Switchable CO2-Responsive Janus Nanoparticle for Lipase Catalysis in Pickering Emulsion.

The use of convertible immobilized enzyme carriers is crucial for biphasic catalytic reactions conducted in Pickering emulsions. However, the intense mechanical forces during the conversion process lead to enzyme leakage, affecting the stability of the immobilized enzymes. In this study, a CO2-responsive switchable Janus (CrSJ) nanoparticle (NP) was developed using silica NP, with one side featuring aldehyde groups and the other side adsorbing N,N-dimethyldodecylamine. A switchable Pickering emulsion catalytic system for biphasic interface reactions was prepared by covalently immobilizing lipase onto the CrSJ NPs. The CO2-responsive nature of the CrSJ NPs allowed for rapid conversion of the Pickering emulsion, and covalent immobilization substantially reduced lipase leakage while enhancing the stability of the immobilization during the conversion process. Impressively, after repeated transformations, the Pickering emulsion still maintains its original structure. Following 10 consecutive cycles of esterification and hydrolysis reactions, the immobilized enzyme's activity remains at 77.7 and 79.5% of its initial activity, respectively. The Km of the CrSJ catalytic system showed no significant change compared to the free enzyme, while its Vmax values were 1.2 and 1.6 times that of the free enzyme in esterification and hydrolysis reactions, respectively.

A chitosan-based antibacterial hydrogel with injectable and self-healing capabilities.

The presence of bacteria directly affects wound healing. Chitosan-based hydrogel biomaterials are a solution as they offer advantages for wound-healing applications due to their strong antimicrobial properties. Here, a double-cross-linking chitosan-based hydrogel with antibacterial, self-healing, and injectable properties is reported. Thiolated chitosan was successfully prepared, and the thiolated chitosan molecules were cross-linked by Ag-S coordination to form a supramolecular hydrogel. Subsequently, the amine groups in the thiolated chitosan covalently cross-linked with genipin to further promote hydrogel formation. In vitro experimental results indicate that hydrogel can release Ag+ over an extended time, achieving an antibacterial rate of over 99% against Escherichia coli and Staphylococcus aureus. Due to the reversible and dynamic feature of Ag-S coordination, an antibacterial hydrogel exhibited injectable and self-healing capabilities. Additionally, the hydrogel showed excellent biocompatibility and biodegradability. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00211-z.

Magnetic macroporous chitin microsphere as a support for covalent enzyme immobilization.

In this study, a novel magnetic macroporous chitin microsphere (MMCM) was developed for enzyme immobilization. Chitin nanofibers were prepared and subsequently subjected to self-assembly with magnetic nanoparticles and PMMA (polymethyl methacrylate). Following this, microspheres were formed through spray drying, achieving a porous structure through etching. The MMCM serves as an effective support for immobilizing enzymes, allowing for their covalent immobilization both on the microsphere's surface and within its pores. The substantial surface area resulting from the porous structure leads to a 2.1-fold increase in enzyme loading capacity compared to non-porous microspheres. The MMCM enhances stability of the immobilized enzymes under various pH and temperature conditions. Furthermore, after 20 days of storage at 4 °C, the residual activity of the immobilized enzyme was 2.93 times that of the free enzyme. Even after being recycled 10 times, the immobilized enzyme retained 56.7 % of its initial activity. It's noteworthy that the active sites of the enzymes remained unchanged after immobilization using the MMCM, and kinetic analysis revealed that the affinity of the immobilized enzymes rivals that of the free enzymes.

Characterization and comparison of carboxymethylation and TEMPO-mediated oxidation for polysaccharides modification.

In this study, carboxymethylation and TEMPO-mediated oxidation were compared for their ability to introduce carboxyl groups to polysaccharides, using cellulose and chitin as model polysaccharides. The carboxyl group contents and changes in the molecular weight of carboxymethylated and TEMPO-oxidized cellulose/chitin were measured. The results revealed that carboxymethylation achieved higher carboxyl group contents, with values of 4.99 mmol/g for cellulose and 4.46 mmol/g for chitin, whereas for TEMPO-oxidized cellulose and chitin, the values were 1.64 mmol/g and 1.12 mmol/g, respectively. As a consequence of TEMPO-mediated oxidation, polysaccharides underwent degradation, leading to a decrease in the molecular weight of 42.46 % for oxidized cellulose and 64.5 % for oxidized chitin. Additionally, the crystallinity of carboxymethylated polysaccharides decreased with an increase in the carboxyl group contents, whereas that of TEMPO-oxidized polysaccharides remained unchanged. Furthermore, TEMPO-mediated oxidation selectively oxidized C6 primary hydroxyls, while carboxylmethylation converted all the hydroxyl groups on the polysaccharides.

Preparation and comparison of dialdehyde derivatives of polysaccharides as cross-linking agents.

Dialdehyde-based cross-linking agents are widely used in the cross-linking of amino group-containing macromolecules. However, the most commonly used cross-linking agents, glutaraldehyde (GA) and genipin (GP), have safety issues. In this study, a series of dialdehyde derivatives of polysaccharides (DADPs) were prepared by oxidation of polysaccharides, and their biocompatibility and cross-linking properties were tested using chitosan as a model macromolecule. The DADPs showed outstanding cross-linking and gelation properties comparable to GA and GP. The DADPs and hydrogels cross-linked with the DADPs exhibited excellent cytocompatibility and hemocompatibility with different concentrations while significant cytotoxicity was observed in GA and GP. The experimental results showed that the cross-linking effect of DADPs increased along with their oxidation degree. The outstanding cross-linking effect of the DADPs show a potential for use in the cross-linking of biomacromolecules with amino groups and could be an adequate alternative to existing cross-linkers.

A Biodegradable Multifunctional Film as a Tissue Adhesive for Instant Hemostasis and Wound Closure.

Here, a multifunctional film (MFF) as an alternative tissue adhesive in the form of an interpenetrating network consisting of self-crosslinked aldehyde-functionalized chitosan (AC) and crosslinked poly(acrylic acid) (PAA) further coordinated with Ag+ is reported. The MFF combines enhanced toughness and stretchability, which is attributed to the synergistic effects of the double-network design. Covalent crosslinking maintains the overall integrity of the MFF matrix, while noncovalent crosslinking dissipates energy under deformation. Upon contact, the MFF quickly dries the tissue surface followed by instant physical crosslinking to the tissue. Subsequent covalent crosslinking between the aldehyde groups of the MFF and the primary amine groups on the surface of the tissue further stabilizes the adhesion. Meanwhile, Ag+ provides strong antibacterial properties to the MFF. Notably, in vivo studies demonstrate that the MFF allows facile and tough attachment to the wet and dynamic surface of rabbit liver and presents superior hemostasis and sealing properties. Furthermore, the MFF can be safely degraded without causing abnormal defects in vivo. The outstanding physicochemical properties of the MFF can potentially be a good alternative to existing sutures or staples and has potential for use in clinical practice.

Design, Preparation, and Evaluation of Enteric Coating Formulation of HPMC and Eudragit L100 on Carboxylated Agarose Hydrogel by Using Drug Tartrazine.

Enteric-coated application on drug is used to prevent the drug from inactivation which are degraded by gastric enzyme. The present study is aimed at achieving controlled drug delivery in acidic medium of gastrointestinal tract (GIT) by enteric coating of hydroxy propyl methylcellulose (HPMC) and Eudragit L100 on carboxylated agarose hydrogel, creating a pH-dependent delivery system. Fourier-transformed infrared spectroscopy (FTIR) was for the detection of carboxylic group on agarose hydrogel, and scanning electron microscope (SEM) was used for the determination surface of prepared formulation. To check the pH sensitivity of enteric-coated formulation, different pH solution was used. It was found that the formulation was not dissolved in 1.2 but dissolve in pH 6.8 similarly; hydrogels lacking coating showed that tartrazine was more dissolved in pH 1.2, and less dissolved at pH 6.8. The release of tartrazine from the hydrogels was measured by using spectrophotometer and using a scanning electron microscope to examine the morphology and surface appearance of hydrogel capsules. This study revealed cracks on coated samples, while noncoated samples showed clear appearance with no cracks. Our findings revealed that this method could be useful for the development of an enteric coating drug delivery system.

Synthesis of Agarose-Based Multistimuli-Responsive Hydrogel Dressing for Accelerated Wound Healing.

Stimuli-responsive hydrogels have drawn increasing research interest in regenerative medicine due to their tunable molecular structures and functional properties for both providing a suitable microenvironment for wound healing and to serve as a sustainable therapeutic. Hence, we developed a stimuli-responsive drug-loaded hydrogel wound dressing for sustained, controlled release of the drug and accelerating wound healing. Hydrogel dressings with stimuli-responsive properties were prepared using carboxymethyl agarose (CMA) with various degrees of substitution and calcium ion crosslinking, followed by the loading of recombinant human epidermal growth factor (Rh-EGF) on the CMA hydrogel. Experimental results indicated that these hydrogel composites showed pH and temperature stimuli-responsive behaviors and released the encapsulated drug sustainedly in various release media. Moreover, the hydrogel dressings exhibited a porous network structure, stable physical properties, and excellent biocompatibility. The investigation in vivo showed that the Rh-EGF-loaded CMA hydrogel dressing significantly enhanced wound healing and reduced inflammatory responses by upregulating the transforming growth factor-beta, vascular endothelial growth factor, and cluster of differentiation 31 (CD31). These results confirm that Rh-EGF-loaded CMA hydrogel dressings possess potential application in accelerating wound healing and tissue regeneration.

An efficient method for chitin production from crab shells by a natural deep eutectic solvent.

Crab shells are an important feedstock for chitin production. However, their highly compact structure significantly limits their use for the production of chitin under mild conditions. Here, a green and efficient approach using a natural deep eutectic solvent (NADES) to produce chitin from crab shells was developed. Its effectiveness in isolating chitin was investigated. The results showed that most proteins and minerals were removed from crab shells and the relative crystallinity of the isolated chitin reached 76%. The quality of the obtained chitin was comparable to chitin isolated by the acid-alkali method. This is the first report on a green method for efficient chitin production from crab shells. This study is expected to open new avenues for green and efficient production of chitin from crab shells.

Characterization of TEMPO-oxidized chitin nanofibers with various oxidation times and its application as an enzyme immobilization support.

Chitin nanofibers have recently received increased attention and are considered to be a promising material for a wide range of applications because of their excellent characteristics. In this study, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-oxidized chitin nanofibers (CNFs) with various oxidation times were prepared and characterized. CNFs with different oxidation times were then utilized for enzyme immobilization, using chymotrypsin as a model enzyme. The effects of oxidation time on enzyme immobilization were explored. Results showed characteristics of chitin nanofibers can be controlled by adjusting oxidation time. CNFs treated with TEMPO for 360 min showed the lowest crystallinity (79.13 ± 1.43%), the shortest length (241.70 ± 74.61 nm), the largest width (12.67 ± 3.43 nm), and the highest transmittance (73.01% at 800 nm). The activity of immobilized enzymes and enzyme loading showed good correlation to the carboxylate content of CNFs. The enzyme efficiency based on CNFs and the content of carboxylate groups peaked at the oxidization time of 60 min. When the additional amount of chymotrypsins (CTs) was 500 or 2000 mg/g carrier, the highest loading amount of CTs was 307.17 ± 4.08 or 726.82 ± 12.05 mg/g carrier, respectively.

A Novel Soluble Squalene-Hopene Cyclase and Its Application in Efficient Synthesis of Hopene.

Hopene is an important precursor for synthesizing bioactive hopanoids with great commercial value. However, the chemical methods for synthesizing hopene are not efficient to date. Hopene is commonly obtained by extracting from plants or bacteria like other terpenoids, but the complicated extraction process is inefficient and unfriendly to the environment. Hopene can be biological synthesized by squalene-hopene cyclase (SHC) from squalene. However, hopene production by SHC remained at a low level until now. In this work, we found a novel SHC named OUC-SaSHC from Streptomyces albolongus ATCC 27414. An easy procedure for expression and purification of OUC-SaSHC was established. The conditions for OUC-SaSHC to convert squalene into hopene are optimized as in 100 mM sodium phosphate buffer (pH 7.0) containing 0.5% Tween 80, 20 mM squalene and 0.14 mg/mL OUC-SaSHC at 30°C. In the scale-up reaction with the final volume of 100 mL, the yield of squalene could be up to 99% at 36 h, and 8.07 mg/mL hopene was produced. Our work showed a great potential of OUC-SaSHC as biocatalyst on scale-up production of hopene, hence improves the SHC-catalyzing enzyme synthesis of hopene from laboratory level to application level.

New type of green extractant for oil production: Citric acid/citric acid sodium extraction system.

Developing green solvents with low toxicity and low energy consumption is an important issue for edible oil production. In this study, a novel extraction system, specifically a citric acid/citric acid sodium mixture, was developed for oil extraction from seed crops. Peanut and pumpkin seeds were used to evaluate extraction efficiency and more than 70% and 57% oils, respectively, were extracted from peanut and pumpkin seeds at 4 °C. After extraction, the oils floated on the surface of the solution and could be separated from the solvent system without evaporation. The extraction of edible oils was achieved without the use of toxic chemicals or energy-intensive equipment. This study provided a green and efficient method, and showed the potential of the proposed citric acid/citric acid sodium extraction system for production of edible oils from natural sources.

Combining Cell Surface Display and DNA-Shuffling Technology for Directed Evolution of Streptomyces Phospholipase D and Synthesis of Phosphatidylserine.

Phospholipids have been widely used in food, medicine, cosmetics, and other fields because of their unique chemical structure and healthcare functions. Phospholipase D (PLD) is a key biocatalyst for the biotransformation of phospholipids. Here, an autodisplay expression system was constructed for rapid screening of mutants, and PLD variants were recombined using DNA shuffling technology and three beneficial mutations were obtained. The results of enzymatic performance and sequence information comparison indicated that C-terminal amino acids exerted a greater impact on the correct folding of PLDs, and N-terminal amino acids are more important for catalytic reaction. The best-performing recombinant enzyme in transphosphatidylation reactions was Recom-34, with a phosphatidylserine content accounting for 80.3% of total phospholipids and a 3.24-fold increased conversion rate compared to the parent enzyme. This study demonstrates great significance for screening ideal biocatalysts, facilitating soluble expression of inclusion body proteins, and identifying key amino acids.

Effects of Comedication and Genetic Factors on the Population Pharmacokinetics of Lamotrigine: A Prospective Analysis in Chinese Patients With Epilepsy.

Lamotrigine (LTG) is a second-generation anti-epileptic drug widely used for focal and generalized seizures in adults and children, and as a first-line medication in pregnant women and women of childbearing age. However, LTG pharmacokinetics shows high inter-individual variability, thus potentially leading to therapeutic failure or side effects in patients. This prospective study aimed to establish a population pharmacokinetics model for LTG in Chinese patients with epilepsy and to investigate the effects of genetic variants in uridine diphosphate glucuronosyltransferase (UGT) 1A4, UGT2B7, MDR1, ABCG2, ABCC2, and SLC22A1, as well as non-genetic factors, on LTG pharmacokinetics. The study population consisted of 89 patients with epilepsy, with 419 concentrations of LTG. A nonlinear mixed effects model was implemented in NONMEM software. A one-compartment model with first-order input and first-order elimination was found to adequately characterize LTG concentration. The population estimates of the apparent volume of distribution (V/F) and apparent clearance (CL/F) were 12.7 L and 1.12 L/h, respectively. The use of valproic acid decreased CL/F by 38.5%, whereas the co-administration of rifampicin caused an increase in CL/F of 64.7%. The CL/F decreased by 52.5% in SLC22A1-1222AA carriers. Patients with the ABCG2-34AA genotype had a 42.0% decrease in V/F, whereas patients with the MDR1-2677TT and C3435TT genotypes had a 136% increase in V/F. No obvious genetic effect of UGT enzymes was found relative to the concentrations of LTG in Chinese patients. Recommended dose regimens for patients with different gene polymorphisms and comedications were estimated on the basis of Monte Carlo simulations and the established model. These findings should be valuable for developing individualized dosage regimens in adult and adolescent Chinese patients 13-65 years of age.

A novel autolysis system for extracellular production and direct immobilization of a phospholipase D fused with cellulose binding domain.

BACKGROUND: Several types of phospholipases have been described in phospholipids modification. The majority of phospholipase D (PLD) superfamily members can catalyze two separate reactions: the hydrolysis of phospholipids to produce phosphatidic acid (PA) and the transphosphatidylation of phosphatidyl groups into various phosphatidyl alcohols to produce modified phospholipids. Transphosphatidylation is a useful biocatalytic method for the synthesis of functional phospholipids from lecithin or phosphatidylcholine (PC), which are both easily accessible. Different PLD coding genes have been cloned from various sources from viral, prokaryotic, and eukaryotic organisms. Despite the catalytic potential of PLD, their low productivity has hampered their practical applications, probably because PLD, which is highly toxic to the host cells, when transformation of the PLD genes into the host cells, degrade PLs in the cell membrane. In this study, we designed a novel two-step expression system to produce and secrete recombinant PLD in extracellular medium, cellulose-binding domains as an affinity fused with PLD for immobilization and purification proteins. RESULTS: The engineered BL21 (DE3) host strain, which harbored the final expression vector pET28a-PLD-CBD-araC-ESN, was induced by IPTG and L-arabinose, the cell density decreased rapidly over a 2 h period and the enzymes released into the extracellular medium accounts owned 81.75% hydrolytic activity. Scanning electron microscopy results showed that there were obvious structural changes on the cell surface. The extracellularly secreted PLD-CBD powder was used to catalyze the transphosphatidylation reaction synthesis of phosphatidylserine, 2.3 U enzymes reacted for 12 h, during which the conversion rate reached 99% with very few by-products being produced. When the fused protein PLD-CBD immobilized on microcrystalline cellulose, the enzymes can be cycle used five times with 26% conversion rate was preserved. CONCLUSIONS: This study introduced an effective method for use in the expression of recombinant proteins and their extracellular secretion that simplifies the steps of sonication and purification and demonstrates great potential in the industrial application of enzymes. Cellulose as the most abundant renewable biomass resources in nature, and the cost is low, used for PLD immobilization make it more simple, effective and sustainable.

Two-Step Separation of Chitin from Shrimp Shells Using Citric Acid and Deep Eutectic Solvents with the Assistance of Microwave.

In this research, a two-step extraction approach was developed for chitin preparation from shrimp shells by utilizing citric acids and deep eutectic solvents (DESs), which effectively removed minerals and proteins. In the first step, minerals of shrimp shells were removed by citric acid, and the demineralization efficiency reached more than 98%. In the second step, the removal of protein was carried out using deep eutectic solvents with the assistance of microwave, and the deproteinization efficiency was more than 88%. The results of scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction analysis (XRD), and thermogravimetric analysis (TGA) showed that the quality of DES-prepared chitin was comparable to that of traditional acid/alkali-prepared chitin. These results were realized without utilizing hazardous chemicals, which are detrimental to the environment. This research indicates that a DES-based preparation approach has the potential for application in the recovery of biopolymers from natural resources.

Efficient enzymatic hydrolysis of ionic liquid pretreated chitin and its dissolution mechanism.

Colloidal chitin, the substrate of chitinase with an open hydrated gel-like structure, can be obtained by treatment using either traditional hydrochloric acid (HCl) or ionic liquid (IL) 1-ethyl-3-methylimidazolium acetate ([Emim][OAc]). IL-pretreated chitin provided an efficient production of N-acetylglucosamine (175.62 mg g chitin) and N,N'-diacetylchitobiose (341.70 mg g chitin) with a conversion of 61.49% at 48 h catalyzed by chitinase from Streptomyces albolongus ATCC 27414. A short time second homogenization treatment after IL pretreatment can increase the conversion to 76.11%. A comprehensive characterization and comparison of chitin with different pretreatments suggested that enzymatic performances were correlated with the structural changes (size of the grains and porosity), high decrease in crystallinity, and high enzyme adsorption. The NMR spectroscopy studies of N-acetylglucosamine solvation in [Emim][OAc] clearly suggest that hydrogen bonding is formed between the hydroxyls of N-acetylglucosamine and both the anions and cations of the IL.

Conformational changes of proteins and oil molecules in fish oil/water interfaces of fish oil-in-water emulsions stabilized by bovine serum albumin.

This study investigated the conformational changes in proteins and oil molecules at fish oil/water interfaces. Results of FT-IR indicate that α-helical content of bovine serum albumin decreased (from 46.1% to 28.4%), and ß-sheet and random coil contents increased (from 15.5% and 13.5% to 18.9% and 28.1%, respectively) after protein adsorption at the fish oil/water interface. Fluorescence emission maximum of emulsions showed a blue shift compared to native proteins. FT-IR spectra of oil molecules showed broadenings in CH stretching vibration peaks of CH3 and CH2 groups of the fish oil. These results combined with structural information of BSA in the protein database led to the following hypothesis: binding occurs between proteins and lipid molecules in the oil-water interface. This study provides insight into the conformational changes of proteins and oil molecules in fish oil/water interfaces and broadens the fundamental understanding of the possible stability mechanisms of emulsions stabilized by proteins.

Green and Facile Production of Chitin from Crustacean Shells Using a Natural Deep Eutectic Solvent.

Natural deep eutectic solvents (NADESs) are sustainable, nontoxic, and biodegradable solvents, which are composed of natural primary metabolites. A green and efficient approach based on choline chloride-malic acid, a NADES, was developed for extracting chitin from crustacean shells, and its effectiveness for demineralization and deproteinization was determined. Fourier transform infrared (FT-IR) spectroscopy, thermogravimetric analysis (TGA), X-ray diffraction (XRD), and scanning electron microscopy (SEM) were used to investigate changes in the chemical composition of extracted chitin. The results revealed that most of the minerals and proteins were removed from the shrimp shells by using a NADES with the assistance of microwave irradiation. The quality of the obtained chitin was superior, and it displayed a relative crystallinity of 71%. All of these results were achieved without using harsh chemicals, which can raise environmental issues. This study provides a green and facile approach for chitin production from crustacean shells and reveals the potential of NADESs for applications in the extraction of biopolymers from natural sources.

Simultaneous cell disruption and lipid extraction in a microalgal biomass using a nonpolar tertiary amine.

A simultaneous cell disruption and lipid extraction method is developed for microalgal biodiesel production using a triethylamine/methanol solvent system. Individually, the pure solvents, i.e. triethylamine and methanol, do not exhibit significant enhancement in lipid extraction, but a 3:7 (v/v) triethylamine/methanol mixture exhibits the highest lipid extraction, corresponding to 150% of the conventional chloroform/methanol (2:1, v/v) solvent extraction. This extraction is equivalent to 92.5% of the total lipids, even when extracted from a wet microalgal biomass with a water content of 80%. The cell surfaces of the microalgae are significantly disrupted without using additional cell disruption reagents and without requiring energy-intensive equipment. The lipid mass transfer coefficient is 1.6 times greater than that of the chloroform/methanol solvent system. It is clearly demonstrated that triethylamine and methanol cooperate well for the cell disruption and lipid extraction.