Biocatalytic systems for cellulose-based biofuels

In this project we have developed a biocatalytic enzymogel which demonstrates a novel type of remote controlled phase-boundary biocatalysis that involves remotely directed binding to and engulfing insoluble substrates, high mobility and stability of the catalytic centers. The enzymogel is designed as a core shell nanoparticle made of a magnetic core and polymer brush. The mobile enzymes reside in the polymer brush scaffold and shuttle between the enzymogel interior and surface of the engulfed substrate in the bioconversion process. Biocatalytic activity of the mobile enzymes is preserved in the enzymogel while the brush-like architecture favors the efficient interfacial interaction when the enzymogel spreads over the substrate and extends substantially the reaction area as compared with rigid particles.

Design of enzymogel nanoparticles includes a core–shell structure with 15 nm g-super paramagnetic inorganic core embedded in a 100±10 nm silica and a 30±5 nm(in the dry state) poly(acrylic acid) (PAA) brush shell (Fig. 1a).  The PAA brush was synthesized by surface-initiated polymerization of tert butylacrylate and the subsequent hydrolysis of poly(tert-butylacrylate). In an aqueous environment in a pH range from 5 to 7 the enzymogel particle has a homogeneous, swollen, negatively charged PAA brush which is clearly observable in cryo-TEM images (Fig. 1b). The PAA shell shrinks at pH 4-5 but still maintains a net negative charge (Fig. 1c). PAA is a weak polyelectrolyte with a monomer unit pKa of 4.5 and is negatively charged in a pH range of 4 to 8 which is optimal for the highest enzymatic activity for most enzymes. Cellulose enzyme has typically isoelectric at pH 4.9 and, thus, attains a moderate positive charge at pH 4.5. It was loaded into the enzymogel particles at pH 4.5 (Fig. 1d). We used cellulase (CEL) enzymes to cleave cellulose molecules and convert them into glucose for biofuel or biochemical production.

In situ Ellipsometry adsorption measurement.

Adsorption at pH 4.5  and desorption at pH 7 kinetics in a 1.4 mg/ml CEL concentration  was monitored with in situ ellipsometry on the surface of a reference sample of the 32 ± 2 nm thick PAA brush grafted to the Si-wafer substrate.  CEL resides in the brush even after multiple washing in buffer at pH 4.5. Limited leakage (about 5%) of CEL was observed after rinsing of the fully loaded enzymogel particles.

Bio-catalytic activity of cellulose was estimated by using two groups based on a cellulose substrate type: (I–V) hydrolysis of insoluble cellulose (filter paper standard) and (VI–VIII hydrolysis of semi-soluble colloidal dispersion of a-cellulose (MM= 9000 gx). The results provide evidence that the enzymogel nanoparticles catalyze hydrolysis of cellulose using a new type of phase boundary catalysis (cases III and VIII); the particles are adsorbed on the surface of cellulose fibers and cleave the cellulose chains by enzymes shuttling between the PAA brush interior and the brush–cellulose interface.

Numerous possible applications including biomedical technologies are illustrated using experiments with a cotton floss (c-h). A droplet of a free CEL solution was deposited on the floss (c) and let to dry. In the second experiment, the droplet of enzymogel solution was deposited on the floss and dried. In both cases concentration of CEL was the same. Then, the floss was immersed in buffer pH 5.5 solution. In 2.5 h, the floss treated with the enzymogel was broken while the one treated with a free enzyme was intact after 14 h. If at the same concentration of CEL the enzymogel was collected on the floss with a magnet, the degradation experiment demonstrated an even faster floss failure after 1 h (g and h).  Thus, application of the enzymogel using magnetic field can be explored for localized highly efficient bio-conversion processes.

Mean diameter of a contact of the enzymogel particles with the cellulose substrate was proved with in situ AFM experiments when single enzymogel nanoparticles were examined on the surface of a cellophane film as model of cellulose substrate. The images of single adsorbed-on-the-substrate nanoparticles using topographical, adhesion and mechanical modulus contrasts are shown:

The spreading of the enzymogel over the substrate is clearly visible on the 3D topography images and topographical cross-sections. A simple estimation shows that the reaction contact area of an enzymogel nanoparticle due to the spreading (engulfing) is about an order of magnitude greater than for a solid particle of the same diameter with a traditional method of surface immobilization (grafting) of enzymes.

In Summary, The enzymogel nanoparticles provides a unique opportunity for industrial enzyme recovery. Because of the high biocatalytic activity of the enzymes that reside in the enzymogel there is no need to release and extract enzymes, so the enzymogel can be used for biomass conversion as integral moieties. After conversion of the cellulosic biomass, the enzymogel nanoparticles can be magnetically extracted and transferred into a freshly loaded bioreactor for reuse. The experiments demonstrated that this methodology provides an about four-fold increase in glucose per enzyme when compared with the traditional one-way use of CEL for cellulose conversion. This work introduces novel phase boundary biocatalysis when encapsulated enzyme retains its mobility in a soft hydrophilic polymer carrier and is capable of hydrolyzing insoluble substrates using highly dynamic behavior of the enzyme and their high affinity to the polymer brush of the enzymogel and to the substrate. The enzyme remains encapsulated in the polymer brush and catalyzes the hydrolysis of the insoluble substrates attached to the enzymogel. The fermentation processes described in the article are illustrated with video files.

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