Changing surface grafting density has an effect on the activity of immobilized xylanase towards natural polysaccharides

Enzymes are involved in various types of biological processes. In many cases, they are part of multi-component machineries where enzymes are localized in close proximity to each-other. In such situations, it is still not clear whether inter-enzyme spacing actually plays a role or if the colocalization of complementary activities is sufficient to explain the efficiency of the system. Here, we focus on the effect of spatial proximity when identical enzymes are immobilized onto a surface. By using an innovative grafting procedure based on the use of two engineered protein fragments, Jo and In, we produce model systems in which enzymes are immobilized at surface densities that can be controlled precisely. The enzyme used is a xylanase that participates to the hydrolysis of plant cell wall polymers. By using a small chromogenic substrate, we first show that the intrinsic activity of the enzymes is fully preserved upon immobilization and does not depend on surface density. However, when using beechwood xylan, a naturally occurring polysaccharide, as substrate, we find that the enzymatic efficiency decreases by 10–60% with the density of grafting. This unexpected result is probably explained through steric hindrance effects at the nanoscale that hinder proper interaction between the enzymes and the polymer. A second effect of enzyme immobilization at high densities is the clear tendency for the system to release preferentially shorter oligosaccharides from beechwood xylan as compared to enzymes in solution.


Plasmids pBMW1 and pBMW2
To minimize the effect of introducing an aromatic residue to the protein sequence of Jo and In, a glutamine residue next to the His tag at the N-terminus of plasmid pBMW1 and pBMW2 was

Plasmid pET28-InNpXyn11A
The region of in gene was amplified by PCR using the following primers introduce an BamHI and an NheI site. The PCR product was introduced by homologous recombination (In-Fusion® HD cloning kit, Clonetech) into a pET28b linearized vector using NcoI and NheI to produce pET28-In. The region of xyn11A gene was amplified by PCR using the following primers: 5' TCG TGA TAA CGT CGA CAA GTT TAC TGT CGG TAA TGG ACA   AAA CCA AC 3' and 5' GTG CGG CCG CAA GCT TGT TAG GCT GGA CTA GAA CCC TTT GGA G 3', which introduce an SalI and an HindIII site. The PCR product was introduced by homologous recombination (In-Fusion® HD cloning kit, Clonetech) into a pET28-In linearized vector using SalI and HindIII to produce pET28-InNpXyn11A. The resulting gene codes for the Nterminal His-tagged fusion protein InNpXyn11A with a short linker of three amino acid between the N-terminal In and C-terminal NpXyn11A domains.

Protein purification
The strain, BL21 (DE3) of Escherichia coli harboring plasmids of interest was cultured to midexponential phase (A600nm 0.6) in Luria-Bertani broth at 37 °C. Recombinant enzyme expression was induced by the addition of isopropyl-β-D-thiogalactopyranoside to a final concentration of 0.5 mM at 37 °C for 4 h. Cells were harvested by centrifugation at 5,000 × g for 10 min, re-suspended in 10 ml of 50 mM sodium phosphate buffer, pH 7 containing 300 mM NaCl 20 mM imidazole and a protease inhibitor cocktail (cOmplete Protease Inhibitor EDTA-free, Roche) and lysed by sonication on ice for 1 min. The lysate was clarified by centrifugation (30 min at 74,000 × g at 4 °C). Proteins were purified by immobilized metal ion affinity chromatography (IMAC) using Talon resin (Clontech) and elution in 50 mM sodium phosphate buffer, pH 7 containing 300 mM NaCl and 150 mM imidazole. The eluted proteins were desalted using a PD-10 desalting columns (GE Healthcare Life Sciences) or extensively dialyzed against 50 mM sodium phosphate buffer, pH 7.
A final round of purification was carried out using a XK16 HiLoad 16/600 75 prep grade gel filtration column (GE Healthcare Life Sciences) connected to an Äkta pure system. Typically, 1 ml of protein was loaded onto the column at 1 ml/min using a 1 ml static loop and finally eluted from the column using 50 mM sodium phosphate buffer, pH 7 containing 150 mM NaCl. Purified proteins were adjudged homogenous by SDS-PAGE. Protein concentrations were determined by measuring absorbance at 280 nm and applying the Lambert-Beer equation. Theoretical molar extinction coefficients were calculated using ProtParam online software 1 .

Enzyme immobilization onto paramagnetic beads
Beads specific surface using BET The nitrogen adsorption-desorption isotherms were collected at 77 K using an adsorption analyzer BELSORP-Mini II (BEL-Japan). Aliquot of 340 µl of beads (20 % v/v) were washed three times with water and dried under vacuum for 16 h at 21 °C, resulting into 0.0523 g of material. From the N2 isotherm, the specific surface area (SBET) was determined by the BET method in 0.10 ⩽ P/P0 ⩽ 0.30 domain. The errors associated with adsorption-desorption analyses were estimated to be 4% for the specific surface.

Protein immobilization on paramagnetic beads
From the crystal structure of the complex 2 , Jo displays four solvent exposed lysine (K162, K168, K173, K213) and one additional lysine is involved in the intra molecular isopeptide bond (K191), whereas In presents eight solvent exposed lysine (K590, K591, K648, K651, K673, K690, K703, K717). Preliminary data clearly highlighted a higher efficiency of specific immobilization when Jo is firstly immobilized on the beads (data not shown). This behavior could be explained regarding the distribution of the solvent exposed lysine on the surface of Jo which are at the opposite of the catalytic residue Lys191, instead of more exhibited around the catalytic Asn695 in the case of In (Fig.   S2). Such lysine distribution may promote appropriate orientation of Jo and enable access to the catalytic Asn695 of In.

An estimation of pNP-X3 diffusion time into the beads
pNP-X3 has a molecular weight of 535.45 g.mol -1 and is about 21×6×6 Å in size. The diffusion coefficient D of a Brownian particle of diameter d can be estimated using the Stokes-Einstein expression D = kBT/6(d/2) with kB the Boltzmann constant, T the temperature, and  the viscosity of the medium (taken as water). At room temperature, this gives a lower limit of D = 2.04 × 10 -10 m 2 .s -1 for pNP-X3 if we use its largest dimension 21 Å as an equivalent diameter d. In addition, we know that the 3D root mean square displacement of a Brownian particle with a diffusion coefficient D in a time t is given by <r 2 > 1/2 = (6Dt) 1/2 . 5 This means that pNP-X3 diffuses a distance r = 10 microns in a time t  r 2 /6D = 0.08 s. If we assume that the diffusion into the beads is not hindered by porosity or obstruction effects, this means that pNP-X3 takes less than one second to fully enter the beads. If, on the other hand, we hypothesize that the diffusion coefficient into the beads is 1% of D because of some strong obstruction effects (this percentage is very exaggerated, see the review of Masaro et al. on the diffusion of tracers in polymer solutions or gels 6 ), the time for diffusing into one bead is still 8 s; which is also less than 1 minute as stated in the article.

Optimal pH and temperature
The effect of pH on free or immobilized NpXyn11A was determined   The dRI chromatogram present three distinct peaks: peak 1 centered at 22 mL of elution, peak 2 at 27 mL, peak 3 at 32 mL. Peak 1 only is well resolved by LS; it represents 15 % of the total mass of beechwood xylan; while peaks 2 and 3 represent 85 % of beechwood xylan. The average molecular weight of each fraction is reported in the table below (Table S4). Most of the xylan polymer is therefore present as chains of molecular weight 250-350 kDa (number average molecular weight). The other part is present as much larger objects (7-14 MDa); presumably clusters of chains associated through non-covalent interactions.
The Rg values are estimated from the intensities scattered at different angles following Zimm's theory. The average Rg calculated for peak 1 is fully reliable. The Rg values for peaks 2 and 3 are less trustworthy and probably overestimated as the LS signal is still polluted by large objects that correspond to peak 1 at these elution times.

Catalytic parameters of InNpXyn11A
In the next figure we provide two examples of the Michaelis-Menten curves from which the catalytic parameters of InNpXyn11A towards beechwood xylan were determined ( Table 4 in     InNpXyn11A in solution (B) on beechwood xylan. A1 to A5 referred to beads 1 to 5 respectively.
B1 to B5 referred as the corresponding equivalent amount of free enzymes.