Transdermal Sustained Release Properties and Anti-Photoaging Efficacy of Liposome-Thermosensitive Hydrogel System.

Drug delivery systems have become a research priority in the biomedical field. The incorporation of liposomes to hydrogels further forms more robust multifunctional systems for more effective and sustained topical drug delivery. In this study, carboxymethyl-modified chitosan/hyaluronic acid (CMC/HA, CMH) thermosensitive hydrogel is developed for sustained transdermal delivery of liposomes. Hydrogels are crosslinked by hydrogen bonding, hydrophobic interaction and electrostatic interaction. The gel properties can be regulated by substitution degree (DS), and when DS = 18.20 ± 0.67% (CMH2), the gel temperature is 37.8 °C, allowing rapid gelation at body temperature (315 s). Moreover, CMH2 hydrogel has suitable spreadability (17.7-57.2 cm2 ), viscosity (2133.4 mPa s) and porous structure, which facilitated its adhesion and application on the skin and liposomes delivery. The hydrogel can retard the liposomes release, and the release rate of ascorbyl glucoside (AA2G) is 33.92-49.35% in 24 h. Hydrogel avoids the rapid clearance of liposomes from the skin and improved the skin retention, achieving the long-term release of bioactive components. Liposome-hydrogel system more efficiently promotes the anti-photoaging effect of AA2G on skin, reducing epidermal thickness, melanin deposition and lipid oxidative damage and increasing collagen density. Therefore, liposome-hydrogel systems are proposed as multifunctional delivery systems for sustained transdermal delivery.

Promoter engineering for efficient production of sucrose phosphorylase in Bacillus subtilis and its application in enzymatic synthesis of 2-O-α-D-glucopyranosyl-L-ascorbic acid.

2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G), a stable glucoside derivative of L-ascorbic acid (L-AA), can be one-step synthesized by sucrose phosphorylase (SPase). In this study, we attempted to produce extracellular SPase in Bacillus subtilis WB800 for the food-grade production of AA-2G. The results showed that the secretion of SPases did not require signal peptide. Promoter and its compatibility to target SPase gene were proved to be the key factors for high-level secretion. The strong promoter P43 and synthetic SPase gene derived from Bifidobacterium longum (BloSPase) were selected due to generate a relatively high extracellular activity (0.94 U/mL) for L-AA glycosylation. A highly active dual-promoter system PsigH-100-P43 was further constructed, which produced the highest extracellular and intracellular activity were 5.53 U/mL and 6.85 U/mL in fed-batch fermentation, respectively. Up to 113.58 g/L of AA-2G could be achieved by the supernatant of fermentation broth and a higher yield of 146.42 g/L was obtained by whole-cells biotransformation. Therefore, the optimal dual-promoter system in B. subtilis is suitable for the food-grade scale-up production of AA-2G.

Optimization of fermentation medium and conditions for enhancing valinomycin production by Streptomyces sp. ZJUT-IFE-354.

Valinomycin is a cyclodepsipeptide antibiotic with a broad spectrum of biological activities, such as antiviral, antitumor, and antifungal activities. However, the low yield of valinomycin often limits its applications in medicine, agriculture, and industry. In our previous report, Streptomyces sp. ZJUT-IFE-354 was identified as a high-yielding strain of valinomycin. In this study, Plackett-Burman design (PBD) and response surface methodology (RSM) were used to optimize components of medium. The optimal medium contained 31 g/L glucose, 22 g/L soybean meal, and 1.6 g/L K2HPO4·3H2O, which could generate 262.47 ± 4.28 mg/L of valinomycin. Then, the culture conditions were optimized by a one-factor-at-a-time (OFAT) approach. The optimal conditions for the strain included a seed age of 24 h, an inoculum size of 8% (v/v), an incubation temperature of 28 °C, an initial pH of 7.2, an elicitor of 0.1% Bacillus cereus feeding at 24 h cultivation, and the feeding of 0.6% L-valine at 36 h cultivation. The final valinomycin production increased to 457.23 ± 9.52 mg/L, which was the highest yield ever reported. It highlights that RSM and OFAT may be efficient methods to enhance valinomycin production by Streptomyces sp. ZJUT-IFE-354.

Engineering of Cyclodextrin Glycosyltransferase through a Size/Polarity Guided Triple-Code Strategy with Enhanced α-Glycosyl Hesperidin Synthesis Ability.

Hesperidin, a flavonoid enriched in citrus peel, can be enzymatically glycosylated using CGTase with significantly improved water solubility. However, the reaction catalyzed by wild-type CGTase is rather inefficient, reflected in the poor production rate and yield. By focusing on the aglycon attacking step, seven residues were selected for mutagenesis in order to improve the transglycosylation efficiency. Due to the lack of high-throughput screening technology regarding to the studied reaction, we developed a size/polarity guided triple-code strategy in order to reduce the library size. The selected residues were replaced by three rationally chosen amino acids with either changed size or polarity, leading to an extremely condensed library with only 32 mutants to be screened. Twenty-five percent of the constructed mutants were proved to be positive, suggesting the high quality of the constructed library. Specific transglycosylation activity of the best mutant Y217F was assayed to be 935.7 U/g, and its kcat/KmA is 6.43 times greater than that of the wild type. Homology modeling and docking computation suggest the source of notably enhanced catalytic efficiency is resulted from the combination of ligand transfer and binding effect. IMPORTANCE Size/polarity guided triple-code strategy, a novel semirational mutagenesis strategy, was developed in this study and employed to engineer the aglycon attacking site of CGTase. Screening pressure was set as improved hesperidin glucoside synthesis ability, and eight positive mutants were obtained by screening only 32 mutants. The high quality of the designed library confirms the effectiveness of the developed strategy is potentially valuable to future mutagenesis studies. Mechanisms of positive effect were explained. The best mutant exhibits 6.43 times enhanced kcat/KmA value and confirmed to be a superior whole-cell catalyst with potential application value in synthesizing hesperidin glucosides.

Effects of Different Pesticides on the Brewing of Wine Investigated by GC-MS-Based Metabolomics.

The application of pesticides is critical during the growth of high-quality grape for wine making. However, pesticide residues have significant influence on the wine flavor. In this study, gas chromatography-mass spectrometry (GC-MS) was performed and the obtained datasets were analyzed with multivariate statistical methods to investigate changes in flavor substances in wine during fermentation. The principal component analysis (PCA) score plot showed significant differences in the metabolites of wine treated with various pesticides. In trials using five pesticides (hexaconazole, difenoconazole, flutriafol, tebuconazole, and propiconazole), more than 86 metabolites were changed. Most of these metabolites were natural flavor compounds, like carbohydrates, amino acids, and short-chain fatty acids and their derivatives, which essentially define the appearance, aroma, flavor, and taste of the wine. Moreover, the five pesticides added to grape pulp exhibited different effects on the metabolic pathways, involving mainly alanine, aspartate and glutamate metabolism, butanoate metabolism, arginine, and proline metabolism. The results of this study will provide new insight into the potential impact of pesticide residues on the metabolites and sensory profile of wine during fermentation.

The optimized biocatalytic synthesis of (S)-methyl 2-chlorobutanoate by Acinetobacter sp. lipase.

Epilepsy is a chronic disease caused by sudden abnormal discharge of brain neurons, leading to transient brain dysfunction. Levetiracetam, developed by the UCB company in Belgium, is an effective drug for the treatment of epilepsy. (S)-Methyl 2-chlorobutanoate is an important chiral building block of levetiracetam, which has attracted a great deal of attention. In this study, a strain of lipase-produced Acinetobacter sp. zjutfet-1 was screened from soil samples. At optimized conditions for fermentation and biocatalysis, the bacterial lipase exhibited high catalytic activity for hydrolysis and stereoselectivity toward racemic methyl 2-chlorobutanoate. When the enzymatic reaction was carried out in 6% of racemic substrate, the enantiomeric excess (e.e.s ) reached more than 95%, with a yield of over 86%. Therefore, this lipase can efficiently resolve racemic methyl 2-chlorobutanoate and obtain (S)-methyl 2-chlorobutanoate, which presents great potential in the industrial production of levetiracetam.

Maleimide structure: a promising scaffold for the development of antimicrobial agents.

Natural compounds bearing maleimide rings are a series of secondary metabolites derived from fungi/marine microorganisms, which are characterized by a general structure -CO-N(R)-CO-, and the R group is normally substituted with alkyl or aryl groups. Maleimide compounds show various biological activities such as antibacterial, antifungal, and anticancer activity. In this review, the broad-spectrum antimicrobial activities of 15 maleimide compounds from natural sources and 32 artificially synthesized maleimides were summarized, especially against Candida albicans, Sclerotinia sclerotiorum, and Staphylococcus aureus. It highlights that maleimide scaffold has tremendous potential to be utilized in the development of novel antimicrobial agents.

Efficient production of valinomycin by the soil bacterium, Streptomyces sp. ZJUT-IFE-354.

A novel strain with antifungal activity against Sclerotinia sclerotiorum was isolated from soil, and identified as Streptomyces sp. ZJUT-IFE-354 using morphological and 16S rDNA sequence analysis. The bioactive metabolite produced by strain ZJUT-IFE-354 was identified and characterized as valinomycin by spectroscopic and chemical methods. The yield of valinomycin was 191.26 mg/L from the culture of Streptomyces sp. ZJUT-IFE-354, which was the highest yield to our knowledge. The in vitro antifungal activity of valinomycin against S. sclerotiorum was investigated as 0.056 ± 0.012 (EC50) and 0.121 ± 0.023 µg/mL (EC95), respectively, which was approximately 10.696- and 30.960-fold more active than that of carbendazim. The results from scanning electron microscopy, cell membrane permeability, and D-sorbitol and ergosterol assay indicated that valinomycin exerted the antifungal activity probably by increasing permeability of fungal cell membrane, leading to mycelial electrolyte leakage, and eventually resulting in the death of S. sclerotiorum. Thus, valinomycin may be a promising antifungal agent to control S. sclerotiorum. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03055-5.

Efficient production of l-menthyl α-glucopyranoside from l-menthol via whole-cell biotransformation using recombinant Escherichia coli.

l-Menthyl α-D-glucopyranoside (α-MenG) is a glycoside derivative of l-menthol with improved water-solubility and new flavor property as a food additive. α-MenG can be synthesized through biotransformation, but its scale-up production was rarely reported. In this study, the properties of an α-glucosidase from Xanthomonas campestris pv. campestris 8004 (Agl-2) in catalyzing the glucosylation of menthol was investigated. Agl-2 can almost completely glycosylate l-menthol (> 99%) when using 1.2 M maltose as glycosyl donor. Accumulated glucose resulted from maltose hydrolysis and transglycosylation caused the inhibition of the glucosylation rate (40% reduction of the glucosylation rate in the presence of 1.2 M glucose) which can be avoided through whole-cell catalysis with recombinant E. coli. Interestingly, in spite of the poor solubility of menthol, the productivity of α-MenG reached 24.7 g/(L·h) in a 2 L catalyzing system, indicating industrialization of the reported approach.

[Application of sucrose phosphorylase in glycosylation].

Water solubility, stability, and bioavailability, can be substantially improved after glycosylation. Glycosylation of bioactive compounds catalyzed by glycoside hydrolases (GHs) and glycosyltransferases (GTs) has become a research hotspot. Thanks to their rich sources and use of cheap glycosyl donors, GHs are advantageous in terms of scaled catalysis compared to GTs. Among GHs, sucrose phosphorylase has attracted extensive attentions in chemical engineering due to its prominent glycosylation activity as well as its acceptor promiscuity. This paper reviews the structure, catalytic characteristics, and directional redesign of sucrose phosphorylase. Meanwhile, glycosylation of diverse chemicals with sucrose phosphorylase and its coupling applications with other biocatalysts are summarized. Future research directions were also discussed based on the current research progress combined with our working experience.

[Application of immobilized glycosidase in the synthesis of glycoside compounds].

Glycoside compounds are widely used in medicine, food, surfactant, and cosmetics. The glycosidase-catalyzed synthesis of glycoside can be operated at mild reaction conditions with low material cost. The glycosidase-catalyzed processes include reverse hydrolysis and transglycosylation, appropriately reducing the water activity in both processes may effectively improve the catalytic efficiency of glucosidase. However, glucosidase is prone to be deactivated at low water activity. Thus, glucosidase was immobilized to maintain its activity in the low water activity environment, and even in neat organic solvent system. This article summarizes the advances in glycosidase immobilization in the past 30 years, including single or comprehensive immobilization techniques, and immobilization techniques combined with genetic engineering, with the aim to provide a reference for the synthesis of glycosides using immobilized glycosidases.

Valinomycin as a potential antiviral agent against coronaviruses: A review.

Human coronaviruses (HCoVs), including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have been resulting in global epidemics with heavy morbidity and mortality. Unfortunately, there are currently no specific medicines that can better treat these coronaviruses. Drug repurposing is an effective and economical strategy for drug discovery from existing drugs, natural products, and synthetic compounds. In this review, the broad-spectrum antiviral activity of valinomycin (VAL), especially its activity against coronaviruses such as SARS-CoV, MERS-CoV, human coronavirus OC43 (HCoV-OC43), were summarized, it highlights that VAL has tremendous potential for use as a novel antiviral agent against SARS-CoV-2.

Glycosyl hydrolase catalyzed glycosylation in unconventional media.

The reversible hydrolytic property of glycosyl hydrolases (GHs) as well as their acceptance of aglycones other than water has provided the abilities of GHs in synthesizing glycosides. Together with desirable physiochemical properties of glycosides and their high commercial values, research interests have been aroused to investigate the synthetic other than the hydrolytic properties of GHs. On the other hand, just like the esterification processes catalyzed by lipases, GH synthetic effectiveness is strongly obstructed by water both thermodynamically and kinetically. Medium engineering by involving organic solvents can be a viable approach to alleviate the obstacles caused by water. However, as native hydrolyases function in water-enriched environments, most GHs display poor catalytic performance in the presence of organic solvents. Some GHs from thermophiles are more tolerant to organic solvents due to their robust folded structures with strong residue interactions. Other than native sources, immobilization, protein engineering, employment of surfactant, and lyophilization have been proved to enhance the GH stability from the native state, which opens up the possibilities for GHs to be employed in unconventional media as synthases. KEY POINTS: • Unconventional media enhance the synthetic ability but destabilize GHs. • Viable approaches are discussed to improve GH stability from the native state. • GHs robust in unconventional media can be valuable industrial synthases.

Antifungal peptides produced by actinomycetes and their biological activities against plant diseases.

Antibacterial peptides are a class of naturally occurring peptides produced by eukaryotes and prokaryotes. Some of them exhibit broad-spectrum antifungal activity. Antifungal peptides (AFPs) can be developed as antibiotic to control fungal infections in agriculture due to their different antifungal mechanisms. As actinomycetes are still one of the most important sources of novel antibiotics, in this review, the mechanisms of action of AFPs are explained. Characterization of several AFPs produced by actinomycetes and their biological activities against plant diseases are summarized. Furthermore, the pathway for total synthesis of naturally occurring cyclodepsipeptide, valinomycin, is proposed. Finally, the pathway for biosynthesis of kutzneride 2 is proposed and the structure-activity relationship of kutznerides is discussed.

Insight into the glycosylation and hydrolysis kinetics of alpha-glucosidase in the synthesis of glycosides.

α-Glucosidase, Agl2, from Xanthomonas campestris was successfully overexpressed in Escherichia coli BL21(DE3) cells and purified with Ni columns. The enzyme exhibits glycosylation abilities towards a wide range of phenolic substrates, including phenol, vanillin, and ethyl vanillin, with maltose as the glycosyl donor. The catalytic properties of the purified enzyme were further investigated. It was observed that the synthesized glycosides started to degrade with prolonged catalytic time, giving an "n"-shaped kinetic profile. To understand such catalytic behavior, the Agl2-catalyzed glycosylation process was investigated kinetically. Based on the obtained parameters, it was concluded that although the substrate conversions are thermodynamically restricted in a batch system, the glycosylation efficiency can be kinetically controlled by the glycosylation/hydrolysis selectivity. Glucose was produced by both glycosylation and hydrolysis, significantly impacting the glycosylation efficiency. This study provides a mechanistic understanding of the α-glucosidase-catalyzed glycosylation process in a water-based system. The developed kinetic model was successful in explaining and analyzing the catalytic process. It is suggested that when α-glucosidase is employed for glycosylation in a water-enriched environment, the catalytic efficiency is mainly impacted by the enzyme's glycosylation/hydrolysis selectivity and glucose content in the catalytic environment.

Optimization of whole-cell biotransformation for scale-up production of α-arbutin from hydroquinone by the use of recombinant Escherichia coli.

α-Arbutin is an effective skin-whitening cosmetic ingredient and hyperpigmentation therapy agent. It can be synthesized by one-step enzymatic glycosylation of hydroquinone (HQ), but limited by the low yield. Amylosucrase (Amy-1) from Xanthomonas campestris pv. campestris 8004 was recently identified with high HQ glycosylation activity. In this study, whole-cell transformation by Amy-1 was optimized and process scale-up was evaluated in 5000-L reactor. In comparison with purified Amy-1, whole-cell catalyst of recombinant E. coli displays better tolerance against inhibitors (oxidized products of HQ) and requires lower molar ratio of sucrose and HQ to reach high conversion rate (> 99%). Excess accumulation of glucose (0.6-1.0 M) derived from sucrose hydrolysis inhibits HQ glycosylation rate by 46-60%, which suggests the importance of balancing HQ glycosylation rate and sucrose hydrolysis rate by adjusting the activity of whole-cell catalyst and HQ-fed rate. Using optimal conditions, 540 mM of final concentration and 95% of molar conversion rate were obtained within 13-18 h in laboratory scale. For industrial scale-up production, 398 mM and 375 mM of final concentration with high conversion rates (~ 95%) were obtained in 3500-L and 4000-L of reaction volume, respectively. These yields and productivities (4.5-4.9 kg kL-1 h-1) were the highest by comparing to the best we known. Hence, high-yield production of α-arbutin by batch-feeding whole-cell biotransformation was successfully achieved in the 5000-L reaction scale.

Characterization of Glucokinase Catalysis from a Pseudo-Dimeric View.

Glucose phosphorylation by glucokinase exhibits a sigmoidal dependency on substrate concentration regardless of its simple structure. Dimorph mechanism suggested the existence of two enzymatic states with different catalytic properties, which has been shown to be plausible by structural analysis. However, the dimorph mechanism gives rise to a complicated or non-explicit non-closed mathematical form. It is neither feasible to apply the dimorph mechanism in effector characterizations. To improve the area of glucokinase study with stronger theoretical support and less complication in computation, we proposed the investigation of the enzyme from a pseudo-dimeric angle. The proposed mechanism started from the idealization of two monomeric glucokinase as a dimeric complex, which significantly simplified the glucose phosphorylation kinetics, while the differences in enzyme reconfiguration caused by variable substrates and effectors have been successfully characterized. The study presented a simpler and more reliable way in studying the properties of glucokinase and its effectors, providing guidelines of effector developments for hyperglycemia and hypoglycemia treatment.

Spherical harmonics based generalized image source method for simulating room acoustics.

Allen and Berkley's image source method (ISM) is proven to be a very useful and popular technique for simulating the acoustic room transfer function (RTF) in reverberant rooms. It is based on the assumption that the source and receiver of interest are both omnidirectional. With the inherent directional nature of practical loudspeakers and the increasing use of directional microphones, the above assumption is often invalid. The main objective of this paper is to generalize the frequency domain ISM in the spherical harmonics domain such that it could simulate the RTF between practical transducers with higher-order directivity. This is achieved by decomposing transducer directivity patterns in terms of spherical harmonics and by applying the concept of image sources in spherical harmonics based propagation patterns. Therefore, from now on, any transducer can be modeled in the spherical harmonics domain with a realistic directivity pattern and incorporated with the proposed method to simulate room acoustics more accurately. We show that the proposed generalization also has an alternate use in terms of enabling RTF simulations for moving point-transducers inside pre-defined source and receiver regions.

Theory and design of compact hybrid microphone arrays on two-dimensional planes for three-dimensional soundfield analysis.

Soundfield analysis based on spherical harmonic decomposition has been widely used in various applications; however, a drawback is the three-dimensional geometry of the microphone arrays. In this paper, a method to design two-dimensional planar microphone arrays that are capable of capturing three-dimensional (3D) spatial soundfields is proposed. Through the utilization of both omni-directional and first order microphones, the proposed microphone array is capable of measuring soundfield components that are undetectable to conventional planar omni-directional microphone arrays, thus providing the same functionality as 3D arrays designed for the same purpose. Simulations show that the accuracy of the planar microphone array is comparable to traditional spherical microphone arrays. Due to its compact shape, the proposed microphone array greatly increases the feasibility of 3D soundfield analysis techniques in real-world applications.

A Kinetic Study of DDGS Hemicellulose Acid Hydrolysis and NMR Characterization of DDGS Hydrolysate.

Liquid hot water (LHW) extraction was used as a pretreatment method to separate the hemicellulose fraction from dried distiller's grain with solubles (DDGS) into liquid phase. Acid hydrolysis using 3.264 % w/w sulfuric acid at 130 °C was performed to convert polysaccharides in LHW extract to monosaccharides. The structure characterization of DDGS in anomeric carbon region based on proton NMR and heteronuclear single quantum coherence (HSQC) during acid hydrolysis was studied in this work. It reveals that the sugar units in DDGS hemicelluloses are constructed with (1-4)-ß-D-xylopyranose and α-L-arabinofuranosyl residues. A kinetic model is included to explain the changing concentration of monomer, oligomer, and sugar units. The model was further tested based on the changing concentration of five carbon sugar units during hydrolysis.