Team:Edinburgh/Proof Of Concept
Proof of concept:
Our concept was that immobilisation not only improves enzyme recoverability but also has the additional benefits of increasing enzyme stability and recoverability, enabling continuous operation. This potentially decreases costs associated with bioconversion processes, which are a major challenge for biorefineries [1]. A recent analysis of cellulosic ethanol biorefineries suggested that commercial enzyme production costs for biofuel had previously been underestimated, and in many cases make the process unprofitable [2]. Realistic cost estimates for cellulases range from $0.68-1.47/gallon ethanol, depending on reaction conditions and feedstocks used [3]. This is a high proportion of the eventual selling price of ~$4.07/gallon, reducing ability to compete with fossil fuel prices. Therefore, a reduction in cost through development of a more efficient, and widely applicable platform for enzyme immobilisation and waste management would be highly valuable.
Enzyme Immobilisation using the silica binding tag
The nature of recalcitrant polymers typically comes from their crystalline nature which has specifically evolved to be resistant to enzymatic and microbial attack. Our hypothesis is that by immobilising the enzyme to a solid surface, the combination of mechanical and enzymatic forces will increase the degradation of the polymer into its constitutive monomers (Figure1). Additionally, the support provided by silica beads is postulated to increase the stability and reusability of the immobilised enzymes.
Figure 1 (A) Cellulase unable to access the cellulose due to the crystalline form. (B) The SuperGrinder: Cellulase immobilised on the silica beads (solid support) allowing enzymes to access cellulose through mechanical force. [graphical scheme created with Biorender]
In aqueous solutions silica (SiO2) displays both siloxane bridges (-Si-O-Si-) and silanol groups (Si-OH) at its surface ( Figure 2). Silanol can dissociate to Si-O- and H+ under neutral or basic conditions, enabling silica binding by electrostatic attractions at pH>7 [4,5].
Figure 2 Silica surface chemistry showing siloxane bridges and dissociation of silanol groups under neutral and basic conditions. Adapted from (Pavan et al, 2019)[4]
With the benefits of silica beads as a solid support for enzymes. We demonstrate the affinity of the binding tag with various silica beads using sfGFP in order to facilitate the detection.
Results
The immobilisation was visualised directly under the blue light to qualitatively monitor sfGFP immobilisation (Figure 3). We found that sfGFP is indeed immobilised via tags we created, namely Car9, L2NC, and L2NC-linker. By comparing to the untagged sfGFP, fluorescence can be observed as a glowing bead immobilised with different tags (Figure 3). sfGFP immobilised on different beads with various tags result in the different binding affinity. This result indicate that L2NC linker is the silica-binding tag with the highest affinity and the immobilisation work best with the Celite545 beads.
Figure 3 sfGFP immobilised with various silica-binding tags on four silica beads: Celite545, 100um silica beads, glass beads acid washed and glass beads 6 mm.
The protein on the Celite beads is again confirmed with the SDS-PAGE comparing with the cell lysate of E.coli BL21(DE3) to investigate the purity of enzymes immobilised on the beads (Figure 4). The SDS-PAGE reveals that many other endogenous enzymes are also immobilised on the beads.
Figure 4 SDS-PAGE of sfGFP immobilised on the silica bead. Protein quantity in supernatant and cell pellet were determined and prepared in 5 ug protein. Enzymes on the beads were de-immobilised without protein quantification but directly boil in the SDS-PAGE sample buffer to denature proteins (1-3) E.coli BL21(DE3) lysate, pellet and enzyme on the beads, (4) Protein marker, (5-7) E.coli BL21(DE3) with overexpressed sfGFP supernatant, cell pellet and enzyme on the bead. (8-10) E.coli BL21(DE3) with overexpressed sfGFP-L2NC linker supernatant, cell pellet and enzyme on the bead. The expected size of sfGFP is 26.67 kDa, and that of sfGFP-L2NC linker is 42.12 kDa.
Improved enzyme stability after immobilisation
The protein stability after immobilisation was demonstrated using the celiite beads. The small particle size is expected to allow more surface area for enzymes to be immobilised and consequently result in the higher signal. From the previous experiment, the silica-binding tag that provides the highest enzyme binding is the L2NC-linker tag, therefore, this experiment carried on with L2NC-linker tag to test the potential of maintaining enzyme activity after immobilisation in a prolonged incubation at 37°C.
The stability of enzyme was tested using both sfGFP and Cex immobilised in a different buffer according to the optimal pH of each protein. The signal from sfGFP was measured with the microplate reader and compared overtime. Although the result shown here (Figure 5) did not directly indicate the stability of sfGFP, we hypothesize that the increasing signal of free sfGFP-L2NC-linker might caused by the addition of L2NC-linker tag which maintained the fluorescence of sfGFP-L2NC-linker even in an accumulate form. The absent of signal from immobilised sfGFP highlighted that the signal detected from immobilised sfGFP-linker is solely from the sfGFP which are successfully immobilised on the bead. Notably, the different magnitude of immobilised sfGFP-L2NC-linker from other free sfGFPs should not be taken into account since we were unable to quantify the amount of protein on the beads and the amount of free sfGFP is the total protein use for immobilisation while the protein in immobilised sample lost through the washing process as unbound proteins.
Figure 5 The sfGFP stability test after immobilisation for up to 30 hours.
Cex enzymatic activity was measured using 0.1 mM 4-MUC to 100 uL of bead suspension and the assay was done after 18 hours of incubation at 37°C (Figure 6). We observed that the enzyme characteristic was slightly changed in the presence of the L2NC-linker tag and greatly affected when immobilised on the silica beads.
Figure 6 The Cex assay of free enzyme, free enzyme with L2NC-linker tag and immobilised enzyme.
However, comparing the stability of enzymes from after immobilisation and after 16 hours of incubating, we observed that the initial velocity of Cex changed by 30.76% and 35.49% for free enzyme cex and cex-L2NC-linker, while only 13.37% changed for the immobilised enzyme (Figure 7). This indicates that Cex is more stable after immobilisation on the silica beads.
Figure 7 The relative initial velocity observed from Cex assay after prolonged incubation. The maintained relative initial velocity imply that enzyme is more stable after immobilisation.
References:
[1] Girelli AM, Astolfi ML & Scuto FR (2020) Agro-industrial wastes as potential carriers for enzyme immobilization: A review. Chemosphere 244: 125368
[2] Liu G, Zhang J & Bao J (2016) Cost evaluation of cellulase enzyme for industrial-scale cellulosic ethanol production based on rigorous Aspen Plus modeling. Bioprocess Biosyst. Eng. 39: 133–140
[3] Klein-Marcuschamer D, Oleskowicz-Popiel P, Simmons BA & Blanch HW (2012) The challenge of enzyme cost in the production of lignocellulosic biofuels. Biotechnol. Bioeng. 109: 1083–1087
[4] Pavan C, Delle Piane M, Gullo M, Filippi F, Fubini B, Hoet P, Horwell CJ, Huaux F, Lison D, Lo Giudice C, Martra G, Montfort E, Schins R, Sulpizi M, Wegner K, Wyart-Remy M, Ziemann C & Turci F (2019) The puzzling issue of silica toxicity: are silanols bridging the gaps between surface states and pathogenicity? Part. Fibre Toxicol. 2019 161 16: 1–10
[5] Ikeda T, Ninomiya K ichi, Hirota R & Kuroda A (2010) Single-step affinity purification of recombinant proteins using the silica-binding Si-tag as a fusion partner. Protein Expr. Purif. 71: 91–95