d-Amino acid oxidases (DAAOs) are flavor enzymes and have been used in resolution of racemic amino acids and manufacturing of pharmaceuticals. However, the evolved H2O2 during the catalysis has deleterious and inhibitory effects. Decomposition of the hydrogen peroxide by catalase (CAT) can eliminate the negative effects. DAAO and CAT are dimeric and tetrameric proteins, respectively. Here, the N-terminus of the DAAO subunits has been specifically ligated to the C-terminus of the CAT subunits with native peptides through intein-mediated in vivo protein splicing. The in vivo splicing has little effect on the secondary structures of the enzymes as confirmed by circular dichroism (CD) spectra, and fluorescence spectra showed that the spliced product DAAO&CAT has a higher stability than DAAO. In the spliced product DAAO&CAT, the DAAO subunits are in close proximity to the CAT subunits, facilitating immediate transfer of H2O2 from one catalytic site to the other, enabling efficient decomposition of the generated H2O2. The reduced cofactors of the DAAO subunits were reoxidized by the evolved molecular oxygen around. Kinetics analysis showed that the d-alanine substrate follows Michaelis–Menten kinetics. The catalytic efficiency of DAAO&CAT is 22.4-fold that of DAAO. Furthermore, the spliced product DAAO&CAT has been encapsulated within a coordination polymer with an encapsulation efficiency of 91.3 ± 2.7%. The encapsulated DAAO&CAT has retained 98.1 ± 3.1% and 94.9 ± 2.9% of the activity of free DAAO&CAT at 30 and 40 °C, respectively.

An easy-to-operate method of using R-ω-transaminase has been developed by fusing it to an elastin-like polypeptide and forming a complex with D-amino acid oxidase.R-ω-Transaminase (R-ω-TA) was fused to an elastin-like polypeptide (ELP) through genetic engineering of the enzyme. The enzyme was purified through reversible phase transition. For the single-enzyme system, in the reaction media, ELP-R-ω-TA self-assembled and formed enzyme clusters of micrometer size, and the substrate, (R)-1-phenylethylamine, also formed droplets of micrometer size. Intimate contact of the enzyme clusters and the substrate droplets provided a microenvironment of high substrate concentration close to the enzyme, facilitating the diffusion of substrate molecules into the active sites. For the two-enzyme system, ELP-R-ω-TA and ELP-fusion D-amino acid oxidase assembled to form two-enzyme complexes, forming clusters with a size much larger size than that of single enzymes. The efficiency of the combined enzymes for producing the product was 99.6 %.The two-enzyme complexes significantly improved the catalytic efficiency. Potentially, the two enzymes forming complex clusters can facilitate the immobilization of the two enzymes together through non covalent methods by entrapping in porous supports.

D-Amino acid oxidase (DAAO) catalyzes oxidative deamination of D-amino acids to yield corresponding α-keto acids, producing hydrogen peroxide (H2O2). D-Amino acid oxidase was genetically modified by fusion to an elastin-like polypeptide (ELP). For enzyme immobilization, multi-walled carbon nanotubes (MWCNTs) were adopted as the model support. MWCNTs were functionalized with hematin. ELP-DAAO was immobilized on the functionalized CNTs by coupling to the hematin. The specific immobilization enabled ELP-DAAO in proximity to the hematin at a molecular distance. The molecular-distance proximity facilitated the immediate decomposition of H2O2 catalyzed by the hematin. The evolved oxygen was efficiently utilized to oxidize the reduced cofactor FDA of DAAO, and H2O2 was produced. The forming of a H2O2 → O2 → H2O2 circle between the DAAO and hematin has been demonstrated to be the driving force to accelerate the deamination reaction. The enzyme kinetics has shown that the ELP-DAAO/hematin-CNT conjugate exhibited a catalytic efficiency more than three times that of free ELP-DAAO, demonstrating its ability in mimicking multi-enzyme catalysis. The methodology for highly specific enzyme immobilization is not restricted to carbon nanotubes, and can be extended easily to other micro and nanomaterials as supports for specific immobilization of oxidases.

Carbon nanotubes (CNTs) were functionalized with sodium hexadecyl sulfate (SHS). The lysozyme adsorbed on the SHS-CNTs exhibited a higher activity than that immobilized on the nonfunctionalized CNTs. To explain the experimental results and explore the mechanism of lysozyme adsorption, large-scale molecular dynamics simulations have been performed for a four-component system, including lysozyme, SHS, CNTs in explicit water. It has been found that the assembled SHS molecules form a soft layer on the surface of CNTs. The interactions between lysozyme and SHS induce the rearrangement of SHS molecules, forming a saddle-like structure on the CNT surface. The saddle-like structure fits the shape of the lysozyme, and the active-site cleft of the lysozyme is exposed to the water phase. Whereas, for the lysozyme adsorbed on the nonfunctionalized CNT, due to the hydrophobic interactions, the active-site cleft of the enzyme tends to face the wall of the CNT. The results of this work demonstrate that the SHS molecules as the interfacial substance have a function of adjusting the lysozyme with an appropriate orientation, which is favorable for the lysozyme having a higher activity.Keywords: carbon nanotubes; lysozyme; molecular dynamics simulation; sodium hexadecyl sulfate

The combination of ionic liquids with lysozyme has a potential application in food processing and analysis. In this work, at acid conditions, the interaction mechanism of the ionic liquid 1-butyl-3-methylimidazolium trifluoromethansulfonate with lysozyme has been investigated by two-dimensional Fourier transform infrared spectroscopy (FTIR). The residues and structures of lysozyme that have preferential interactions with the ionic liquid have been identified. The interaction mechanism can explain experimental results at acidic conditions where it was found that the presence of the ionic liquid is positively correlated to the enhanced enzymatic activity of the hen egg white lysozyme.Keywords: Interaction mechanism; Ionic liquid; Lysozyme; Two-dimensional FTIR spectra

Magnetic multiwalled carbon nanotubes (M-MWNTs) have been functionalized with mixed surfactants consisting of a sugar-based surfactant and an ionic surfactant. The synergistic effect of the two surfactants results in more surfactants adsorbed and the formation of a more disordered structure of assembled surfactants. These two aspects facilitate simultaneous hydrogen bonding and electrostatic interactions with proteins. It has been demonstrated that the M-MWNTs functionalized with mixed surfactants adsorbed more proteins than the M-MWNTs functionalized with single surfactants. The M-MWNTs functionalized with mixed surfactants have been tested for the adsorption of two proteins, bovine serum albumin (BSA) with a low isoelectric point and lysozyme with a high isoelectric point, and similar results have been obtained. Hence, carbon nanotubes functionalized with mixed surfactants have a wide range of potential applications for protein adsorption.

Carboxyl-functionalized graphene oxide (GO–COOH) was utilized to immobilize lipase. Fourier transform infrared (FTIR), UV–visible, and X-ray photoelectron (XPS) spectra were measured to characterize lipase immobilization. At the optimal temperature of 40 °C, the immobilized lipase retains 80% of the hydrolytic activity of the native lipase. For catalyzing the enantioselective reaction in the organic solvent heptane, at 50 °C (optimal), the catalysis efficiency of the immobilized lipase is 1.6 times that of native lipase, and the immobilized lipase retains the selectivity of the native lipase. This work demonstrates that graphene oxide is a suitable support for immobilization of lipase for catalysis in organic solvent.

Maltose substituted β-cyclodextrin (M-β-CD) is an important drug carrier due to its excellent water solubility and good compatibility. In this work, dehydrocholic acid (DHA) was taken as the model drug; the inclusion of M-β-CD/DHA was studied through molecular dynamics simulations. The effect of the maltosyl residue of M-β-CD on the interactions of M-β-CD with DHA, M-β-CD with water, and DHA with water were analyzed. Based on the results, the difference between the complex of M-β-CD/DHA and that of β-CD/DHA can be explained and understood.

Amino-cyclodextrin was covalently attached to multiwalled carbon nanotubes (MWNTs). The functionalized MWNTs have a good dispersibility in water. The lipase was adsorbed onto the functionalized MWNTs. The immobilized lipase was utilized for the resolution of the model compound (R, S)-1-phenyl ethanol in heptane, the ionic liquid [Bmim]PF6 as well as the heptane/[Bmim]PF6 mixture. In the reaction media, the enzymatic activity of the immobilized lipase is much higher than that of the native lipase. In comparison to the catalysis in the ionic liquid and heptane, when using the mixture of heptane/[Bmim]PF6 as the reaction medium, the catalysis by the immobilized lipase at the heptane–ionic liquid interface exhibited a higher catalysis activity. This is due to two aspects: the continuous diffusion of substrate from the heptane phase to the ionic liquid phase; the simultaneous extraction of product from the ionic liquid phase. In addition, the interfacial enzymatic catalysis facilitates the reuse of the immobilized lipase and the ionic liquid.Graphical abstractHighlights► The functionalized carbon nanotubes were used to immobilize the lipase. ► The interfacial enzymatic catalysis by the immobilized lipase exhibited advantages. ► They include the significantly enhanced enzymatic catalysis activity. ► And easy recovery of product and reuse of the immobilized lipase and ionic liquid.

•The interaction mechanism of SDBS with lysozyme has been investigated.•Microenvironmental change in and around the active site region induced by SDBS has been revealed and explained.•HP-β-CD is used to detach SDBS from the inactivated enzyme, and complete recovery of enzymatic activity is achieved.Circular dichroism spectra reveal that sodium dodecyl benzene sulfonate (SDBS) at low concentrations can effectively prevent the aggregation of lysozyme molecules, while SDBS at high concentrations can lead to conformational and structural change of the protein. SDBS is able to inhibit the enzymatic activity of lysozyme in a highly efficient dose-dependent manner. The interaction mechanism of SDBS with lysozyme has been investigated by measuring optical spectra. Based on fluorescence and UV–vis spectra, microenvironmental change in and around the active site region induced by SDBS has been revealed and explained. Two-dimensional FTIR spectra have been analyzed to identify the secondary structures and residues of lysozyme, which have a preferential interaction with SDBS. Hydroxypropyl β-cyclodextrin (HP-β-CD) was used to detach SDBS from the inactivated enzyme, and complete recovery of enzymatic activity was achieved. Thus, the enzymatic activity of lysozyme can be regulated by SDBS and HP-β-CD.Hydroxypropyl β-cyclodextrin detaches SDBS from the inactivated enzyme.

Angiotensin I-converting enzyme (ACE) is a key therapeutic target for combating hypertension and related cardiovascular diseases. ACE inhibitory peptides offer the prospect of enhanced potency, high specificity, and no or low side effect. The ACE inhibitory tripeptides LKP and IKP differ from each other by one amino acid but their inhibitory potencies for ACE differ significantly. To uncover the molecular mechanism underlying this phenomenon, we have investigated the tripeptide/ACE complexes through molecular dynamics simulations coupled with quantum mechanical simulations. Comparative structural analysis has identified a hydrophobic subsite in the active site of cACE comprising hydrophobic residues Val379, Val380, Phe457, Phe527, and Ala418. The interactions of the side chains of Leu and Ile with the hydrophobic residues determine the binding positions of N-terminal residues of the tripeptides, that influence the interaction of the residues of tripeptides with the active site of cACE. This work presents the molecular mechanism of the interactions between the inhibitory tripeptides and ACE, and deciphers the structural basis for the high affinity LKP inhibition of ACE.Highlights► MD simulations have identified a hydrophobic subsite in the active site of cACE. ► The interactions of the side chains of the tripeptides with the hydrophobic residues determine the binding positions of the tripeptides. ► This work presents the molecular mechanism of the interactions between the inhibitory tripeptides and ACE.

A new approach has been developed for the functionalization of multi-walled carbon nanotubes (MWNTs). The self-assembly of a sugar-based amphiphile on MWNTs was carried out in amino acid solutions. The functionalization of MWNTs has been investigated by means of UV–vis, Raman spectra, FTIR, XPS, XRD, and HRTEM. It has been found that amino acids can promote the self-assembly of the amphiphile on MWNTs and regulate the amount and conformation of the assembled amphiphile. The effect of amino acids on the aggregation of the sugar-based amphiphile on MWNTs was analyzed through FTIR spectra. The specific interactions of f-MWNTs with the protein concanavalin A were investigated through the measurement of UV–vis spectra. This work demonstrates that, the functionalized MWNTs with disaccharide groups on their exterior surface have a good dispersibility in water and are biocompatible, and may have a potential application of biomolecular recognition.Graphical abstractHighlights► A new method for functionalization of carbon nanotubes has been demonstrated. ► The adsorption of a sugar-based surfactant on MWNTs is carried out in amino acid solutions. ► Amino acid solutions can promote the adsorption of the surfactant on MWNTs. ► Amino acid solutions can regulate the amount and conformation of the assembled surfactant on MWNTs. ► The functionalized MWNTs (f-MWNTs) have a good dispersibility in water and are biocompatible.

For producing pure hydrogen from biomass gasification with steam as the gasifying agent, an intensified process is proposed. In the process, a bubbling fluidized bed gasifier is used for the steam gasification of biomass, a steam reformer is used for upgrading the biosyngas, and an H2-membrane water-gas-shift (HMWGS) reactor is used for converting the carbon monoxide and separating the hydrogen. Pure hydrogen is the product of the process. High-purity CO2 can be produced and taken as a coproduct. To better simulate the proposed process, models for the steam-blown fluidized bed gasifier and catalytic steam reformer have been developed. The simulation results have been compared with the experimental data, and good agreement has been obtained. The analysis of the proposed process is carried out in terms of the amount of pure hydrogen per kilogram of biomass and the overall thermodynamic efficiency of the process. Factors affecting the pure hydrogen production and thermodynamic efficiency have been studied and discussed.

The method previously developed is applied to determine the parameters of the statistical associating fluid theory (SAFT) equation of state for amino acids. With the parameters determined, the SAFT equation of state is applied to model the amino acid solubility in water with very good precision, including l-tyrosine, l-leucine, l-aspartic acid, l-tryptophan, l-glutamic acid, l-alanine, dl-alanine, dl-valine, l-phenylalanine, dl-serine, l-proline, l-serine, and glycine. On the basis of the results of binary systems of amino acid/water, the solubility of l-alanine and l-leucine in water at high pressures up to 3500 bar is simulated; the amino acid solubilities in an aqueous solution is modeled, including systems dl-alanine/dl-valine/H2O at 25 °C, dl-alanine/dl-serine/H2O at 25 °C, l-glutamic acid/l-aspartic acid/H2O at 25, 40, and 60 °C, l-serine/l-aspartic acid/H2O at 25, 40, and 60 °C, and l-serine/l-glutamic acid/H2O at 25, 40, and 60 °C, and good agreement between modeling results and data are obtained.

Water activity, oxygen solubility and density of aqueous solutions of sugar and sugar alcohols have been modeled with the statistical associating fluid theory (SAFT). The modeling is accomplished by extending the previously developed method to determine the SAFT parameters for sugar and sugar alcohols. For the aqueous solutions of sorbitol/water, xylitol/water, mannitol/water, xylitol/sorbitol/water and xylitol/mannitol/water, the water activity has been predicted. The solubilities of oxygen in water and in aqueous solutions of glucose/water, fructose/water, sucrose/water, maltose/water and mannitol/water have been modeled. The density predictions for mannitol/sucrose/water, mannitol/sorbitol/water and mannitol/sorbitol/sucrose/water have been carried out. All the modeling results show that, by using the previously developed method to determine the SAFT parameters, the SAFT model has been able to model the water activity, oxygen solubility and density with very good accuracy, and the SAFT model can be taken as a suitable tool for describing sugar and sugar containing solutions.

The density of aqueous solutions of amino acids has been modeled with the statistical associating fluid theory (SAFT) equation of state. The modeling is accomplished by extending the previously developed new method to determine the SAFT parameters for amino acids. The modeled systems include α-alanine/H2O, β-alanine/H2O, proline/H2O, l-asparagine/H2O, l-glutamine/H2O, l-histidine/H2O, serine/H2O, glycine/H2O, alanine/H2O/sucrose, dl-valine/H2O/sucrose, arginine/H2O/sucrose, serine/H2O/ethylene glycol, and glycine/H2O/ethylene glycol. The density of binary solutions of amino acids has been correlated or predicted with a high precision. And then the density of multicomponent aqueous solutions of amino acids has been modeled based on the modeling results of binary systems, and a high accuracy of density calculations has been obtained. Finally, the water activities of dl-valine/H2O, glycine/H2O, and proline/H2O have been predicted without using binary interaction parameters, and good results have been obtained.

An integrated process has been proposed for the production of ultrapure hydrogen from biomass gasification with air. The process consists of an air-blown bubbling fluidized bed gasifier, a steam reformer, and a water-gas-shift membrane reactor. A non-isothermal model has been developed to simulate the fluidized bed gasifier, and a one-dimensional model has also been developed to simulate the steam reformer. The simulation results are compared with the experimental data, and good agreement is obtained. Based on the simulation results, the thermodynamic analysis of the integrated process is carried out. The simulation and analysis provide a quantitative tool for gaining insight into the process.

In a previous work, the parameters of the statistical associating fluid theory (SAFT) equation of state for amino acids were determined by using the method developed. The solubility of amino acids in water was modeled. In this work, the SAFT equation of state has been applied to describe the solubility of amino acids in aqueous alcohol solutions. The systems include dl-alanine/ethanol/water, glycine/ethanol/water, dl-valine/ethanol/water, dl-serine/ethanol/water, glycine/1-propanol/water, glycine/2-propanol/water, l-alanine/2-propanol/water, l-leucine/ethanol/water. Binary interaction parameters between amino acid and alcohol are needed by the SAFT model to get good modeling results.

A new approach has been developed for the non-covalent functionalization of multiwalled carbon nanotubes (MWNTs), which allows a presentation of carbohydrate on their surface by hydrophobic interactions. The approach is based on the self-assembly of a sugar-based amphiphile on MWNTs in alcohol/water mixtures, which has been investigated by means of ultraviolet (UV), Raman spectra, Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), and high-resolution transmission electron microscopy (HRTEM). It has been demonstrated that alcohols not only can promote the self-assembly of the amphiphile on MWNTs but also can regulate the amount and conformation of the assembled amphiphile. The adsorption of bovine serum albumin (BSA) onto functionalized MWNTs has been studied and characterized with circular dichroism (CD) spectra. The results showed that the conformation of adsorbed BSA has been well preserved. It has been demonstrated that, the functionalized MWNTs with disaccharide groups on their exterior surface have a good dispersibility in water and are biocompatible, may have a potential application of molecular recognition.Highlights► We demonstrate a new method for functionalization of carbon nanotubes. ► The self-assembly of a sugar-based amphiphile on MWNTs is carried out in alcohol solutions. ► Alcohol solutions can promote the self-assembly of the amphiphile on MWNTs. ► Alcohol solutions can regulate the amount and conformation of the assembled amphiphile on MWNTs. ► The functionalized MWNTs (f-MWNTs) have a good dispersibility in water and are biocompatible. ► f-MWNTs may have a potential application of molecular recognition.

•d-amino acid oxidase (ELP-DAAO) and R-ɷ-transaminases (ELP-RTA) were separately immobilized on the polydopamine-coated MnO2 nanorods.•The immobilized ELP-RTA catalyzed the conversion of (R)-1-phenylethylamine with pyruvate as an amine acceptor.•The immobilized ELP-DAAO catalyzed the conversion of d-alanine to pyruvate, the generated hydrogen peroxide was decomposed by the MnO2 nanorods.•The immobilized enzymes achieved a conversion of 98 ± 1.8% in comparison to 69.6 ± 1.2% by free enzymes.R-ɷ-transaminases transfer an amino group from an amino donor (e.g. (R)-1-phenylethylamine) onto an amino acceptor (e.g. pyruvate), resulting a co-product (e.g. d-alanine). This work intends to immobilize R-ɷ-Transaminase on MnO2 nanorods to achieve multienzyme catalysis. R-ɷ-Transaminase (RTA) and d-amino acid oxidase (DAAO) have been fused to an elastin-like polypeptide (ELP) separately through genetic engineering of the enzymes. ELP-RTA and ELP-DAAO have been separately immobilized on polydopamine-coated MnO2 nanorods. When the two immobilized enzymes were used together in one pot, the transformation of (R)-1-phenylethylamine was catalyzed by the immobilized ELP-RTA, and the co-product d-alanine was converted back to pyruvate under the catalysis of the immobilized ELP-DAAO, achieving the recycling of pyruvate in situ. Thus pyruvate was maintained at a low concentration in order to reduce its negative effect. On the other hand, the generated H2O2 of ELP-DAAO was decomposed by the MnO2 nanorods, and the evolved oxygen oxidized the reduced cofactors of ELP-DAAO. Forming the circles of hydrogen peroxide → oxygen → hydrogen peroxide accelerated the deamination reaction. The highly efficient conversion of the co-product d-alanine back to pyruvate accelerated the forming of the pyruvate → d-alanine → pyruvate cycle between the two immobilized enzymes. The coordination of the pyruvate → d-alanine → pyruvate and hydrogen peroxide → oxygen → hydrogen peroxide cycles accelerated the transformation of (R)-1-phenylethylamine. As a result, As a result, the immobilized enzymes achieved a conversion of 98 ± 1.8% in comparison to 69.6 ± 1.2% by free enzymes.