The concomitant recycling of enzymes and co-factors is a big challenge in biocatalysis. In this study, co -immobilization of the enzyme and the co-factor pyridoxal 5 '-phosphate was achieved using chitin-based mi-crospheres as the support. Chitin/chitosan composite microspheres were prepared and then modified with polyethyleneimine. Three pyridoxal 5 '-phosphate-independent enzymes were used to demonstrate that the self-sufficient biocatalysts based on chitin/chitosan composite microspheres and polyethyleneimine modified mi-crospheres displayed good generalizability for high recycling efficiency of both enzyme and pyridoxal 5 '-phos-phate. Furthermore, the self-sufficient biocatalysts were applied in the continuous flow production of cadaverine and maintained good continuous catalysis without the exogenous addition of pyridoxal 5 '-phosphate, suggesting that this co-immobilization platform shows promise for application in industrial biocatalysis.

Enzyme cofactors are essential in biocatalysis for many enzymes. However, their high commercial cost limits the industrial application of cofactor-dependent enzymes. We overcame this challenge by constructing a self-sufficient biocatalyst that co-immobilised pyridoxal 5'-phosphate (PLP) and L-lysine decarboxylase (LDC) using natural polymer chitin as the functional material. First, degree of deacetylation (DDA) of chitin was regulated to enhance PLP adsorption and immobilisation. Then, the enzyme was immobilised via fusion of a chitin-binding domain. Chitin with 36% DDA efficiently co-immobilised LDC and PLP. Laser scanning confocal microscope and scanning electron microscope proved that the LDC and PLP were immobilised on the chitin. The self-sufficient biocatalyst retained over 55% of its original activity after five cycles. The proposed strategy provides a new way to achieve co-immobilisation of PLP-dependent enzymes.

The cell membrane, as the supporting structure of cells, maintains the stability of the internal microenvironment of cells for whole-cell catalysis, but is also a barrier in transmembrane transport of substrate and product. However, methods for improving cell membrane permeability are usually accompanied with reduced membrane structural strength. Here, we show that a self-assembled biocomposite formed by Mg2+ ions and sodium deoxycholate (NaDC) can form sea urchin-shaped 3D complexes, incorporating living microbial cells. Cell membrane permeability was significantly improved by a fused structure between cell and Mg(DC)2 nanoparticles without membrane damage. Due to with hydrophobic mesopores, hydrophobic core and disseminated structure, the biocomposite greatly improved catalytic activity (up to 427.3%) of an environment-sensitive P450 enzyme expressed in Escherichia coli, which catalyzed hydroxylation with aromatic hydrocarbon. We also show that the biocomposite successfully improved the membrane permeability of live Bacillus cereus and Saccharomyces cerevisiae, making it a promising application platform for improving membrane permeability in whole-cell catalysis.

In this study, a cytochrome P450 monooxygenase CYP116B3 from Rhodococcus ruber was engineered to convert naphthalene selectively into 1-naphthol. The substrate recognition sites (SRSs) of CYP116B3 were identified using structure-based computational analysis. Subsequently, site-directed and saturation mutagenesis were conducted on SRS1, SRS2, and SRS3. The improved resulting triple mutant (E88C-N199Q-Q209A) was characterized. A final 1-naphthol titer of 8.26 mg/L/h was achieved, which is 14-fold higher than the control. The docking results suggest that the conformational changes produced by directed evolution improved the substrate binding environment. The results presented here hold promise for 1-naphthol production by P450 enzymes.

Research on the biocathode-based bioelectrochemical system (BES) has attracted extensive attention because of its ability to increase the electricity-driven production of high-value fuels or chemicals by relying on microbial cells as catalysts. An extracellular electron transfer (EET) that makes electrical connections to microorganisms plays a key role in the BES. Compared with the better understanding of the EET-to-anode connection, the understanding of the mechanism and elements involved in inward EET from cathodes to microbes remains limited. Additionally, the low capability of the EET limits its applications in BESs for producing chemicals. Here, we introduced the Mtr pathway into Escherichia coli cells by expressing ccmABCDEFGH from E. coli and mtrABC from Shewanella oneidensis. Through selection by electrochemical pressure, the evolved E. coli could use electricity to increase the production of succinate in direct BES and enhance the electroactivity. In addition, in studying the mechanism of inward EET, menaquinone was found to be one of the key components of inward EET, and it is essential for the fumarate reduction reaction. Lastly, the intracellular NADH and ATP levels showed that there were differences in the energy conservation coupling between the electron transfer routes of the inward Mtr pathway and the electron mediator.

Metal-organic frameworks (MOFs) for enzyme encapsulation-induced biomimetic mineralization under mild reaction conditions are commonly microporous and hydrophobic, which result in a rather high mass transfer resistance of the reactants and restrain the enzyme catalytic activity. Herein, we prepared a type of hierarchical porous and hydrophilic MOF through the biomimetic mineralization of enzymes, zinc ions, 2-methylimidazole, and lithocholic acid. The hierarchical porous structure accelerated the diffusion process of the reactants and the increased hydrophilicity conferred interfacial activity and increased the enzyme catalytic activity. The immobilized enzyme retained higher catalytic activity than the free enzyme and exhibited enhanced resistance to alkaline, organic, and high-temperature conditions. The nanobiocatalyst was reusable and showed long-term storage stability.

Artemisia selengensis straw is an agricultural residue with great potential as a renewable resource because it is rich in lignocellulose. In this study, A. selengensis straw was used as a source of hemicelluloses (ASH) and cellulose nanocrystals (ASCNC) to produce biodegradable films. Different content levels of ASCNC were used as additives to improve composite films with ASH and polyvinyl alcohol (PVA). Various mechanical and hydrophobic properties of the films were analyzed. The composite films enhanced by ASCNC exhibited greater strength and were more effective as a barrier to water vapor when compared to that of the control ASH/PVA film. The tensile strength of the composite film was increased 80.1% to 36.21 MPa with ASCNC loading exceeding 9%, and the water vapor transmission rate decreased 15.45% when 12% ASCNC was added. Furthermore, the ASCNC-enhanced ASH/PVA composite film reduced a greater amount of light transmission than the control film.

Microbial electrosynthesis (MES) or electro-fermentation (EF) is a promising microbial electrochemical technology for the synthesis of valuable chemicals or high-value fuels with aid of microbial cells as catalysts. By introducing electrical energy (current), fermentation environments can be altered or controlled in which the microbial cells are affected. The key role for electrical energy is to supply electrons to microbial metabolism. To realize electricity utility, a process termed inward extracellular electron transfer (EET) is necessary, and its efficiency is crucial to bioelectrochemical systems. The use of electron mediators was one of the main ways to realize electron transfer and improve EET efficiency. To break through some limitation of exogenous electron mediators, we introduced the phenazine-1-carboxylic acid (PCA) pathway from Pseudomonas aeruginosa PAO1 into Escherichia coli. The engineered E. coli facilitated reduction of fumarate by using PCA as endogenous electron mediator driven by electricity. Furthermore, the heterologously expressed PCA pathway in E. coli led to better EET efficiency and a strong metabolic shift to greater production of reduced metabolites, but lower biomass in the system. Then, we found that synthesis of adenosine triphosphate (ATP), as the “energy currency” in metabolism, was also affected. The reduction of menaquinon was demonstrated as one of the key reactions in self-excreted PCA-mediated succinate electrosynthesis. This study demonstrates the feasibility of electron transfer between the electrode and E. coli cells using heterologous self-excreted PCA as an electron transfer mediator in a bioelectrochemical system and lays a foundation for subsequent optimization.

Microenvironment modification within nanoconfinement can maximize the catalytic activity of enzymes. Phospholipase A1 (PLA1) has been used as the biocatalyst to produce high value L-α-glycerylphosphorylcholine (L-α-GPC) through hydrolysis of phosphatidylcholine (PC). We successfully developed a simple co-precipitation method to encapsulate PLA1 in a metal–surfactant nanocomposite (MSNC), then modified it using alkalescent 2-Methylimidazole (2-Melm) to promote catalytic efficiency in biphasic systems. The generated 2-Melm@PLA1/MSNC showed higher catalytic activity than PLA1/MSNC and free PLA1. Scanning electron microscopy and transmission electron microscopy showed a typical spherical structure of 2-Melm@PLA1/MSNC at about 50 nm, which was smaller than that of 2-Melm@MSNC. Energy disperse spectroscopy, N2 adsorption isotherms, Fourier transform infrared spectrum, and high-resolution X-ray photoelectron spectroscopy proved that 2-Melm successfully modified PLA1/MSNC. The generated 2-Melm@PLA1/MSNC showed a high catalytic rate per unit enzyme mass of 1.58 μmol mg-1 min-1 for the formation of L-α-GPC. The 2-Melm@PLA1/MSNC also showed high thermal stability, pH stability, and reusability in a water–hexane biphasic system. The integration of alkaline and amphiphilic properties of a nanocomposite encapsulating PLA1 resulted in highly efficient sequenced reactions of acyl migration and enzymatic hydrolysis at the interface of a biphasic system, which cannot be achieved by free enzyme.

Artificial metalloenzymes that combine the advantages of natural enzymes and metal catalysts have been getting more attention in research. As a proof of concept, an artificial nanometalloenzyme (CALB-Shvo@MiMBN) was prepared by co-encapsulation of metallo-organic catalyst and enzyme in a soft nanocomposite consisting of 2-methylimidazole, metal ions, and biosurfactant in mild reaction conditions using a one-pot self-assembly method. The artificial nanometalloenzyme with lipase acted as the core, and the metallo-organic catalyst embedded in micropore exhibited a spherical structure of 30-50 nm in diameter. The artificial nanometalloenzyme showed high catalytic efficiency in the dynamic kinetic resolution of racemic primary amines or secondary alcohols compared to the one-pot catalytic reaction of immobilized lipase and free metallo-organic catalyst. This artificial nanometalloenzyme holds great promise for integrated enzymatic and heterogeneous catalysis.

1,5-Pentamethylene diisocyanate, a novel aliphatic diisocyanate formed from bio-based 1,5-pentamethylenediamine, has been used as a hard segmented material to synthesize polyurethane. In this study, several waterborne polyurethane (WPU) dispersions have been successfully prepared by a prepolymer process from 1,5- pentamethylene diisocyanate poly(polyether) with different NCO/OH ratios and 1,6-hexanediol (HDO)/dimethylol propionic acid (DMPA) molar ratios. The Fourier transform infrared (FTIR) spectra, thermogravimetric analysis, differential scanning calorimetry, X-ray diffraction, and a mechanical tensile test were used to investigate the structures, thermal stability, phase separation, crystallinity, mechanical properties, and adhesive performance of the WPU dispersions. The FTIR results indicate that the degree of hydrogen bonding and the numbers of urea groups increase as the NCO/OH ratio and HDO/DMPA molar ratio increase. Furthermore, the phase separation increases and the thermal stability decreases as the NCO/OH ratio increases or the HDO/DMPA molar ratio decreases. Finally, WPU3.0-2.4 (NCO/OH = 3, HDO/DMPA = 2.4) exhibits a maximum tensile strength and shear strength, pointing to its possible use as an adhesive. These results could provide a very valuable reference for industrial applications of WPU.