em>yn</em>B gene was successfully amplified by polymerase chain reaction (PCR) technique using a pair of primers flanking the gene sequence and chromosomal DNA of the W23 strain as a template. The <em>xyn</em>B gene inserted in recombinant plasmid was confirmed by PCR detection using primers pair’s specific for <em>xyn</em>B gene and for the vector, then continued by restriction analyses. The result showed that transformants clone 9 and 10 bear the recombinant pMMB-<em>xyn</em>B plasmid. The xylanase activity of <em>xyn</em>B gene in <em>Escherichia coli</em> Origami clone 10 was detected by sodium-dodecyl-sulfate polyacrylamide gel analyses and with addition of isopropyl-β-D-thio-galactoside (IPTG) as an inducer. The protein seem to be over-expressed as intra- and extra-cellular protein detected on SDS-PAGE gel. Result from xylan degrading activity on Luria-Bertani-xylan-IPTG plate with addition of Congo Red, showed that the cells with pMMB-<em>xyn</em>B recombinant plasmid have clear zone around the colonies while the transformant bearing an empty plasmid showed no clear zone. It could be concluded that the <em>xyn</em>B gene of <em>Bacillus subtilis</em> subsp.spizizenii W23 has been successfully been cloned on pMMB67EH plasmid and over-expressed in the <em>Escherichia coli</em> Origami cells as intra- and extra-cellular protein, as observed on SDS-PAGE gel analysis. The protein has activity on xylan degradation.</p>

Abstract No. 129 - Development of a novel bispecific antibody for targeted immunotherapy of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is an ideal target for novel immunotherapies due to its otherwise immunosuppressive intratumoral environment. While the efficacy of bispecific antibodies for immunotherapy is well established for hematological malignancies, their limited circulatory half-lives make them challenging to use for solid tumors. Intratumoral delivery can bypass this issue, potentially making transcatheter-administered bispecific antibody therapy an exciting new paradigm for HCC treatment. We have developed a bispecific antibody that can simultaneously bind to both HCC cells and cytotoxic T cells in order to enhance T-cell mediated tumor lysis. The bispecific antibody targeting the HCC surface antigen glypican-3 (GPC-3) and the T-cell antigen CD-3 was designed as a fusion protein (IDT DNA) and expressed recombinantly in E. coli expression system (Origami Cells) according to previous reports. [1] Specific binding of the antibody against both HCC cells (HepG2) and T cells (Jurkat) was confirmed using cell-surface ELISA assays. [2] Bispecific-triggered activation of T cells (donor CD4 cells) was assessed in vitro using IFN-gamma release ELISA. The bispecific antibody is designed as a 33kD fusion protein consisting of a GPC-3 binding peptide (RLNVGGTYFLTTRQ), previously shown to bind specifically to HCC cells, connected to a CD3-binding single variable fragment domain (ScFV), derived from an OKT3 antibody. [3] The antibody was successfully expressed and purified. It binds to both HCC cells (31.6nM, SD 0.008nM) and T cells (13.2nM, SD 0.006nM) with affinities that are comparable to values reported for other bispecific constructs. [4] The binding to both cell lines were specific and can be inhibited by using excess of either pure GPC-3 binding peptide or full-length OKT3 antibody. The activity of the bispecific antibody is demonstrated by its ability to activate T cells in vitro, resulting in robust IFN-gamma release as measured using ELISA (0.196 Bispecific vs 0.008 Cell Only Control, p = 0.0003). A novel bispecific antibody targeting GPC-3 and CD-3 was successfully synthesized and validated in vitro demonstrating the potential for targeted immunotherapy of HCC.

Uncovering the deformation mechanisms of origami metamaterials by introducing generic degree-four vertices.

Origami-based design holds promise for developing new mechanical metamaterials whose overall kinematic and mechanical properties can be programmed using purely geometric criteria. In this article, we demonstrate that the deformation of a generic degree-four vertex (4-vertex) origami cell is a combination of contracting, shearing, bending, and facet-binding. The last three deformation mechanisms are missing in the current rigid-origami metamaterial investigations, which focus mainly on conventional Miura-ori patterns. We show that these mechanisms provide the 4-vertex origami sheets and blocks with new deformation patterns as well as extraordinary kinematical and mechanical properties, including self-locking, tridirectional negative Poisson's ratios, flipping of stiffness profiles, and emerging shearing stiffness. This study reveals that the 4-vertex cells offer a better platform and greater design space for developing origami-based mechanical metamaterials than the conventional Miura-ori cell.

Heterologous expression, protein folding and antibody recognition of a neurotoxin from the Mexican coral snake Micrurus laticorallis

BackgroundThe cysteine-rich neurotoxins from elapid venoms are primarily responsible for human and animal envenomation; however, their low concentration in the venom may hamper the production of efficient elapid antivenoms. Therefore, the aim of the present study was to produce fully active elapid neurotoxic immunogens for elapid antivenom production.MethodCysteine-rich neurotoxins showed recombinant expression in two strains of E. coli, and were purified using affinity chromatography and reverse-phase HPLC (rpHPLC).ResultsThe cDNA of the four disulfide-bridged peptide neurotoxin Mlat1 was cloned into a modified expression vector, pQE30, which was transfected into two different E. coli strains. The recombinant toxin (HisrMlat1) was found only in inclusion bodies in M15 strain cells, and in both inclusion bodies and cytoplasm in Origami strain cells. The HisrMlat1 from inclusion bodies from M15 cells was solubilized using guanidine hydrochloride, and then purified by rpHPLC. It showed various contiguous fractions having the same molecular mass, indicating that HisrMlat1 was oxidized after cell extraction forming different misfolded disulfide bridge arrangements without biological activity. In vitro folding conditions of the misfolded HisrMlat1 generated a biologically active HisrMlat1. On the other hand, the HisrMlat1 from the cytoplasm from Origami cells was already soluble, and then purified by HPLC. It showed a single fraction with neurotoxic activity; so, no folding steps were needed. The in vitro folded HisrMlat1 from M15 cells and the cytoplasmic soluble HisrMlat1from Origami cells were indistinguishable in their structure and neurotoxicity. Rabbit polyclonal antibodies raised up against biologically active HisrMlat1 recognized the native Mlat1 (nMlat1) from the whole venom of M. laticorallis. In addition, HisrMlat1 was recognized by horse polyclonal antibodies obtained from the immunization of elapid species from sub-Saharan Africa.ConclusionHisrMlat1 shows increased biological activities compared to the native peptide, and may be used as an immunizing agent in combination with other toxic components such phospholipases type A2 for elapid antivenom production.

Microbial host selection and periplasmic folding in Escherichia coli affect the biochemical characteristics of a cutinase from Fusarium oxysporum

A cutinase from the mesophilic fungus Fusarium oxysporum (FoCut5a) was functionally expressed in different hosts and their recombinant products were characterized regarding their activity, thermostability and tolerance in organic solvents. The cutinase gene cut5a was expressed in the BL21 and Origami 2 Escherichia coli strains and the resulting protein was folded either in the cytoplasm or in the periplasmic space, with the aim of correct formation of disulfide bonds. Increase of thermostability occurred when the enzyme was expressed in the oxidative cytoplasm of Origami 2. All expression products showed maximum enzyme activity at 40 °C, while thermostability increased by 73% when expressed in the Origami strain compared to the cytoplasmic expression in BL21 cells. The melting temperature of each protein construct was determined by fluorescence spectroscopy showing an additional transition at about 63 °C for enzymes expressed in Origami cells, indicating the co-presence of a different thermostable species. Kinetic studies performed on three p-nitrophenyl synthetic esters of aliphatic acids (C2, C4, C12) indicated that this cutinase shows higher affinity for the hydrolysis of the butyl ester.