Relationships of crystallinity and reaction rates for enzymatic degradation of poly (ethylene terephthalate), PET.

Biocatalytic degradation of plastic waste is anticipated to play an important role in future recycling systems. However, enzymatic degradation of crystalline poly (ethylene terephthalate) (PET) remains consistently poor. Herein, we employed functional assays to elucidate the molecular underpinnings of this limitation. This included utilizing complementary activity assays to monitor the degradation of PET disks with varying crystallinity (XC), as well as determining enzymatic kinetic parameters for soluble PET fragments. The results indicate that an efficient PET-hydrolase, LCCICCG, operates through an endolytic mode of action, and that its activity is limited by conformational constraints in the PET polymer. Such constraints become more pronounced at high XC values, and this limits the density of productive sites on the PET surface. Endolytic chain-scissions are the dominant reaction type in the initial stage, and this means that little or no soluble organic product are released. However, endolytic cuts gradually and locally promote chain mobility and hence the density of attack sites on the surface. This leads to an upward concave progress curve; a behavior sometimes termed lag-phase kinetics.

Heterogeneous biocatalytic system for effective decolorization of textile dye effluent.

The current physicochemical methods for decolorizing toxic synthetic dyes are not sustainable to halt the environmental damage as they are expensive and often produce concentrated sludge, which may lead to secondary disposal problems. Biocatalysis (microbes and/or their enzymes) is a cost-effective, versatile, energy-saving and clean alternative. The most common enzymes involved in dye degradation are laccases, azoreductases and peroxidases. Toxic dyes could be converted into less harmful byproducts through the combined action of many enzymes or the utilization of whole cells. The action of whole cells to treat dye effluents is either by biosorption or degradation (aerobic or anaerobic). Using immobilized cells or enzymes will offer advantages such as superior stability, persistence against harsh environmental conditions, reusability and longer half-lives. This review envisages the recent strategies of immobilization and bioreactor considerations with the immobilized system as the effective treatment of textile dye effluents. Packed bed reactors are the most popular heterogeneous biocatalytic reactors for dye decolorization due to their efficiency and cost-effectiveness.

Reaction Pathways for the Enzymatic Degradation of Poly(Ethylene Terephthalate): What Characterizes an Efficient PET-Hydrolase?

Bioprocessing of polyester waste has emerged as a promising tool in the quest for a cyclic plastic economy. One key step is the enzymatic breakdown of the polymer, and this entails a complicated pathway with substrates, intermediates, and products of variable size and solubility. We have elucidated this pathway for poly(ethylene terephthalate) (PET) and four enzymes. Specifically, we combined different kinetic measurements and a novel stochastic model and found that the ability to hydrolyze internal bonds in the polymer (endo-lytic activity) was a key parameter for overall enzyme performance. Endo-lytic activity promoted the release of soluble PET fragments with two or three aromatic rings, which, in turn, were broken down with remarkable efficiency (kcat /KM values of about 105  M-1 s-1 ) in the aqueous bulk. This meant that approximatly 70 % of the final, monoaromatic products were formed via soluble di- or tri-aromatic intermediates.

In situ modulation of enzyme activity via heterogeneous catalysis utilizing solid electroplated cofactors.

During product isolation the received bioreceptors often do not exhibit a sufficient biochemical activity due to multistep dissociation and loss of cofactors. However, for bioelectrochemical applications the presence of cofactors is necessary for a successful oxidative or reductive conversion of the substrates to the products. Herein, we show how the immobilization of the required electroplated cofactors in a design of amperometric electrodes can in situ assist the activity of apo-enzymes. Compared to conventional approaches used in enzyme engineering this tailored nanoengineering methodology is superior from economic point of view, labor and time costs, storage conditions, reduced amount of waste and can fill the gap in the development of tuned bioelectrocatalysts.

A Versatile Chemoenzymatic Nanoreactor that Mimics NAD(P)H Oxidase for the In Situ Regeneration of Cofactors.

Herein, we report a multifunctional chemoenzymatic nanoreactor (NanoNOx) for the glucose-controlled regeneration of natural and artificial nicotinamide cofactors. NanoNOx are built of glucose oxidase-polymer hybrids that assemble in the presence of an organometallic catalyst: hemin. The design of the hybrid is optimized to increase the effectiveness and the directional channeling at low substrate concentration. Importantly, NanoNOx can be reutilized without affecting the catalytic properties, can show high stability in the presence of organic solvents, and can effectively oxidize assorted natural and artificial enzyme cofactors. Finally, the hybrid was successfully coupled with NADH-dependent dehydrogenases in one-pot reactions, using a strategy based on the sequential injection of a fuel, namely, glucose. Hence, this study describes the first example of a hybrid chemoenzymatic nanomaterial able to efficiently mimic NOx enzymes in cooperative one-pot cascade reactions.

Co-Immobilization and Co-Localization of Multi-Enzyme Systems on Porous Materials.

The immobilization of multi-enzyme systems on solid materials is rapidly gaining interest for the construction of biocatalytic cascades with biotechnological applications in industry. The heterogenization and control of the spatial organization across porous materials of the system components are essentials to improve the performance of the process providing higher robustness, yield, and productivity. In this chapter, the co-immobilization and co-localization of a bi-enzymatic bio-redox orthogonal cascade with in situ cofactor regeneration are described. An NADH-dependent alcohol dehydrogenase catalyzes the asymmetric reduction of 2,2,2 trifluoroacetophenone using an NADH regeneration system consisting of a glutamate dehydrogenase and glutamic acid. Three different spatial organizations of the enzymes were compared in terms of cofactor-recycling efficiency. Furthermore, we demonstrated how the co-localization and uniform distribution (by controlling the enzyme immobilization rate) of the main and recycling dehydrogenases inside the same porous particle lead to enhance the cofactor-recycling efficiency of the bi-enzymatic bio-redox systems.

A tailor-made, self-sufficient and recyclable monooxygenase catalyst based on coimmobilized cytochrome P450 BM3 and glucose dehydrogenase.

Cytochrome P450 monooxygenases (P450s) promote hydroxylations in a broad variety of substrates. Their prowess in C-H bond functionalization renders P450s promising catalysts for organic synthesis. However, operating P450 reactions involve complex management of the main substrates, O2 and nicotinamide adenine dinucleotide phosphate (NAD(P)H) reducing equivalents against an overall background of low operational stability. Whole-cell biocatalysis, although often used, offers no general solution to these problems. Herein, we present the design of a tailor-made, self-sufficient, operationally stabilized and recyclable P450 catalyst on porous solid support. Using enzymes as fusion proteins with the polycationic binding module Zbasic2 , the P450s BM3 (from Bacillus megaterium) was coimmobilized with glucose dehydrogenase (type IV; from B. megaterium) on anionic sulfopropyl-activated carrier (ReliSorb SP). Immobilization via Zbasic2 enabled each enzyme to be loaded in controllable amount, thus maximizing the relative portion of the rate limiting P450 BM3 (up to 19.5 U/gcarrier ) in total enzyme immobilized. Using lauric acid as a representative P450 substrate that is poorly accessible to whole-cell catalysts, we demonstrate complete hydroxylation at low catalyst loading (≤0.1 mol%) and efficient electron coupling (74%), inside of the catalyst particle, to the regeneration of NADPH from glucose (27 cycles) was achieved. The immobilized P450 BM3 showed a total turnover number of ∼18,000, thus allowing active catalyst to be recycled up to 20 times. This study therefore supports the idea of practical heterogeneous catalysis by P450s systems immobilized on solid support.

Cross-linked cytochrome P450 BM3 aggregates promoted by Ru(II)-diimine complexes bearing aldehyde groups.

Cross-linked enzyme aggregate (CLEA) methodology has been applied to immobilize cytochrome P450 BM3 variants (F87A and 21B3) with peroxygenase activity. Several Ru(II)-diimine complexes were found to be suitable cross-linking agents, surpassing the traditional glutaraldehyde and dextran aldehyde. They offer modular numbers of aldehyde functionalities and a more rigid framework than their organic counterparts. The F87A CLEAs display significant activity loss compared to the protein in solution. Meanwhile, for the 21B3 CLEAs, high activity recovery (up to 95%) is obtained. In order to minimize enzyme leaching from the CLEA, sodium cyanoborohydride was used to reduce the CLEAs imine bonds. The reduced CLEAs were active for several rounds of reactions leading to an overall increase in protein activity of 170% compared to the free protein in solution.

Enzymatic Hydrophobic Modification of Jute Fibers via Grafting to Reinforce Composites.

Horseradish peroxidase (HRP)/H2O2 system catalyzes the free-radical polymerization of aromatic compounds such as lignins and gallate esters. In this work, dodecyl gallate (DG) was grafted onto the surfaces of lignin-rich jute fabrics by HRP-mediated oxidative polymerization with an aim to enhance the hydrophobicity of the fibers. The DG-grafted jute fibers and reaction products of their model compounds were characterized by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC). The results clearly indicated the grafting of DG to the jute fiber by HRP. Furthermore, the hydrophobicity of jute fabrics was determined by measuring the wetting time and static contact angle. Compared to the control sample, the wetting time and static contact angle of the grated fabrics changed from ~1 s to 1 h and from ~0° to 123.68°, respectively. This clearly proved that the hydrophobicity of jute fabrics improved considerably. Conditions of the HRP-catalyzed DG-grafting reactions were optimized in terms of the DG content of modified jute fabrics. Moreover, the results of breaking strength and elongation of DG-grafted jute/polypropylene (PP) composites demonstrated improved reinforcement of the composite due to enzymatic hydrophobic modification of jute fibers.

Enzyme directed formation of un-natural side-chains for covalent surface attachment of proteins.

The covalent immobilization of proteins onto surfaces is an essential aspect of several fields of research, including proteomics, sensing, heterogeneous biocatalysis, and more broadly biotechnology. Site-specific, covalent attachment of proteins has been achieved in recent years by the use of expanded genetic codes to produce proteins with controlled placement of un-natural amino acids bearing bio-orthogonal functional groups. Unfortunately, the complexity of developing such systems is impractical for most laboratories; hence, a less complicated approach to generating un-natural amino acid side-chains has been employed. Utilizing a straightforward reaction with formylglycine generating enzyme, we use the site-specific modification of engineered proteins to yield un-natural amino acid side-chains for protein immobilization. Using this approach, we demonstrate the controlled immobilization of various enzymes onto a variety of amine coated surfaces. Our results reveal reusability of the immobilized enzymes via this strategy, and furthermore, we find the activity of the immobilized enzymes to remain even after a month of use indicating significant stability of the linkage.