Study of the complex coacervation mechanism between the lysing enzyme from T. harzianum and polyallylamine hydrochloride

Accepted Manuscript Study of the complex coacervation mechanism between the lysing enzyme from T. harzianum and polyallylamine hydrochloride

Houda Bey, Wala Gtari, Adel Aschi PII: DOI: Reference:

S0141-8130(18)35223-1 https://doi.org/10.1016/j.ijbiomac.2018.11.266 BIOMAC 11143

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

30 September 2018 20 November 2018 28 November 2018

Please cite this article as: Houda Bey, Wala Gtari, Adel Aschi , Study of the complex coacervation mechanism between the lysing enzyme from T. harzianum and polyallylamine hydrochloride. Biomac (2018), https://doi.org/10.1016/ j.ijbiomac.2018.11.266

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ACCEPTED MANUSCRIPT Study of the complex coacervation mechanism between the Lysing Enzyme from T. Harzianum and Polyallyamine Hydrochloride

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Houda Bey, Wala Gtari, Adel Aschi* Université de Tunis El Manar, Faculté des Sciences de Tunis, LR99ES16 Laboratoire

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Physique de la Matière Molle et de la Modélisation Électromagnétique, 2092, Tunis, Tunisia

ABSTRACT

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Complex coacervation was achieved by mixing the Lysing Enzyme from T. Harzianum (LYS) with Polyallyamine Hydrochloride (PAH). We show in this work that the study electrostatic

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complexes conformation can lead to the formation of dense complexes. We systematically

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investigated the effects of pH and the mass ratio on the structure and properties of the complex. The different transition phases (pHc, pHφ1, and pHφ2) have been determined using dynamic light scattering, zeta potential and turbidimetric measurements. The

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interpolymeric bonds may be ionic or physical, depending on the pH of the system. For a pH value of 4.9, the mixture system [LYS]/ [PAH] gives raise the formation of coacervate

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droplets. The effects of temperature on the structure of coacervate droplets are studied by small angle light scattering (SALS).

KEYWORDS: Intramolecular electrostatic complexes; Complex coacervation; Coacervate droplets; Dynamic light scattering; Small angles static light scattering.

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ACCEPTED MANUSCRIPT INTRODUCTION Enzyme−polyelectrolyte interaction plays a very important unit in molecular and cellular biology. In addition, these complexes can also be generating novel soft biomaterials [1-7]. The study of the behavior of these complexes fluids is commonly used in the food industry, cosmetics, medicine, and biological fields because of their structure. The interplay of a

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variety of physical forces, thermodynamic environment, the amino acid sequence, and protein structure governs these interactions. Complex coacervation is currently described as

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a liquid-liquid phase separation mechanism resulting from the formation of intramolecular

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electrostatic complexes between macromolecules.

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The aggregation of these initially soluble complexes leads to the formation of insoluble intermolecular complexes which, after flocculation and coalescence, form liquid droplets

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called coacervates. Many authors like Dubin et .al [8, 9], De Kruif et. Al [7,10] have shown that the enzyme /polyelectrolyte complex can be given rise to the formation of intermolecular

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electrostatic complexes and complex coacervation. This formation of coacervate is

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influenced by several conditions including pH, ionic strength, total polymer concentration, polymer composition, and the ratio of the two polymers.

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In a variety of soft matter phases (coacervates and transparent gels), several studies have

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identified the influence of different parameters on enzymatic-polyelectrolyte systems such as charge density, molecular mass, pH, mass ratio, concentration [7,8,10].

Our aim is to identify the interactions caused by the complex formation between Lysing Enzyme of T. Harizanuim/polyallyamine hydrochloride (LYS/PAH). The Lysing Enzyme of T. Harizanuim was used as biocontrol agents against plant pathogenic fungii as due to its excellent properties. [11] The polyallyamine hydrochloride is a weak polycation at neutral pH by virtue of its amine group; it is in particular used in the food and cosmetics industries.

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ACCEPTED MANUSCRIPT In fact, this study is based on the variation of the existing interaction force between these two biopolymers, modifying the mass ratio of the mixture and the total concentration of the biopolymers. In this paper, we determine the different phases of separation (pHc, pHφ1 and pHφ2) on the basis of turbidity and dynamic light scattering (DLS) measurements. These techniques are very powerful tools which give information on the structure and changes in

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the molecular size of the complexes in solution. This work is an extension of the results obtained by Aschi et al., [12,13] which followed the conformational changes of the

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complexes.

Our results show the formation of liquid droplets called coacervates at pH = 4.9. The effect of

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the temperature of the coacervate droplets is studied by small angles static light scattering (SALS). This technique makes it possible to determine the radius of gyration Rg and the

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fractal dimension Df of the macromolecules studied in solution.

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MATERIALS AND METHODS

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Materials

Lysing Enzymes from Trichoderma harzianum (L1412, Mw = 25 KDa, residues = 226) and poly(allylamine hydrochloride (283215, Mw=17.5KDa) were purchased from Sigma

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Aldrich. The Lysing enzymes from Trichoderma harzianum are also known as Glucanex. It

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has a compact structure of molecular weight Mw = 25 kDa with a single chain of 226 amino acids. It contains 7% α helices and 54% β sheets. These enzymes are known to have a common isoelectric pH, pI= 4.9 ± 0.2 [11]. Ultrapure water (Millipore) is used to prepare all of the aqueous solutions. The concentrations are defined as the weight of the chemicals per total weight of the solution.

Sample Preparation. LYS/PAH (Pr:Pl) aqueous solution was prepared at different mass ratios r = [Lys] / [PAH] where (Pr:Pl) 10:2 (%g/g); 20:2 (% g/g) at a fixed concentration of PAH (0.2%). The 3

ACCEPTED MANUSCRIPT mixture solution was then titrated with 0.1N HCl or 0.1 N NaOH to adjust the pH. Complex coacervate samples were prepared by mixing the mass ratios r= 1: 2; 5: 2; 10: 2; 20: 2 at a fixed concentration of PAH (0.2%) with 0.1N HCl or 0.1 N NaOH to adjust the pH to pI. These samples were stored in glass bottles (trace amount of Sodium azide (500 ppm) was added in

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order to avoid bacterial contamination).

pH Measurement

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The pH was measured with a precision pH meter (a multi Consort C862 scanner

 NaOH   LYS 

and  

 HCl  .  LYS 

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the medium. Where  

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resolution up to 0.001 pH units). By varying, the neutralization rate α and β can fix the pH of

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Turbidity Measurement

The turbidity of mixtures of LYS and PAH at different pH was determined from the

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transmittance using a UV/Vis with 1 cm path length at the fixed wavelength of 633 nm. The

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transmittance T of the samples was determined from the relation: T = It/I0, where It is the transmitted light intensity of solution and I0 is the light intensity for solvent only.

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Zeta Potential Measurements

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The zeta potential of the particles in the solutions (LYS, PAH, and complex LYS/PAH) was measured using Zetasizer Nano ZS (Malvern Instruments, Malvern, U.K.). Three rounds of assays were averaged. All measurements were performed at 25 °C.

Optical Microscopy An optical microscope (Leica TCS SPE, Germany) equipped with a lens (PLANAPO 5.0x/0.50 LWD) and an eyepiece tube (LEICA 10447367 063x) with transmission lighting was used to obtain images for the coacervate droplets LYS-PAH at pH = 4.9. The coacervate

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ACCEPTED MANUSCRIPT mixture was placed on a glass slide to image the droplets. The temperature was fixed to 45 o

C.

Dynamic light scattering (DLS) The assembly of dynamic light scattering (DLS) was realized in our laboratory. The

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measurements were carried out by a light source constituted by a He-Ne laser with a wavelength λ = 633 nm and a power of 22 mW to emit monochromatic light. A sample holder

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equipped with a rotation/translation system (CRTU) which allows the spatial exploration of

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the sample. The incident beam is focused at the sample level. After selecting the scattering angle of 90°, the scattered intensity is transmitted to the photomultiplier (PMT) via an optical

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fiber. This intensity will be amplified using an amplifier and then sent to a "Flexo type" digital correlator capable of measuring the autocorrelation function. The hydrodynamic radius was

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determined as described in Bey et al. [11]

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Ultra Small Angle Light Scattering (USALS)

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The technique of Small Angle Light Scattering (SALS) was illustrated by a quantitative characterization method whose aim was to follow the changes in particle size (the correlation length). This technique has been performed in our laboratory, similar to that by Ferri et.al.[16]

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This device was equipped with a coherent light source (He-Ne) of wavelength λ = 633 nm

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and power 20mW (Cube Coherent Inc.). After the laser, there is a diaphragm that works like a pinhole. An adjustable support in height was placed to ensure the placement of our sample. The incident intensity was then attenuated by using neutral density filters (Newport Co.). A beam stop of 1.5 mm diameter is used to prevent the main beam from directly affecting the detector. The CCD camera (Pulnix TM 6470 America, Sunnyvale, CA) with a resolution of 1000 x 1000 pixels was used to record the broadcast results with a frequency of one photo every 300 seconds.

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ACCEPTED MANUSCRIPT In fact, the CCD captures a two-dimensional image offers a range of diffusion angles (θ) of between 1.5° and 6.1 °. The range of diffusion vectors (q) ranges from 0.1 – 0.8 μm-1. The fractal colloid aggregates Df and the radius of gyration Rg were determined as described in Gtari et al. [17].

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Computational Methods We used the Adaptive Poisson-Boltzmann Solver (APBS) program to calculate the

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electrostatic potential,[18] which can be read by the Chimera software.[19] We downloaded

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the amino acid sequence of the enzyme from the RCSB Protein Data Bank (PDB)[20] under the identification code 4h7m. We emphasize that the limiting step in electrostatic potential

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calculations is the addition of the missing atomic coordinates to the molecular structures taken from the PDB. To solve this technical difficulty, we developed the PDB2PQR software.

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[21, 22]

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RESULTS AND DISCUSSION

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Effect of the ratios LYS/PAH on the complex structure Effect of pH

The mass ratio enzyme/polyelectrolyte has a great importance on the phase separation

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phenomenon and on the electrostatic interactions. Many authors have shown that, at a given

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pH, the mass ratio is responsible for the neutralization of the charges between the biopolymers.[23] In our study, we varied mass ratio of enzyme/PAH mixtures, where (Pr: Pl) 10:2 (%g / g), 20:2 (%g / g) in the pH range (2-10) by dropwise addition of 0.1 M HCl or 0.1M NaOH. All these solutions have been studied in the absence of salt ([NaCl] = 0 mM).

Figure 1.a shows the change in turbidity (100% - T) versus pH and mass ratio r. We have noticed in this figure four phases marked (A, B, C, D) separated by three critical pH. In addition, critical pH transition points (pHc, pHφ1, pHφ2) were deduced by the intersection of two tangents of the curve.[24] 6

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These phases were obtained with the change of turbidity (Fig. 1.a) and hydrodynamic radius obtained by the dynamic light scattering (Fig. 1.b). In region A, where (pH > pHc  7.12), the Coulomb repulsion forces between the negative charges of the enzyme and the negative charges of the polyelectrolyte prohibited the complex formation, what is translated in a

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macroscopic manner by a constant value of the turbidity. The region B corresponds to pH pHφ1≤pH≤ pHc, there is a slight increase in turbidity, which indicates the formation of a

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soluble complex, still called primary complex. This phenomenon thus involves the attraction

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between charges of polycations and the opposed patches on the surface of the enzyme.[12,13, 25-27] In the neighborhood of the isoelectric point (pI=4.9), there is a

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significant increase in turbidity, which shows that complex large sizes or coacervates are formed in the region C (pHφ2
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point of the number of charges brought by the two polymers corresponds to the pHmax or pH optimal of electrical equivalence. In the region D (pH ≤ 4), the mixture LYS/PAH presents a

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loss of turbidity, what indicates the abolition of the separation phase from pHφ2. The curve of

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the turbidity of LYS (Fig. 1.a) has a small peak due to the aggregation of enzymes. Whence, the enzyme did not aggregate when the pH reaches a lower value than pHc. In our case, the interactions are repulsive or attractive depending on the sign of the charge of the protein.

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This is due in fact to the pH which controlling the degree of ionization in the entire range of

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the pH. In summary, the increase in turbidity between pHφ1 and pHφ2 indicates the presence of a large-sized complex or droplets of coacervate. A close inspection of the complex coacervates under the optical microscope (Fig. 2) reveals that large droplets seem to have formed inside these phases. The droplets were made up from uniformly sized ( 1m), approximately spherical particles. The complex coacervation is formed by the interaction between the two oppositely charged polyelectrolytes in the solutions, which can result from electrostatic interactions between the oppositely charged polymers. So, a gain of entropy occurs due to the release of small

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ACCEPTED MANUSCRIPT counterions and water molecules. These complex coacervates can be applicable in microencapsulate of drugs, in biomedical and food [28].

Still, figure 1.a shows a pick around isoelectric pH. This increase in turbidity is induced by a decrease of the electrostatic repulsion between the molecules of proteins.[29-32] Thus, when

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the number of particles formed increases, we also observe an influence on the size of the formed aggregates.[33] Indeed, several counter ions were easily released by increasing the

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enzyme concentration in solution, which protects the sites of the charges on enzyme

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surfaces. De Kruif et al. [34] observed that the performance of coacervation decreased due to the effect of mass action and the concentration of biopolymer. Schmitt et al.[35] also

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showed that the increase in polymer concentration reduces the effect of the pH on the

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complex coacervation. These works confirm what we have observed in figure 1.a.

The results obtained by dynamic light scattering were analyzed by the REPES method to

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obtain information on the size of the complex (Fig. 3.a.b). We determined the change in

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hydrodynamic radius Rh of the complex particles within the limits of each region cited before. We note that the hydrodynamic radius of the mixture of LYS-PAH increases sharply until it reaches the maximum value for pH = pHmax. This confirms previous results obtained by

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turbidimetric analysis. In fact, at pHmax large aggregates in the solution are presented. By

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continuing acidification, the hydrodynamic radius also decreases so strongly until it reaches an almost minimal value for a pH < pHφ2 and remains constant thereafter. Around the isoelectric pH  4.9, we can conclude that the complexes combine to form coacervates then dissociate into small molecules which look like compact structures (Fig. 2). These results are consistent with the observations made by the measures of the turbidity in this pH range. Here, the size of the coacervate obtained from the optical microscope is larger than the size obtained by DLS measurements due to the rearrangement of the complex structure in time. In addition, the coacervate phase reorganizes with time to form a more homogeneous and transparent phase (48 Hours in our case). Moreover, the structure of the coacervates is 8

ACCEPTED MANUSCRIPT dynamic and the time scale varies from seconds to days to form precipitate [36]. Indeed, this phenomenon has been observed with other complexes formed by oppositely charged biopolymers,[37-39] which is once again an explanation within the framework of the theory of Zhang and B. Shklovskii.[40, 41]

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We extended our work by studying the electrostatic potential of Lysing enzyme (PDB ID code 4h7m). We used the Adaptive Poisson-Boltzmann Solver (APBS) program to monitor the pH

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effect on the electrostatic potential, [19] which we can read it in the Chimera software.[20]

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The electrostatic potential helps to identify the functional sites situated at the protein surface and can be visualized by color coding as a function of potential values. The aim of this work

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is to know precisely how the polyelectrolyte binds with the enzyme at different pH transition. Figure 1.b shows the electrostatic potential contour around the protein as a function of pH

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and corresponding net charges. The electrostatic potential is given in units of KBT/e and the, two isosurfaces are shown, red: −1KBT/e and blue: +1 KBT/e. we have noticed that the net

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charge Zp on the surface of the protein changes when the pH changes and this result is in

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conformity with that which will be shown in detail in figure 4. The enzyme chain at basic pH is illustrated by little patches in blue. This shows how the protein binds with the polyelectrolyte composed by negative net charges. At pH close to pI, Lysine enzyme shows high positive

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lobe, as seen in figure 1.b.

Influence of density charge The mechanism of coacervates is essentially influenced by the charge density of the two biopolymers. Indeed, the process of forming the complex is mainly due to electrostatic interactions between the enzyme and the polyelectrolyte. An indirect manner of following the enzyme charges is to study the evolution of their zeta potential versus pH. The figure 4 also shows that PAH carries zero charges ( 0 mV) at pH value near 7, this value is close to that found in the literature.[42]

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ACCEPTED MANUSCRIPT The zeta potential of isolated LYS and LYS-PAH mixture is found to be zero around pH 4.8 and 6.7 respectively, which means that charge neutralization is total under these conditions. The presence of enzymatic aggregates during the complex coacervation of the Lysing enzyme from Trichoderma harizanuim and polyallyamine hydrochloride modifies the structure, the size and surface properties of coacervates.[43] Consequently, the change of

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charges of the enzyme according to the pH is an essential element to understand the

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complexation process.[11]

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Effect of mass ratio on the complex formation

The figures 5.a and 5.b show in detail the turbidity and phase diagram respectively of the

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association of the LYS-PAH for the different ratio (r = Pr:Pl = 1: 2; 5: 2; 10: 2; 20: 2) and for pHc, pHφ1 and pHφ2 in the absence of salt. In these figures, the value of pHc remains almost

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constant around pHc ~ 7,0. We, therefore, conclude that the formation of the soluble complex is controlled by the association of the monomers between a single chain of polyelectrolyte

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and a number of the enzyme.[13] When the number of protein attached to a chain of

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polyelectrolyte increases, we find that the charge necessary to achieve neutralization of the complex decreases and which results in an increase of pHφ1.[25, 44] On the other hand, we observe a slight decrease at pHφ2. This is due to the fact that not all protein molecules

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contribute to the formation of aggregates, i.e. a more acidic pH was necessary to obtain

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positive charges on the Lysing enzyme and to compensates the negative charges of the PAH. Therefore, the complexation occurs readily at very high mass ratios. Indeed, recent studies have investigated the interaction of protein-polyelectrolyte of which the purpose is to discuss the borders of the phases. For example, Mattison et al.[45] have studied the complexation of BSA with the PDMDAAC and found that the total biopolymer concentration had no remarkable effect on pHc and pHφ1.

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ACCEPTED MANUSCRIPT Influence of mass ratio on the diffusion coefficient (Dcomp) of the complex The effect of the mass concentration of enzyme can help us to understand the mechanism of the association of the LYS on PAH. The figure 5.c presents the variation of apparent translational diffusion coefficient D versus LYS/PAH mass ratio and shows a linear behavior for all three pH transitions (pHc, pH, and pH. The linear behavior of the diffusion

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coefficient has been recognized especially in the case of the dilute concentration regime of a single polymer solution. When the interactions between the particles are low, the

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translational diffusion coefficient and the hydrodynamic second virial coefficient kd can be

(1)

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𝐷 = 𝐷𝑐𝑜𝑚𝑝 (0)(1 + 𝑘𝑑 𝐶𝑃𝐴𝐻 )

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determined by the following equation:[12,46]

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where Dcomp(0) is the diffusion coefficient of the complex at finite dilution and kd is the hydrodynamic virial coefficient of the complex. In fact, in the solution, positive and negative for

kd

represent

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the

repulsive

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intermolecular

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values

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interactions.[47]

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form aggregates.

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The kd values of the LYS-PAH mixture are negative indicating that the complexes tend to

By a simple study, we can determine the number of proteins (npr) attached to the polyelectrolyte skeleton from the following relationship:[12,46]

𝐷𝑐𝑜𝑚𝑝 (0) 𝐷𝑃𝐴𝐻 (0)

Mpr

= 1 + npr M

(2)

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where npr is the number of adsorbed proteins in the PAH skeleton and Mpr is the molar mass of the protein.

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In fact, since kd depends on thermodynamic effects, the interpretation may require other information. For this, we have estimated the sign of the second coefficient of virial A 2,12 responsible for the LYS–PAH interaction. The kd in Eq (1) can be expanded as follows:

(3)

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𝑘𝑑 = 2𝑀𝑃𝐴𝐻 (𝐴2,22 + 𝑟𝐴1,12 ) − 𝑣̅

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solution, 𝑣̅ is partial specific volume and r =CLYS/CPAH.

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where kd = 2MPAH A2, 22 is the second virial coefficient of pair self-interaction for pure PAH

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The values of kd, DCOMP (0) and npr are summarized in Table 1. The maximum value of npr is obtained in the case of pH < pH, thus, the complex aggregation process is more favorable.

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Whereas in the phases pHφ2 < pH < pHφ1 and pHφ1 < pH
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positive charges of Lysine enzyme. According to the Table 1, the npr increases by

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acidification. This result is consistent with the process of the formation of electrostatic complexes controlled by the acidity of the medium. Indeed, npr is very weak in a basic

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medium and it increases by acidification. Therefore, the potential of the electrostatic attractive interaction between the protein and the polyelectrolyte became increasingly

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favorable by increasing the positive charges of the protein.

Influence of temperature on coacervates formation Generally, the temperature plays an important role during the molecular interactions in many scientific questions. In this section, we propose to understand and characterize the conformation of obtained coacervates (Fig. 2) under the influence of temperature. We studied by small angles light scattering (SALS) technique, the structures of coacervates formed by LYS and PAH in a temperature range from 25 °C to 65 °C. The weight ratio of

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ACCEPTED MANUSCRIPT mixture LYS-PAH (Pr: Pol) (20: 2) prepared at pH = 4.9 and a salt concentration of 0 mM. The measurements of small angles light scattering are shown in figure 6.a; note, four temperature values (40-50 °C) are not shown in figure 6.a to avoid superimposition “overlap” between the curves. We determined the variation of the intensity I(q) as a function of temperature. Moreover, as can be seen in figure 6.a, the scattering curve of the coacervates

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obtained at different temperature shows mainly the Guinier regime, i.e. the scattered intensity is independent of q and this implies that the obtained droplets are still too small within the

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chosen temperature range.

We determined in the Guinier regime, qRg <1 and for molecules strongly diffused, the

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average radius of gyration Rg. The radius of gyration is estimated from small angles light scattering data and imaging results. The inset in figure 6.a shows the variation of ln [I(q)]

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versus q² allowing to determine the radius of gyration from the slope (-Rg2 / 3). The values of the radius of gyration and fractal dimension Df obtained from analyses are summarized in

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Table 2. In our case, mixtures of LYS and PAH can form a heterogeneous complex

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systems.[4] So, to study the evolution of the internal structure of LYS–PAH complexes by determining the fractal dimension (Df) from the slope at large scattering vectors values (q,

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μm−1) using the power-law I(q) ∼ q−Df.

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The fractal dimensions of the droplets coacervates are computed from the figure 6. a. In fact, the power law is valid only when the aggregate size is much larger than the primary particle size. Indeed, as shown in table2, the fractal dimensions of the obtained droplets coacervates increase from 1.35 ± 0.10 (at 25 °C) to 1.82 ± 0.10 (at 65 °C). For colloidal systems, the fractal dimension gives a description of particulate aggregates formed by either, diffusion limited (DLCA) or reaction-limited cluster aggregation (RLCA).[48] For T< 50 °C, the fractal dimensions (Df= 1.35) are almost weak compared to those obtained at a higher temperature and can be disregarded, since at law temperatures the number of primary particles making up the aggregates is not large enough so that the aggregates 13

ACCEPTED MANUSCRIPT possess fractal structure. Diffuse LYS-PAH aggregates formed initially (Df∼1.35) a fractal aggregates then reorganized into more compact structures exhibiting dimension Df~2. The value of the Df equal to 1.82 ± 0.1 corresponds to the diffusion-limited cluster aggregation mechanism.

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Figure 6.b illustrates the likely size (diameter = 2Rg) of droplets coacervate versus temperature. This figure shows that the diameter for the LYS/PAH mixture has a value of the

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order of 7.3 m at 45°C. This confirms the result obtained by the microscopy technique (Fig.

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2) and allows us to deduct first of all that the process of association between the polypeptide chains is so favored where the tendency to form aggregates at low temperatures.[49]

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Secondly, we consider that this fact is probably due to the low hydrophobicity of LYS/PAH at low temperatures. Indeed, for increasing temperatures the aggregate size increases. This

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fact is the origin of the increased hydrophobic effects which tend to minimize the contact with the solvent. The supramolecular structures resulting from the assembly between the LYS

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and PAH as a function of the temperature assemble to form spherical structures. The size of

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formed complex molecules increases until reaching the maximum value of diameter = 7.40 m in the vicinity of 35 °C. Therefore, beyond this temperature, we can consider that the

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association process between biopolymers continuous, the hydrophobic effects increase [49] and the size decreases to reach a minimum value at 65 °C. Hence, the effects of the

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excluded volume are reduced and the enzyme begins to acquire its compact structure. Still, these findings imply the existence of repulsive interactions and an increase in hydrophobic interactions within the LYS-PAH system.

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ACCEPTED MANUSCRIPT CONCLUSION We showed that complex coacervation is a phase separation phenomenon resulting from electrostatic interactions between the two polymers, mainly with an electrostatic nature bearing opposite charges. We followed the development of complex coacervation of the system Lysing enzyme from Trichoderma Harizanuim/polyallyamine hydrochloride by

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estimating the ratio effect. The dependence of the weight ratio of the diffusion coefficient (or hydrodynamic radius) of the complex LYS/PAH shows that by increasing the protein

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concentration the size of coacervates droplet becomes even bigger. We determined the

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number of proteins that adsorb to the polyelectrolyte skeleton in each phase. The study of the development of inhomogeneities during the complex coacervation leads to the formation

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of microcapsules which depends on rearrangement electrostatic aggregates initially formed phase soluble complexes. Therefore, these aggregates appear to correspond to an

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intermediate structure between the aggregates formed at a lower temperature and spherical

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structures formed at higher temperatures.

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ACCEPTED MANUSCRIPT Acknowledgments - We thank Luca cipelletti from Laboratory of Charles Coulomb University of Montpellier, France, for the help with (SALS) software.

ABBREVIATIONS

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PAH, poly(allylamine hydrochloride; LYS, Lysing enzyme from Trichoderma harizanuim;

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APBS, Adaptive Poisson-Boltzmann Solver; 4h7m, Code Protein Data Bank.

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ACCEPTED MANUSCRIPT FIGURES CAPTIONS: Figure.1: Effect of the ratios LYS/PAH on the complex structure. (a) Measurement of the turbidity of the complex as a function of the pH and at different mass ratios of the LYS / PAH mixture, NaCl = 0 mM. The interrupted line corresponds to the limit of the 4 regions. (b) Variation of hydrodynamic radius as a function of pH and mass ratio (●) r = 20: 2, (■) r =

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10: 2, [NaCl] = 0 mM. The interrupted line corresponds to the limit of the 4 regions electrostatic potential contour (+1 kT/e (blue) and−1 kT/e (red)) around the Lysing Enzyme

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from T. Harzianum at ionic strength 0 mM. In region D : pH=3.4 (zp∼+19.8) , region C: pH=

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calculation was based on PDB ID code 4h7m.

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4,9(zp∼0.00), region B: pH= 7.2 (zp∼−7.3), region A :pH=8.2 (zp∼−16.93). The pH

Figure.2: Micrograph droplets of complex coacervation LYS/PAH at pH = 4.9 and T  45°C,

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scale bar 10μm.

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Figure. 3 : Evolution hydrodynamic radius RH of the mixture LYS / PAH function of pH.

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REPES analysis of DLS measurements performed at 25◦C with (a) r = 20: 2, (b) r = 10: 2, using 0.1 M of HCl and 0.1 M of NaOH.

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Figure. 4: Values Zeta potential (mV) for homogeneous and mixed systems LYS / PAH (●)

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Pr: Pl = 20: 2 (■) LYS (♦) PAH, [NaCl] = 0 mM.

Figure.5: Turbidimetric titration LYS-PAH mixtures depending on the pH. (a) Total concentration (20: 2, 10: 2, 5:2, 1: 2 ratio, 0mM NaCl). (b) The phase diagram in the function of the mass ratio of LYS / PAH mixture, (●) pHc, (■) pHφ1, (♦) pHφ2. (c) Determination of the coefficient of apparent diffusion mixture LYS / PAH in three regions according to (C LYS / CPAH) CPAH = 0.2%, [NaCl] = 0mM. (●) pH> pHc, (■) pHφ1
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ACCEPTED MANUSCRIPT Figure 6: Impact of temperature on coacervates formation. (a) I(q) vs q (μm−1) at different temperature with ratio r=10 (20:2) at pH=4.9. The inset presents the variation of ln (I (q)) as a function of q2 in Guinier regime qRg<1. (b) Variation of the coacervate diameter (2  Rg) of

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droplets coacervates as a function of temperature T.

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ACCEPTED MANUSCRIPT TABLES CAPTIONS:

Table 1: Interactions Parameters kd and diffusion coefficient Dcomp (0) of Complex LYS/PAH and Number of Proteins npr attached in PAH

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Table 2: diameter (2  Rg) and fractal dimension Df of complex measured at different

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Temperature.

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10 μm

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Diameter (2  Rg) (µm)

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Table 1: Interactions Parameters kd and diffusion coefficient Dcomp (0) of Complex LYS/PAH and Number of Proteins npr attached on PAH Dcomp(0)

kd (10-3m3g-1)

(10-12 m2s-1)

A2,12

npr

(10-9 mol m3g-2)

1.78

-0.129

-4.3

359.27

pH1
1.6

-0.118

-3.93

172.36

pH>pHc

1.33

-0.042

-1.40

8.09

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pH< pH1

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Table 2: Diameter 2Rg and fractal dimension Df of complex measured at different Temperature. T (°C) Df 2Rg (m) 6.83  0.18

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7.30  0.12

1.35  0.30

7.40  0.10

1.28  0.35

7.34  0.19

1.32  0.32

7.31  0.18

1.43  0.20

6.90  0.11

1.66  0.18

6.49  0.08

1.74  0.16

6.47  0.09

1.82  0.15

6.36  0.07

1.82  0.15

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1.63  0.20

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Graphical abstract

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