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1. One-pot synthesis of enzyme@metal–organic material (MOM) biocomposites for enzyme biocatalysis

   https://doi.org/DOI https://doi.org/10.1039/D1GC00775K  

   Pan, Y.; Li, H.; Lenertz, M.; Han, Y.; Ugrinov, A.; Kilin, D.; Chen, B.; Yang, Z. (March 2021, Green chemistry)

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   Metal–organic frameworks/materials (MOFs/MOMs) are advanced enzyme immobilization platforms that improve biocatalysis, materials science, and protein biophysics. A unique way to immobilize enzymes is co-crystallization/co-precipitation, which removes the limitation on enzyme/substrate size. Thus far, most enzyme@MOF composites rely on the use of non-sustainable chemicals and, in certain cases, heavy metals, which not only creates concerns regarding environmental conservation but also limits their applications in nutrition and biomedicine. Here, we show that a dimeric compound derived from lignin, 5,5′-dehydrodivanillate (DDVA), co-precipitates with enzymes and low-toxicity metals, Ca2+ and Zn2+, and forms stable enzyme@Ca/Zn–MOM composites. We demonstrated this strategy on four enzymes with different isoelectric points (IEPs), molecular weights, and substrate sizes. Furthermore, we found that all enzymes displayed slightly different but reasonable catalytic efficiencies upon immobilization in the Ca–DDVA and Zn–DDVA MOMs, as well as reasonable reusability in both composites. We then probed the structural basis of such differences using a representative enzyme and found enhanced restriction of enzymes in Zn–DDVA than in Ca–DDVA, which might have caused the activity difference. To the best of our knowledge, this is the first aqueous-phase, one-pot synthesis of a lignin-derived “green” enzyme@MOF/MOM platform that can host enzymes without any limitations on enzyme IEP, molecular weight, and substrate size. The different morphologies and crystallinities of the composites formed by Ca–DDVA and Zn–DDVA MOMs broaden their applications depending on the problem of interest. Our approach of enzyme immobilization not only improves the sustainability/reusability of almost all enzymes but also reduces/eliminates the use of non-sustainable resources. This synthesis method has a negligible environmental impact while the products are non-toxic to living things and the environment. The biocompatibility also makes it possible to carry out enzyme delivery/release for nutritional or biomedical applications via our “green” biocomposites. 

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2. Cascade/Parallel Biocatalysis via Multi-enzyme Encapsulation on Metal–Organic Materials for Rapid and Sustainable Biomass Degradation

   https://doi.org/https://doi.org/10.1021/acsami.1c12209  

   Li, Q.; Pan, Y.; Li, H.; Lenertz, M.; Reed, K.; Jordahl, D.; Bjerke, T.; Ugrinov, A.; Chen, B.; Yang, Z. (September 2021, ACS applied materials interfaces)

   null (Ed.)

   Multiple-enzyme cooperation simultaneously is an effective approach to biomass conversion and biodegradation. The challenge, however, lies in the interference of the involved enzymes with each other, especially when a protease is needed, and thus, the difficulty in reusing the enzymes; while extracting/synthesizing new enzymes costs energy and negative impact on the environment. Here, we present a unique approach to immobilize multiple enzymes, including a protease, on a metal–organic material (MOM) via co-precipitation in order to enhance the reusability and sustainability. We prove our strategy on the degradation of starch-containing polysaccharides (require two enzymes to degrade) and food proteins (require a protease to digest) before the quantification of total dietary fiber. As compared to the widely adopted “official” method, which requires the sequential addition of three enzymes under different conditions (pH/temperature), the three enzymes can be simultaneously immobilized on the surface of our MOM crystals to allow for contact with the large substrates (starch), while MOMs offer sufficient protection to the enzymes so that the reusability and long-term storage are improved. Furthermore, the same biodegradation can be carried out without adjusting the reaction condition, further reducing the reaction time. Remarkably, the simultaneous presence of all enzymes enhances the reaction efficiency by a factor of ∼3 as compared to the official method. To our best knowledge, this is the first experimental demonstration of using aqueous-phase co-precipitation to immobilize multiple enzymes for large-substrate biocatalysis. The significantly enhanced efficiency can potentially impact the food industry by reducing the labor requirement and enhancing enzyme cost efficiency, leading to reduced food cost. The reduced energy cost of extracting enzymes and adjusting reaction conditions minimize the negative impact on the environment. The strategy to prevent protease damage in a multi-enzyme system can be adapted to other biocatalytic reactions involving proteases. 

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3. Tetrapyrrolic Surface Coatings for Applications in Photoelectrosynthetic Fuel Production

   Moore, Gary; Beiler, Anna; Khusnutdinova, Diana; Wadsworth, Brian (January 2018, ECS Meeting Abstracts)

   Hybrid materials capable of linking light capture and conversion technologies with the ability to drive reductive chemical transformations are attractive as components in photoelectrosynthetic cells. [1] We have recently reported methods of applying molecular surface coatings composed of metalloporphyrin redox catalysts onto solid-state substrates that are either conductive or semi-conductive. [2-5] The metalloporphyrin catalysts used in this work are capable of activating electrochemical transformations including the conversion of protons to hydrogen and carbon dioxide to carbon monoxide. In one approach, metalloporphyrin precursors are prepared via a novel synthetic strategy to yield a macrocycle with a pendent 4-vinylphenyl surface attachment group at the beta-position of the porphyrin ring structure. [2] This modification allows use of a photo-induced immobilization chemistry to attach intact metalloporphyrins to a range of (semi)conducting surfaces. In addition, we have shown that initial application of thin-film polymer surface coatings can provide a molecular interface for assembling metalloporphyrin catalysts in a subsequent wet chemical treatment step. [3] In this presentation, spectroscopic characterization of these materials coupled with electrochemical analysis will be presented. These findings offer an improved understanding of the structure and function relationships governing this class of materials. 1. A. M. Beiler, D. Khusnutdinova, S. I. Jacob, G. F. Moore, Ind. & Eng. Chem. Research, 55, 5306-5314 (2016); DOI: 10.1021/acs.iecr.6b00478 2. D. Khusnutdinova, A. M. Beiler, B. L. Wadsworth, S. I. Jacob, G. F. Moore, Chem. Sci., 8, 253-259 (2017); DOI: 10.1039/c6sc02664h 3. A. M. Beiler, D. Khusnutdinova, B. L. Wadsworth, G. F. Moore, Inorg. Chem., 56, 12178 (2017); DOI: 10.1021/acs.inorgchem.7b01509 4. A. M. Beiler, D. Khusnutdinova, S. I. Jacob, G. F. Moore, ACS Appl. Mater. Interfaces, 8, 10038-10043 (2016); DOI: 10.1021/acsami.6b01557 5. B. L. Wadsworth, A. M. Beiler, D. Khusnutdinova, S. I. Jacob, G. F. Moore, ACS Catal. 6, 8048-8057 (2016); DOI: 10.1021/acscatal.6b02194 

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4. Finned hierarchical MOFs supported on cellulose for the selective adsorption of n -hexane and 1-hexene

   https://doi.org/10.1039/D1CC04548B  

   Yin, Xinyang; Zha, Jie; Gregor, Skyler; Ding, Shengzhe; Alsuwaidi, Ahmad; Zhang, Xueyi (December 2021, Chemical Communications)

   Finned hierarchical MOF particles (Cu(BDC) nanosheets as fins grown perpendicularly on Ni 2 (BDC) 2 (DABCO) hexagonal prisms) were synthesized on modified cellulose support. The hierarchical MOF particles exposed the open Cu( ii ) sites on Cu(BDC) to enable selective 1-hexene/ n -hexane separation. 

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5. Supercharging Carbohydrate Binding Module Alone Enhances Endocellulase Thermostability, Binding, and Activity on Cellulosic Biomass

   https://doi.org/10.1021/acssuschemeng.3c06266  

   DeChellis, Antonio; Nemmaru, Bhargava; Sammond, Deanne; Douglass, Jenna; Patil, Nivedita; Reste, Olivia; Chundawat, Shishir_P S (March 2024, ACS Sustainable Chemistry & Engineering)

   Lignocellulosic biomass recalcitrance to enzymatic degradation necessitates high enzyme loadings, incurring large processing costs for the production of industrial-scale biofuels or biochemicals. Manipulating surface charge interactions to minimize nonproductive interactions between cellulolytic enzymes and plant cell wall components (e.g., lignin or cellulose) via protein supercharging has been hypothesized to improve biomass biodegradability but with limited demonstrated success to date. Here, we characterize the effect of introducing non-natural enzyme surface mutations and net charge on cellulosic biomass hydrolysis activity by designing a library of supercharged family-5 endoglucanase Cel5A and its native family-2a carbohydrate binding module (CBM) originally belonging to an industrially relevant thermophilic microbe, Thermobifida fusca. A combinatorial library of 33 mutant constructs containing different CBM and Cel5A designs spanning a net charge range of −52 to 37 was computationally designed using Rosetta macromolecular modeling software. Activity for all mutants was rapidly characterized as soluble cell lysates, and promising mutants (containing mutations on the CBM, Cel5A catalytic domain, or both CBM and Cel5A domains) were then purified and systematically characterized. Surprisingly, often endocellulases with mutations on the CBM domain alone resulted in improved activity on cellulosic biomass, with three top-performing supercharged CBM mutants exhibiting between 2- and 5-fold increase in activity, compared to native enzyme, on both pretreated biomass enriched in lignin (i.e., corn stover) and isolated crystalline/amorphous cellulose. Furthermore, we were able to clearly demonstrate that endocellulase net charge can be selectively fine-tuned using a protein supercharging protocol for targeting distinct substrates and maximizing biocatalytic activity. Additionally, several supercharged CBM-containing endocellulases exhibited a 5–10 °C increase in optimal hydrolysis temperature, compared to native enzyme, which enabled further increase in hydrolytic yield at higher operational reaction temperatures. This study demonstrates the first successful implementation of enzyme supercharging of cellulolytic enzymes to increase hydrolytic activity toward complex lignocellulosic biomass-derived substrates. 

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