Solutions of a pure organic solvent acidified with 1% sulfuric acid, and some of their aqueous mixtures were used for lignin extraction in the Pinus radiata sawdust delignification. Organic acid solvents including acetic, lactic and citric acids as well as non-acidic compounds such as γ-valerolactone, 2-methyltetrahydrofuran, glycerol and ethylene glycol were studied. Crude lignin extractions yields (%) ranging between ca. 5-50% were obtained, from which ethylene glycol (33%), γ-valerolactone (48%) and propylene carbonate (52%) showed the greatest effectiveness. The effect of added water on the lignin extraction was investigated in mixtures of an organic solvent with a variable water content (75%w/w, 50% w/w, 25%w/w and 10% w/w) where it was observed that the yield of extraction decreased with the increased water content. Moreover, the purity of extracted lignins were analyzed by spectroscopic methods (UV and IR). Kamlet-Taft solvent polarity parameters, were determined with the solvatochromic probes 4-nitroaniline, N,N-diethyl-4-nitroaniline, Nile Red and 6-propionyl-2-N,N-dimethylaminonaphthalene (PRODAN), and then correlated to lignin extraction yields to explain the influence of the solute-solvent interactions on biomass delignification. A reasonable correlation was found between the medium polarizability-dipolarity π* and the effectiveness of the solvent mixture on the extraction of lignin wood.

The wide-spread use of laccases in industry is often limited due to the enzyme inactivation over time at conditions which exceeds the operating conditions of the enzymes, which are neutral pH and ambient temperatures (30-40 °C). Natural Deep Eutectic Solvents (NADESs) have attracted considerable attention as reaction media in biocatalysis due to their promising compatibility with enzymes and sustainable derivation. In this contribution we demonstrate the possibility of applying aqueous NADESs as incubation media to alter the activity and inhibit thermal inactivation of laccase T. versicolor. For example we show that by incubating 0.25 g L-1 laccase in an aqueous 25 wt% betaine-xylitol based NADES at 70 °C for 15 min, the measured residual activity of laccase is a near 10 fold greater than the measured residual activity of laccase when incubated without the NADES. Moreover, the comparison of the residual activities of the enzyme in presence betaine, xylitol or NADES is clearly showing the advantage of using a NADES over its individual components. The drastic enhancement of the enzyme thermostability by pre-incubation of laccase in NADES media showcases a facile, cheap and green method of boosting the stability laccase.

Ulva intestinalis, a tubular green seaweed, is a rich source of nutrient, especially sulphated polysaccharides. Sulphated polysaccharides from U. intestinalis were extracted with distilled water, 0.1N HCl, and 0.1N NaOH at 80°C for 1, 3, 6, 12, and 24h to study the effect of the extraction solvent and time on their chemical composition and antioxidant activity. Different types of solvents and extraction time had a significant influence on the chemical characteristics and antioxidant activity (p<0.05). Monosaccharide composition and FT-IR spectra analyses revealed that sulphated polysaccharides from all solvent extractions have a typical sugar backbone (glucose, rhamnose, and sulphate attached at C-2 or C-3 of rhamnose). Sulphated polysaccharides extracted with acid exhibited greater antioxidant activity than did those extracted with distilled water and alkali. The results indicated that solvent extraction could be an efficacious method for enhancing antioxidant activity by distinct molecular weight and chemical characteristic of sulphated polysaccharides.

Natural deep eutectic solvents (NADES) play a role as alternative media to water in living organisms. They are formed by mixing two or more natural compounds in certain ratios producing a liquid having a lower melting point than those of the individual NADES components. Although, the eutectics medium bring several advantages as enhanced solubility of non-polar substrates and/or products, however, these advantages would often be limited by a lower stability of biocatalysts in these systems. To examine this matter, biochemical characterization, thermal stability and tertiary structure of laccase from Bacillus HR03 was investigated as a model in betaine and choline based NADES. In eutectics containing choline, a sudden drop in enzyme activity and stability was observed. Betaine based eutectics exhibited a better media for the laccase stability in comparison with the aqueous buffer and choline chloride eutectics. The enzyme highest activity was observed in 20% (v/v) glycerol:betaine (2:1). Among betaine based eutectics, the enzyme exhibited its highest stability in sorbitol:betaine:water (1:1:1) and glycerol:betaine (2:1) compared to the aqueous buffer at 80 and 90°C. Associated conformational changes caused by solvents were monitored using fluorescence technique. Finally, the effects of NADES on the enzyme activity and stability were discussed.

Lignin is the most abundant heterogeneous aromatic polymer on earth to produce a large number of value-added chemicals. Besides, the separation of lignin from the lignocellulosic biomass is essential for cellulosic biofuel production. For the first time, we report a cosolvent-based approach to understand the dissolution of lignin with 61 guaiacyl subunits at the molecular level. Atomistic molecular dynamics simulations of the lignin were performed in 0%, 20%, 50%, 80%, and 100% 1-Ethyl-3-Methylimidazolium Acetate (EmimOAc) systems. The lignin structure was significantly destabilized in both 50%, and 80% EmimOAc cosolvents, and pure EmimOAc systems leading to the breakdown of intrachain hydrogen bonds. Lignin-OAc and lignin-water hydrogen bonds were formed with increasing EmimOAc concentration, signifying the dissolution process. The OAc anions mostly solvated the alkyl chains and hydroxy groups of lignin. Besides, the imidazolium head of Emim cations contributed to solvation of methoxy groups and hydroxy groups, whereas ethyl tail interacted with the benzene ring of guaiacyl subunits. Effective dissolution was obtained in both the 50% and 80% EmimOAc cosolvent systems. Overall, our study presents a molecular view of the lignin dissolution focusing on the role of both cation and anion, which will help to design efficient cosolvent-based methods for lignin dissolution.

Xylosidases with tolerance to high concentration of salts, organic solvents, and enzyme hydrolytic products are preferential for industrial application but were rarely reported. In this study, a novel xylosidase XYL21 belong to glycoside hydrolase 39 was characterized with optimal temperature of 45 °C and optimal pH of 5.50. Different to other GH39 xylosidases, XYL21 had excellent tolerance to salts, the activity of which is not inhibited but slightly increased in 0.50-1.50 M NaCl. It is also tolerant to organic solvents, especially retaining 105.18% relative activity even in the presence of 15.00% (v/v) ethanol. Moreover, XYL21 was insensitive to the final lignocellulose hydrolysis products including glucose, xylose, arabinose, mannose and galactose, which retains 111.36% and 53.49% relative activity in 0.30 and 0.90 M xylose, respectively. Further structural modeling analysis indicated that its excellent tolerance may be attributed to its high structural flexibility caused by the high proportion of random coils. Furthermore, XYL21 had a wide substrate specificity to catalyze xylan and xylo-oligosaccharides, and it significantly cooperated with xylanase to improve the hydrolysis efficiency with 1.52-fold. Considering these unique properties, XYL21 is a good candidate for both basic research and various potential industrial applications such as seafood processing and bioethanol production.

Lipase is a versatile enzyme found in microorganisms, animals and plants. It has applications in a wide variety of fields ranging from the food industry to the pharmaceutical. For these applications, mainly microbial lipases are exploited in great detail. On the other hand lipases from the plant source have been characterized to a much lesser extent. Although many plant lipase sequences have been reported in UniProtKB, till date there is no report on the crystal structure of any plant lipase. In view of very limited availability of structural information on plant lipases, in this study, we modeled the three-dimensional structure of seven plant lipases and studied the conformational changes under four different solvents at two different temperatures. Most lipases have a lid domain and its movement is implicated in the interfacial activation of lipases. Among the 56 conditions tested in this study, some lipases at certain condition exhibit the lid domain movement thus implying the functional importance. Laborious purification and minimal yield are the likely reasons for poor characterization of plant lipases. In this scenario, the results of computational studies on plant lipases under different environmental conditions will provide useful data for subsequent in vitro functional studies.

Nanoencapsulation of saffron extract (SE) components into the rapeseed lecithin nanoliposomes were performed by sonication of their aqueous dispersions as a green process. Dynamic light scattering (DLS) results exhibited that empty and SE loaded nanoliposomes (SENL) had average sizes in range of 118-138 nm, negative zeta potentials (-32.0 to -46.8 mV) and polydispersity index (PDI) less than 0.3 during storage for 28 days at 4 °C. Encapsulation efficiency of crocin was approximately 30%. The 70% of crocin released from SENLs within 5 h in PBS solution. Pullulan-based films were fabricated by incorporation of empty and SE loaded nanoliposomes into pullulan solution through casting method. The mechanical resistance and thermal stability of the films reduced by addition of nanoliposomes. FTIR and thermal characterizations indicated that SE was successfully encapsulated in the nanoliposomes and film matrix with high thermal stability. Incorporation of nanoliposomes enhanced the oxygen barrier properties of the films, while it didn't significantly affect the water vapor permeability (WVP) of the films. The obtained edible films or coatings can provide additional benefits due to unique flavor and color of saffron. In addition, the utilization of SE, can provide benefits for health-allegation from SE antioxidant capacity.

Herein, we develop a novel smart cotton swab as a diagnostic assay for onsite monitoring of sweat pH changes toward potential applications in monitoring human healthcare and drug exam. Anthocyanin (Ac) can be extracted from Brassica oleracea var. capitata f. rubra using a simple procedure. Then, it can be used as a direct dye into cotton fibers using potash alum as mordant (M) to fix the anthocyanin dye onto the surface of the cotton fabric (Cot). This was monitored by generating mordant/anthocyanin nanoparticles (MAcNPs) onto the fabric surface. The cotton sensor assay demonstrated colorimetric changes in the ultraviolet-visible absorbance spectral analysis associated with a blueshift from 588 to 422 nm with increasing the pH of a perspiration simulant fluid. The biochromic performance of the dyed cotton diagnostic assay depended essentially on the halochromic activity of the anthocyanin spectroscopic probe to demonstrate a color change from pink to green due to intramolecular charge transfer occurring on the anthocyanin chromophore. After dyeing, no significant defects were detected in air-permeability and bend length. High colorfastness was investigated for the dyed cotton fabrics.

A protein precipitation technique was optimized to produce biophysically stable 'protein microbeads', applicable to highly concentrated protein formulation. Initially, production of BSA microbeads was performed using rapid dehydration by vortexing in organic solvents followed by cold ethanol treatment and a vacuum drying. Out of four solvents, n-octanol produced the most reversible microbeads upon reconstitution. A Shirasu porous glass (SPG) membrane emulsification technique was utilized to enhance the size distribution and manufacturing process of the protein microbeads with a marketized human IgG solution. Process variants such as dehydration time, temperature, excipients, drying conditions, and initial protein concentration were evaluated in terms of the quality of IgG microbeads and their reversibility. The hydrophobized SPG membrane produced a narrow size distribution of the microbeads, which were further enhanced by shorter dehydration time, low temperature, minimized the residual solvents, lower initial protein concentration, and addition of trehalose to the IgG solution. Final reversibility of the IgG microbeads with trehalose was over 99% at both low and high protein concentrations. Moreover, the formulation was highly stable under repeated mechanical shocks and at an elevated temperature compared to its liquid state. Its in vivo pharmacokinetic profiles in rats were consistent before and after the 'microbeadification'.

Topical applications that reduce systemic toxic effects while increasing therapeutic efficacy are a promising alternative strategy. The aim of this study was to provide an enhanced transdermal delivery of low molecular weight heparin (LMWH) through the stratum corneum by using cationic carrier as a novel permeation enhancer. Recent studies have shown that heparin-conjugated biomaterials can be effective in inhibiting tumor growth during cancer treatment due to their high ability to bind growth factors. Paclitaxel (PCL) was co-encapsulated into the same cationic carrier for the purpose of improving of therapeutic efficacy for a combined cancer treatment with LMWH. In vitro and in vivo studies showed that the LMWH and PCL release was significantly affected by polymer molecular weight and block composition. Skin penetration tests have indicated that larger amounts of LMWH were absorbed from LMWH-gel conjugate through SC, than aqueous formula. However, it was found that the plasma transition of LMWH released from gel conjugate was lower compared to the plasma concentration of LMWH released from aqueous solution. It is recommended that PCL-loaded LMWH-conjugated core/shell hydrogels can be used as promising drug release systems for transdermal applications that can improve therapeutic efficacy and reduce side effects in a combined cancer treatment.

Esterases are widely applied in industrial processes due to their versatility, regio- and enantioselectivity, lack of cofactors and stability in organic solvents. Bacillus licheniformis, a microorganism frequently used in industrial and biotechnological applications such as dairy, baking, beverage, pulp and paper, detergent and cosmetics production, organic synthesis and waste management, is a promising source of esterases. Here we describe the biochemical and biophysical characterization of B. licheniformis carboxylesterase BlEst1 and its SAXS-derived molecular envelope. BlEst1 has optimal hydrolytic activity against p‑nitrophenyl acetate at pH 7.0 and 40 °C. Furthermore, BlEst1 is stable in different organic solvents such as methanol, isopropanol and butanol. The BlEst1 homology model reveals a typical α/β hydrolase core with an adjacent auxiliary domain, snuggly fitting the experimental low-resolution SAXS molecular envelope. Moreover, BlEst1 maintained considerable part of its activity in the presence of up to 5 M NaCl and its thermal stability was significantly enhanced by the presence of salt, revealing its halotolerant character. The ability to work under harsh conditions makes BlEst1 an interesting candidate for industrial applications.

Alginate and its derivatives are annually produced approximately 30,000 tons or more and are applied to various industries as they are natural polymers. The global market for alginate and its derivatives has been growing steadily. There is little research compared to other enzymes produced through biomass degradation or modification. An alginate lyase, MtAl138, from Microbulbifer thermotolerans DAU221 was cloned and identified in Escherichia coli BL21 (DE3). MtAl138 contains a highly conserved motif (R538TELR, Q607IH609, and YFKAGVY716NQ), which indicates that it belongs to the polysaccharide lyase family 7 (PL7). MtAl138, with a molecular weight of 77 kDa worked optimally at 45 °C and pH 7.4. MtAl138 showed twice as much activity as when there was no NaCl when there was between 100 and 600 mM NaCl. Moreover, its activity increased in organic solvents such as benzene, hexane, methanol, and toluene. Based on the thin layer chromatography analyses, MtAl38 is an endo-type enzyme that produces di-, tri-, or tetrasaccharides from polyG and polyM. This study provided that MtAl138 is an endoenzyme that showed outstanding enzymatic activity at concentrated salt solutions and organic solvents, which makes it a reasonably attractive enzyme for use in various industries.

Fennel seeds were recognized as a promising polyphenol oxidase (PPO) source upon investigating some edible green plants (carob, jujube, coriander, fennel, and licorice). The fennel PPO enzyme was purified by three-phase partitioning and biochemically characterized in detail for the first time. The purification fold and activity recovery values were determined as 20-fold and 120%, respectively. Its molecular weight was 27.8 kDa. The temperature for the selected substrates (catechol, 4-tert-butylcatechol, 4-methylcatechol, and pyrogallol) was 30 °C, while the optimum pH value varied from 5.0 to 7.0 depending on the substrate. The kcat/Km values exhibited that the enzyme presented the best activity towards catechol among the substrates used. Sodium metabisulfite, ascorbic acid, benzoic acid, l-cysteine, thiourea, β-mercaptoethanol, and glutathione prominently inhibited PPO activity. A remarkable decrease in PPO activity was observed at elevated concentrations of organic solvents, but in cases of the solvents with polarity indexes ≥5.1, the residual activity maintained more than 75% of its original activity up to 10% (v/v). Consequently, the current study suggested that fennel seeds could be used in various industrial sectors to produce low-cost polyphenol oxidase enzymes with an agricultural origin.

If natural proteases are used in organic synthesis, they are often inactivated or give a low rate of reaction in non-aqueous or aqueous-organic media. Therefore, to reveal the molecular mechanism governing the stability of proteases in organic solvents and increase protease stability in those systems is of intriguing interest. In the present study, the activity and conformational changes of an organic solvent-stable protease (OSP) from Bacillus sphaericus DS11 in different concentrations of methanol were investigated by measuring fluorescence, UV-Vis spectra, circular dichroism (CD), and conducting molecular dynamics (MD) simulations. The OSP expanded with increasing methanol concentration. The methanol molecules were able to enter into the OSP, leading to microenvironmental changes around the aromatic amino acids. More hydrophobic groups were exposed to the solvents at high methanol concentrations, and the original hydrophobic interaction in the protein decreased, thus resulting in the secondary and tertiary structure change in the OSP. Our results provide helpful insight into the molecular mechanism of the OSP tolerance to organic solvent and indicate directions for future work to design and engineer proteases that are stable at high organic solvent concentrations.

An identified cold-adaptive, organic solvents-tolerant alkaline α-amylase (HP664) from Catenovulum sp. strain X3 was heterologously expressed and characterized in E. coli, and it was further applied to starch saccharification for biohydrogen production. The recombinant HP664 belongs to a member of glycoside hydrolase family 13 (GH13), with a molecular weight of 69.6kDa without signal peptides, and also shares a relatively low similarity (49%) to other reported amylases. Biochemical characterization demonstrated that the maximal enzymatic activity of HP664 was observed at 35°C and pH 9.0. Most metal ions inhibited its activity; however, low polar organic solvents (e.g., benzene and n-hexane) could enhance the activity by 35-50%. Additionally, HP664 also exhibited the catalytic capability on various polysaccharides, including potato starch, amylopectin, dextrin and agar. In order to increase the bioavailability of starch for H2 production, HP664 was utilized to elevate fermentable oligosaccharide level, and the results revealed that the maximal hydrolytic percentage of starch was up to 44% with 12h of hydrolysis using 5.63U of HP664. Biohydrogen fermentation of the starch hydrolysate by Clostridium sp. strain G1 yielded 297.7mL of H2 after 84h of fermentation, which is 3.73-fold higher than the control without enzymatic treatment of HP664.

A new core-shell pH-responsive nanocarrier was prepared based on magnetic nanoparticle (MNP) core. Magnetic nanoparticles were first modified with hyperbranched polyglycerol as the first shell. Then the magnetic core was decorated with doxorubicin anticancer drug (DOX) and covered with PEGylated carboxymethylcellulose as the second shell. Borax was used to partially cross-link organic shells in order to evaluate drug loading content and pH-sensitivity. The structure of nanocarrier, organic shell loadings, magnetic responsibility, morphology, size, dispersibility, and drug loading content were investigated by IR, NMR, TG, VSM, XRD, DLS, HR-TEM and UV-Vis analyses. In vitro release investigations demonstrated that the use of borax as cross-linker between organic shells make the nanocarrier highly sensitive to pH so that more that 70% of DOX is released in acidic pH. A reverse pH-sensitivity was observed for the nanocarrier without borax cross-linker. The MTT assay determined that the nanocarrier exhibited excellent biocompatibility toward normal cells (HEK-293) and high toxicity against cancerous cells (HeLa). The nanocarrier also showed high hemocompatibility. Cellular uptake revealed high ability of nanocarrier toward HeLa cells comparable with free DOX. The results also suggested that low concentration of nanocarrier has a great potential for use as contrast agent in magnetic resonance imaging (MRI).

The lipase gene from Psychrobacter celer PU3 was cloned into pET-28a(+) expression vector and overexpressed in E. coli BL21 (DE3) pLysS cells. The purified Psychrobacter celer lipase (PCL) was characterized as an alkaline active enzyme and has a molecular mass of around 30 kDa. The PCL was active even at a low temperature and the optimum range was observed between 10 and 40 °C temperatures. MALDI-TOF and phylogenetic analysis ensured that Psychrobacter celer PU3 lipase (PCL) was closely related to P. aureginosa lipase (PAL). MD simulation results suggest that temperature change did not affect the overall structure of PCL, but it might altered the temperature-dependent PCL functional changes. R1 (129-135 AA) and R2 (187-191 AA) regions could be important for temperature-dependent PCL function and they fluctuated much at 35 °C temperature. PMSF completely inhibited PCL lipase activity and it demonstrates the presence of serine residues in the active site of PCL. PCL is moderately halophilic and most of the tested organic solvents found to be inhibiting the lipase activity except the solvents ethanol and methanol. PCL activity was increased with surfactants (SDS and CTAB) and bleaching agents (hydrogen peroxide). The effect of different metal ions on PCL resulted that only mercuric chloride was found as the enhancer of the lipase activity. Antibiofilm property of PCL was evaluated against pathogenic Vibrio parahaemolyticus isolated from the diseased shrimp and MIC value was 500 U. PCL significantly altered the morphology and biofilm density of V. parahaemolyticus and the same was observed through scanning electron microscope (SEM) and confocal laser scanning microscope (CLSM) imaging. RT-PCR analysis revealed that the mRNA expression level of biofilm, colony morphology and major toxin-related (aphA, luxS, opaR, tolC, toxR) genes of V. parahaemolyticus were significantly downregulated with PCL treatment.

Investigating the drug-AChE binding mechanism is vital in understanding its cogent use in medical practice against Alzheimer's disease (AD). The production and accumulation of oligomers of β-amyloid is a central event in the neuropathology of AD. Beside the inhibition of assembly process, modulation of the aggregation process of these proteins towards minimally toxic pathways may be a possible therapeutic strategy for AD. Hence, the present study aims to examine the effect of multifunctional fused tricyclic 7-hydroxy 4-methyl coumarin analogs (HMC1-5) on the self-induced aggregation of β-amyloid using Thioflavin T (ThT) assay, scanning electron microscopic study, AlamarBlue and immune blotting assays and also the binding mechanism with AChE by fluorescence emission, conformational, molecular docking and molecular dynamic simulation studies under physiological pH 7.4. The ThT assay, FE-SEM study, cell line and western blots establish that the HMC1-5 molecules could irreversibly disrupt preformed Aβ42 fibrils, accelerate the aggregates into micro size co-assembled structures, and effectively eliminate the cytotoxicity of Aβ1-42. Fluorescence emission studies indicating a strong binding affinity between HMC1-5 and AChE with the binding constants of 1.04 × 105, 3.57 × 104, 1.97 × 104, 3.07 × 104 and 2.95 × 104 M-1, respectively and binding sites number found to be 1. CD studies disclosed a partial unfolding in the secondary structure of AChE upon binding with HMC1-5. Docking analysis inferred that the HMC1-5 were bound through hydrophobic and hydrophilic interactions to the AChE active site. Molecular dynamics simulations emphasized the stability of AChE-HMC1-5 complexes throughout the 100 ns simulations, and the local conformational changes of the residues of AChE validate the stability of complexes. These results provide new and unique complementary approach for modulating the biological effects of the Aβ aggregates by coumarin analogs and new insights for further in vivo investigations as novel anti AD agents.

A new bacterial lipolytic enzyme Est903 was obtained from paper mill sludge via metagenomic approach. Est903 displayed moderate similarities to two lipolytic enzymes from Rhodopirellula islandica and contained a distinctive pentapeptide motif (GFSAG) that differed from those of all known fourteen families of bacterial lipolytic enzymes. Est903 was regarded as from a new bacterial lipolytic enzyme family through multiple sequence alignment and phylogenetic analysis. The recombinant Est903 showed the highest activity for ρ-nitrophenol butyrate. The pH optimum and temperature optimum of the recombinant enzyme was 9.0 and 51 °C, respectively. Also, this enzyme displayed moderate thermostability, high activity under alkaline conditions, and good tolerance against several organic solvents. In addition, the compatibility test and washing performance analysis revealed that Est903 had good compatibility with commercial laundry detergent and high cleaning ability of oil stains. These good properties make Est903 a potential candidate in organic synthesis or detergent industry.