Meso-structured onion-like silica (Meso-Onion-S) was synthesized and used as a host of enzyme immobilization. Meso-Onion-S has a 200–300 nm sized primary meso-structured onion building unit, and each onion unit has highly curved mesopores of 10 nm diameter in a multishell structure. Nanoscale enzyme reactors (NERs) in Meso-Onion-S were prepared via a two-step process of enzyme adsorption and subsequent enzyme cross-linking, which effectively prevents the leaching of cross-linked enzyme aggregates from highly curved mesopores of Meso-Onion-S. As a result, NERs in Meso-Onion-S significantly improved the enzyme stability as well as the enzyme loading. For example, NER of lipase (NER-LP) was stable under rigorous shaking for 40 days, while the control sample of adsorbed LP (ADS-LP) with no enzyme cross-linking showed a rapid inactivation due to rigorous enzyme leaching under shaking. Stable NER-LP was successfully employed to produce biodiesels and fatty acid methyl esters, from the LP-catalyzed transesterification of soybean oil with methanol. Interestingly, the specific activity of NER-LP was 23 and 10 times higher than those of free LP and ADS-LP, respectively, revealing the importance of LP stabilization in the form of NER-LP in the presence of organic solvents.

We developed a process for one-pot CO2 conversion and utilization based on simple conversion of CO2 to bicarbonate at ambient temperature with no energy input, by using the cross-linking-based composites of carboxylated polyaniline nanofibers (cPANFs) and carbonic anhydrase. Carbonic anhydrase was immobilized on cPANFs via the approach of magnetically separable enzyme precipitate coatings (Mag-EPC), which consists of covalent enzyme attachment, enzyme precipitation, and cross-linking with amine-functionalized magnetic nanoparticles. Mag-EPC showed a half-life of 236 days under shaking, even resistance to 70% ethanol sterilization, and recyclability via facile magnetic separation. For one-pot CO2 conversion and utilization, Mag-EPC was used to accelerate the growth of microalga by supplying bicarbonate from CO2, representing 1.8-fold increase of cell concentration when compared to the control sample. After two repeated uses via simple magnetic separation, the cell concentration with Mag-EPC was maintained as high as the first cycle. This one-pot CO2 conversion and utilization is an alternative as well as complementary process to adsorption-based CO2 capture and storage as an environmentally friendly approach, demanding no energy input based on the effective action of the stabilized enzyme system.

Subtilisin Carlsberg (SC) was immobilized and stabilized on magnetically-separable mesoporous silica (Mag-MSU-F) in the form of nanoscale enzyme reactors (NERs) based on the ship-in-a-bottle mechanism. Stabilized NERs of SC (NER-SC) were freeze-dried and successfully used for the transesterification of N-acetyl-L-phenylalanine ethyl ester with n-propanol in isooctane. Magnetic separation of Mag-MSU-F facilitated the repeated usages of stable NER-SC. This is the first demonstration for the use of stable and magnetically-separable NERs in an organic solvent, which has the potential for environmentally-friendly synthesis using enzymes in organic solvents.Enzymes are environmentally-friendly biocatalysts that can be used for chemical transformation processes including the production of biochemicals, bioalcohols and biodiesels. [1][2][3][4][5] Enzymes offer chemo-, regio-and stereo-selectivities under mild reaction conditions, such as ambient temperature and pressure, and physiological pH. [6][7][8] In particular, the change of medium from water to organic solvents enables synthetic reactions such as esterification, transesterification, aminolysis and thiotransesterification that are suppressed in aqueous solution. 9 As a result, the organic solvent enzymology has provided numerous synthetic and processing advantages. 10 Despite these advantages, one of the potential problems is the structural denaturation of native enzymes in organic solvents, leading to the poor enzyme stability and lowered enzyme activity. 11 As a potential solution, the stabilization and immobilization of enzymes has been proposed for successful nonaqueous enzymology with enhanced activity by preserving its native conformation. 12,13 † Electronic supplementary information (ESI) available. See

Carbonic anhydrases convert CO2 to bicarbonate at a high turnover rate up to 106 s–1, but their actual applications in CO2 conversion processes are hampered by their poor stability. This study reports highly loaded and stabilized bovine carbonic anhydrase (bCA) upon being immobilized onto electrospun polymer nanofibers in the form of enzyme precipitate coating (EPC). The EPC protocol, consisting of enzyme covalent attachment, precipitation, and cross-linking, maintained 65.3% of initial activity even after being incubated in aqueous solution at room temperature under shaking at 200 rpm for 868 days. EPC also showed strong resistance to the treatment of the metal chelation agent, ethylenediaminetetraacetic acid, and molecular dynamic simulation was carried out to elucidate the prevention of metal leaching from the active site of bCA upon being cross-linked in the form of EPC. Highly stable EPC with high bCA loading was employed for the conversion of bubbling CO2 to bicarbonate, and the bicarbonate solution was utilized as a carbon source for expedited microalgae growth in a separate bioreactor. The addition of EPC in the bubbling CO2 reactor resulted in 134 and 231% accelerated microalgae growths compared to the controls with and without 25 mM sodium bicarbonate, respectively. EPC with high enzyme loading and unprecedentedly successful stabilization of enzyme stability has a great potential to be used for the development of various enzyme-mediated CO2 conversion and utilization technologies.

The construction of a trypsin column for rapid and efficient protein digestion in proteomics is described. Electrospun and alcohol-dispersed polymer nanofibers were used for the fabrication of highly stable trypsin coatings, which was prepared by a two-step process of covalent attachment and enzyme crosslinking. In a comparative study with the trypsin coatings on as-spun and non-dispersed nanofibers, it has been observed that a simple step of alcohol dispersion improved not only the enzyme loading but also the performance of protein digestion. In-column digestion of enolase was successfully performed in less than twenty minutes. By applying the alcohol dispersion of polymer nanofibers, the bypass of samples was reduced by filling up the column with well-dispersed nanofibers, and subsequently, interactions between the protein and the trypsin coatings were improved, yielding more complete and reproducible digestions. Regardless of alcohol-dispersion or not, trypsin coatings showed better digestion performance and improved performance stability under recycled uses than covalently-attached trypsin, in-solution digestion, and commercial trypsin beads. The combination of highly stable trypsin coatings and alcohol-dispersion of polymer nanofibers has opened up a new potential to develop a trypsin column for on-line and automated protein digestion.