Printable enzyme-embedded materials for methane to methanol conversion

Abstract

An industrial process for the selective activation of methane under mild conditions would be highly valuable for controlling emissions to the environment and for utilizing vast new sources of natural gas. The only selective catalysts for methane activation and conversion to methanol under mild conditions are methane monooxygenases (MMOs) found in methanotrophic bacteria; however, these enzymes are not amenable to standard enzyme immobilization approaches. Using particulate methane monooxygenase (pMMO), we create a biocatalytic polymer material that converts methane to methanol. We demonstrate embedding the material within a silicone lattice to create mechanically robust, gas-permeable membranes, and direct printing of micron-scale structures with controlled geometry. Remarkably, the enzymes retain up to 100% activity in the polymer construct. The printed enzyme-embedded polymer motif is highly flexible for future development and should be useful in a wide range of applications, especially those involving gas-liquid reactions.

Figure 1. Schematic of pMMO encapsulation in hydrogel and activity assay.

(a) Schematic of PEG-pMMO hydrogel fabrication. Membrane-bound pMMO is mixed with PEGDA 575 and photoinitiator and exposed to ultraviolet light to crosslink the material. (b) The resulting gel, shown in the vial, is immersed in buffer containing the reducing agent NADH and is exposed to CH₄ and air. The resulting methanol and protein are quantified to determine the specific activity of the material.

Figure 2. The effect of PEGDA and pMMO content on pMMO retention and activity.

The effect of PEGDA percentage by volume during polymerization on (a) pMMO retention (by weight) and (b) enzyme activity when fabricated with 150 μg pMMO. (c) Amount of pMMO that is retained in the PEGDA 575 hydrogel as a function of the amount of pMMO included during polymerization. (d) Activity of PEG-pMMO and pMMO control as a function of pMMO (μg) included during the activity assay. Each experiment was repeated in triplicate with N=2 and error bars represent the s.d. across the set of three experiments.

Figure 3. The effect of reusing the PEG-pMMO hydrogel over multiple cycles.

(a) pMMO activity in the PEG-pMMO hydrogel during the course of 20 methane activity assay cycles. (b) Cumulative amount of methanol (nmoles) produced per mg of pMMO for both as-isolated membrane-bound pMMO and PEG-pMMO over 20 cycles of the methane activity assay. Error bars represent the s.d. from the average of four replicates.

Figure 4. Continuous methanol production using a flow-through bioreactor design.

(a) Schematic and image of the flow-through bioreactor and the two (thin and thick) silicone lattice structures used to support the PEG-pMMO hydrogel membrane (scale bar, 1 cm). (b) Amount of methanol (nmole) produced per mg of pMMO in the PEG-pMMO hydrogel bioreactor showing continuous MeOH production for 2 h in thin versus thick membrane at 45 °C. Error bars represent the s.d. of three samples.

Figure 5. PEG-pMMO structures printed using PμSL.

Printing PEG-pMMO structures with PμSL allows high-resolution (features on the order of tens of μm) and flexibility in bioreactor component design. (a) Printed PEG-pMMO grid structure with small feature size (scale bar, 500 μm). (b) Large area PμSL was used to print cylinders with varying surface area to volume ratios on a shorter timescale with reduced resolution (scale bar, 1 mm). (c) The dependence of PEG-pMMO activity on surface area to volume ratio (N=3, error bars represent s.d., statistical significance determined by pairwise t-test where *P<0.1, **P<0.05). Inset: printed cylinders with surface area to volume ratios of (left to right) 1.47, 1.67, 1.93 and 2.33 (scale bar, 10 mm).