Aqueous Two-phase Systems Research Articles

Aqueous two‐phase systems as a novel alternative in the primary recovery of human mesenchymal cells obtained from adipose tissue

AbstractIn recent years, therapies based on regenerative medicine have gained wide interest owing to their potential in organ transplantation and in the treatment of multiple diseases. These cell‐based therapies rely mainly on the therapeutic potential of stem cells as a result of their self‐renewal, differentiation, capacities and immunosuppressive properties. Adipose tissue contains a large number of mesenchymal stem cells compared to other tissues and is easily accessible through noninvasive techniques; hence the primary recovery of adipose‐derived mesenchymal stem cells (ASC) has become a critical step. Aqueous two‐phase systems (ATPS) have shown promise as a separation technique for mesenchymal stem cells (MSC) as a result of their scalability, cost‐effectiveness and compatibility. In this study, we investigated the partitioning behavior of the stromal vascular fraction cells, including ASC, in different ATPS compositions [polyethylene glycol, dextran (DEX), Ficoll and Ucon). Among the three ATPS tested, most of the ASC consistently migrates to the top phase as indicated by immunostaining studies for CD73, CD90 and CD105 stem cell markers. Nevertheless, the Ficoll–DEX system retrieves the greatest quantity of ASC recovered in the top phase (89% of total ASC), whereas the Ucon–DEX system effectively eliminates the most contaminating cells (38%). Our findings provide insights into the conditions under which ASC and contaminant cells concentrate in opposite phases in different ATPS, which can contribute to the development of efficient and scalable separation systems for MSC in regenerative medicine and tissue engineering applications. © 2024 Society of Chemical Industry (SCI).

Efficient Separation of Re (VII) and Mo (VI) by Extraction Using E-1006–Ammonium Sulfate Aqueous Two-Phase System

Aqueous two-phase extraction (APTE) stands out as an environmentally friendly technique for the separation of metal ions. The separation of Re (VII) and Mo (VI) in an aqueous solution was investigated using a novel aqueous two-phase system (ATPS) consisting of isodecanol polyoxyethylene ether (E-1006), ammonium sulfate, and water. A phase diagram of this system was developed, and the effects of pH, temperature, extraction time, the concentrations of E-1006 and (NH4)2SO4, and metal ions on the separation of Re (VII) and Mo (VI) were examined. The results show that at pH 7.0, Mo (VI) had almost transformed into the (NH4)2SO4-rich phase, while Re (VI) was extracted into the E-1006-rich phase. The increase in temperature induces a transition of Mo (VI) to the salt-rich phase, which is unfavorable for the extraction of Re (VII). The increase in the concentrations of E-1006 and (NH4)2SO4 has a positive effect on the separation of rhenium and molybdenum. Overall, the ATPS consisting of 200 g/L of E-1006, 200 g/L of (NH4)2SO4, and water yields an extraction efficiency of 97.2% for Re and a high separation factor of 2700 for Re (VII) and Mo (VI) from a mixture of 0.1 g/L of Re (VII) and 5 g/L of Mo (VI) at pH 7.0 and 323.15 K. Separation studies of the simulated leaching solution show that the extraction efficiency for Re (VI) is 99.1% and the separation factor of Re (VII) and Mo (VI) is 5100.

Effect of Salt Mixtures on Aqueous Two-Phase System Composed of PEG 35000: Experiment and Correlation

Effect of Salt Mixtures on Aqueous Two-Phase System Composed of PEG 35000: Experiment and Correlation

Ultrasonication followed by aqueous two-phase system for extraction, on-site modification and isolation of microalgal starch with reduced digestibility

Microalgae are new and sustainable sources of starch with higher productivity and flexible production modes than conventional terrestrial crops, but the downstream processes need further development. Here, ultrasonication (with power of 200 W or 300 W and duration of 10, 15, 20, or 25 min) was applied to simultaneously extract and modify starch from a marine microalga Tetraselmis subcordiformis for reducing the digestibility, and an aqueous two-phase system (ATPS) of ethanol/NaH2PO4 was then used to isolate the starches with varied properties. Increasing ultrasonic duration facilitated the partition of starch into the bottom pellet, while enhancing the ultrasonic power was conducive to the allocation in the interphase of the ATPS. The overall starch recovery yield reached 73 ∼ 87 % and showed no significant difference among the ultrasonic conditions tested. The sequential ultrasonication-ATPS process successfully enriched the starch with purities up to 65 % ∼ 88 %, which was among the top levels reported in microalgal starch isolated. Ultrasonication produced more amylose which was mainly fractionated into the interface of the ATPS. The digestibility of the starch was altered under different ultrasonic conditions and varied from different ATPS phases as well, with the one under the ultrasonic power of 200 W for 15 min at the bottom pellet having the highest resistant starch content (RS, 39.7 %). The structural and compositional analysis evidenced that the ultrasonication-ATPS process could exert impacts on the digestibility through altering the surface roughness and fissures of the starch granules, modulating the impurity compositions (protein and lipid) that could interact with starch, and modifying the long- and short-range ordered structures. The developed ultrasonication-ATPS process provided novel insights into the mechanism and strategy for efficient production of functional starch from microalgae with a potential in industrial application.

Partitioning recombinant chitinase from Nicotiana benthamiana by an aqueous two-phase system based on polyethylene glycol and phosphate salts

The objectives of this study were to purify 42 kDa chitinase derived from Trichoderma asperellum SH16 produced in Nicotiana benthamiana by a polyethylene glycol (PEG)/salt aqueous two-phase system (ATPS). The specific activities of the crude chitinase and the partially purified chitinase from N. benthamiana were about 251 unit/mg and 386 unit/mg, respectively. The study found the 300 g/L PEG 6000 + 200 g/L potassium phosphate (PP) and 300 g/L PEG 6000 + 150 g/L sodium phosphate (SP) systems had the highest partitioning efficiency for each salt in primary extraction. However, among the two types of salt, PP displayed higher efficiency than SP, with a partitioning coefficient K of 4.85 vs. 3.89, a volume ratio V of 2.94 vs. 2.68, and a partitioning yield Y of approximately 95 % vs. 83 %. After back extraction, the enzymatic activity of purified chitinase was up to 834 unit/mg (PP) and 492 unit/mg (SP). The purification factors reached 3.32 (PP) and 1.96 (SP), with recovery yields of about 59 % and 61 %, respectively. SDS-PAGE and zymogram analysis showed that the recombinant chitinase was significantly purified by using ATPS. The purified enzyme exhibited high chitinolytic activity, with the hydrolysis zone's diameter being around 2.5 cm–3 cm. It also dramatically reduced the growth of Sclerotium rolfsii; the colony diameter after treatment with 60 unit of enzyme for 104 spores was only about 1 cm, compared to 3.5 cm in the control. The antifungal effect of chitinase suggests that this enzyme has great potential for applications in agricultural production as well as postharvest fruit and vegetable preservation.

Reconfigurable aqueous 3D printing with adaptive dual locks.

Using aqueous two-phase systems (ATPSs) for three-dimensional (3D) printed complex structures has attracted considerable attention in the field of biomedicine. In this study, we present an unusual approach to constructing reconfigurable 3D printed structures within an aqueous environment. Inspired by biological systems, we introduce both specific and nonspecific interactions to anchor functionalized nanoparticles to the water-water interface, thereby imparting adaptive dual locks of structural integrity and permeability to the 3D printed liquid structures. Using state-of-the-art in situ liquid-liquid interfacial atomic force microscopy imaging, we successfully demonstrate various morphologies of interfacial films formed at the ATPS interface. In addition, by incorporating d-glucose or sodium alginate into the systems, the dual locks can be easily manipulated. Our study paves a pathway for 3D printing multiresponsive all-aqueous systems with controllable structures and permeability, showing promising implications for the development of smart drug delivery systems and in vivo reactions.

Separation of polyphenolic compounds from Citrus aurantium L. peel by deep eutectic solvents and their recovery using a new DES-based aqueous two-phase system

This study aims to investigate the extraction of polyphenolic compounds (PCs) from Citrus aurantium L. peel using Deep Eutectic Solvents (DESs) and ultrasound-assisted extraction (UAE). A screening process evaluated various DESs based on Choline Chloride (ChCl) with polyol, acids, and amides. ChCl-1,4-butanediol performs highest among the tested DESs and the benchmark solvent (70 % ethanol and water). The extraction efficiency, measured by total phenolic content (TPC) using the Folin − Ciocalteu method, was influenced by temperature, time, liquid-to-solid ratio, and water content. Optimal conditions were found at a temperature of 40 °C, extraction time of 30 min, water content of 49.95 %, and 18.97:1 solvent to biomass ratio with a yield of 7.85 mg gallic acid equivalent per gram of peel.Under these conditions, we employed an Aqueous Two-Phase System (ATPS) consisting of salt and DES to recover polyphenols from DES extracts. Various salts (K2HPO4, K3PO4, Na2SO4, LiCL, BaCl2, Na3cit, and NaCl) were assessed for their potential to form the ATPS. Among them, only K2HPO4, and K3PO4 demonstrated the ability to induce phase separation. The effect of the salt concentration was carefully investigated on the partitioning of the PCs. K3PO4 demonstrated a higher performance compared to K2HPO4 with an extraction efficiency of 88.54 %. An ATPS containing DES (0.67 % w/w) and K3PO4 (0.32 % w/w) proved advantageous for back-extraction. Water-immiscible organic solvents, including n-hexane, ethyl acetate, n-butanol, and isoamyl alcohol, were assessed as back-extraction agents. Notably, n-butanol achieved an impressive polyphenol recovery efficiency of 98.11 %.

Purification of exopolysaccharide produced from Lactobacillus spp. using ionic-liquid as adjuvant in alcohol/salt-based aqueous two-phase system for its antidiabetic properties

Diabetes is a chronic, metabolic disease characterized by hyperglycemia resulting from either insufficient insulin production or impaired cellular response to insulin. Exopolysaccharides (EPS) produced by Lactobacillus spp. demonstrated promising therapeutic potential in terms of their anti-diabetic properties. Extraction and purification of EPS produced by Lactobacillus acidophilus and Limosilactobacillus reuteri were performed using ethanol precipitation, followed by alcohol/salt based aqueous two-phase system (ATPS). The purification process involved ethanol precipitation followed by an alcohol/salt-based ATPS. The study systematically investigated various purification parameters in ATPS, including ethanol concentration, type and concentration of ionic liquid, type and concentration of salt and pH of salt. Purified EPS contents from L. acidophilus (63.30 μg/mL) and L. reuteri (146.48 μg/mL) were obtained under optimum conditions of ATPS which consisted of 30 % (w/w) ethanol, 25 % (w/w) dipotassium hydrogen phosphate at pH 10 and 2 % (w/w) 1-butyl-3-methylimidazolium octyl sulfate. The extracted EPS content was determined using phenol sulphuric acid method. In α-amylase inhibition tests, the inhibitory rate was found to be 92.52 % (L. reuteri) and 90.64 % (L. acidophilus), while in α-glucosidase inhibition tests, the inhibitory rate was 73.58 % (L. reuteri) and 68.77 % (L. acidophilus), based on the optimized parameters selected in ATPS. These results suggest that the purified EPS derived from the postbiotics of Lactobacillus spp. hold promise as potential antidiabetic agents.

Aqueous two-phase system based on hexafluoroisopropanol and acetonitrile for homogeneous liquid-liquid microextraction of cationic dyes


 
 
 
 This work demonstrates that an aqueous two-phase system (ATPS) is formed when a small amount of acetonitrile (ACN) is added to the aqueous hexafluoroisopropanol (HFIP) solution. The effect of ACN amount on the volume of the formed HFIP/ACN phase was investigated. It was also shown that relatively hydrophilic methylene blue was completely extracted into the HFIP/ACN phase whereas its extractability with conventional solvents (hexane, CH2Cl2 and CHCl3) was significantly lower. The obtained results suggest that the HFIP/ACN phase exhibits a relatively high polarity and should be a good choice for the extraction of moderately or even highly polar compounds from aqueous samples. Finally, the developed ATPS was applied for the homogeneous liquidliquid microextraction of four cationic dyes from river water samples prior to HPLC analysis. Under optimised extraction conditions, the enrichment factors were around 150. Calibration curves were linear (R2 ≥ 0.9959) for the concentration level between 0.2–0.5 and 50.0 μg/L and the detection limits were in the range 0.05–0.18 μg/L. The recoveries of the dyes for the spiked water samples were 88.6–98.5%, with the relative standard deviation values less than 9.6%.
 
 
 

The Landscape of Exosomes Biogenesis to Clinical Applications.

Exosomes are extracellular vesicles that originate from various cells and mediate intercellular communication, altering the behavior or fate of recipient cells. They carry diverse macromolecules, such as lipids, proteins, carbohydrates, and nucleic acids. Environmental stressors can change the exosomal contents of many cells, making them useful for diagnosing many chronic disorders, especially neurodegenerative, cardiovascular, cancerous, and diabetic diseases. Moreover, exosomes can be engineered as therapeutic agents to modulate disease processes. State-of-art techniques are employed to separate exosomes including ultracentrifugation, size-exclusion chromatography and immunoaffinity. However, modern technologies such as aqueous two-phase system as well as microfluidics are gaining attention in the recent years. The article highlighted the composition, biogenesis, and implications of exosomes, as well as the standard and novel methods for isolating them and applying them as biomarkers and therapeutic cargo carriers.

Green and efficient method to acquire high-value phycobiliprotein from microalgal biomass involving deep eutectic solvent-based ultrasound-assisted extraction

Phycoerythrin (PE) is a phycobiliprotein holding great potential as a high-value food colorant and medicine. Deep eutectic solvent (DES)-based ultrasound-assisted extraction (UAE) was applied to extract B-PE by disrupting the resistant polysaccharide cell wall of Porphyridium purpureum. The solubility of cell wall monomers in 31 DESs was predicted using COSMO-RS. Five glycerol-based DESs were tested for extraction, all of which showed significantly higher B-PE yields by up to 13.5 folds than water. The DES-dependent B-PE extraction efficiencies were proposedly associated with different cell disrupting capabilities and protein stabilizing effects of DESs. The DES-based UAE method could be considered green according to a metric assessment tool, AGREEprep. The crude extract containing DES was further subjected to aqueous two-phase system, two-step ammonium sulfate precipitation, and ultrafiltration processes. The final purified B-PE had a PE purity ratio of 3.60 and a PC purity ratio of 0.08, comparable to the purity of commercial products.

Determination of indoxyl sulfate by spectrofluorimetric method in human plasma through extraction with deep eutectic solvent

A rapid and efficient analytical method was established to quantify indoxyl sulfate (IS) in plasma through extraction technique with a deep eutectic solvent (DES) and spectrofluorimetric method. DES (choline chloride: urea) was mixed with plasma samples for the extraction of IS, followed by the addition of dipotassium hydrogen phosphate (K2HPO4) solution to form an aqueous two-phase system. The fluorescence intensity of IS which was first extracted to the DES-rich-phase and then back-extracted into the salt-rich-phase, was measured by spectrofluorimetric method. Some key factors such as pH, centrifugation speed and time, the volume ratio of DES/salt, and salt concentration were optimized. Under the optimized conditions, the suggested method had a dynamic range between 20 and 160 µg/mL with a coefficient of determination (R2) of 0.99. Precision (relative standard deviation) was less than 15% and accuracy (% relative recovery) was ± 15% at the nominal concentration level. In addition, results showed that IS levels in real samples were higher than 40 µg/mL which was compatible with reported IS levels in end-stage renal disease (ESRD) patients. Overall, all the results reflect the fact that the presented analytical method can potentially be used for the determination of IS in real plasma samples.

In situ ionic liquid-assisted mechanochemical extraction and aqueous two-phase system enrichment of hydrophobic flavonoids from rose

An effective in situ ionic liquid-assisted mechanochemical extraction (MCE) and aqueous two-phase system (ATPS) enrichment method was established. An ATPS can be formed to accomplish the enrichment of the compounds while realizing the MCE for medicinal crop, aiming to establish a pretreatment method for quantitative analysis of active compounds in complex matrices. The effects of ionic liquid amount, milling time, salt amount, vortex time and pH value were investigated, and the optimal conditions for the response surface method were obtained as follows: 1-butyl-3-methylimidazolium bromide amount of 250.37 mg, milling time of 1.99 min and salt amount of 4.38 g. After obtaining the optimal conditions, the method has the correlation coefficients of 0.9963–0.9996, and the limits of detection for the analytes were ranged from 0.05 to 0.08 ng/mL. Besides, the recoveries of rose samples were 84–119% at spiked levels of 0.1 and 1 µg/mL. Compared with other extraction methods, this method synchronously combines MCE and ATPS enrichment techniques, which is environmentally friendly and shortens the extraction time, and can effectively extract hydrophobic flavonoids from roses without any organic solvents.

Extracting and purifying polyphenols from coffee pulp (Coffee arabica L.) using aqueous two‐phase systems

AbstractCoffee pulp, the major by‐product of the beverage industry, is considered as an environmental threat, but also possesses high amounts of bioactive compounds. The extraction of these compounds by using conventional methods and solvents usually accompanies impurities. Here, an aqueous two‐phase system (ATPS) was optimized to extract and purify polyphenols from coffee pulp. Key affecting factors include phase system composition (ethanol and ammonium sulfate), extraction time and temperature while the quality of product was evaluated based on total phenolic content (TPC) and antioxidant activity (AA). The final optimized conditions were at 50 °C for 60 min using the ATPS of ethanol 37.7% (w/w) and ammonium sulfate 25% (w/w). TPC and AA at the optimal conditions were 434.63 ± 25.40 mg GAE/g dw and 622.71 ± 9.65 mg TE/g dw, respectively, which were higher than the results of the extract only used ethanol as the extraction solvent. The results showed that ATPS was a valuable method to extract and purify coffee bioactive compounds.

Engineering heterogeneous hierarchical hydrogels based on Artemisia sphaerocephala Krasch polysaccharide/whey protein isolate fibrils aqueous two-phase emulsion

Emulsion-templating techniques have been used to develop structurally anisotropic hydrogels. This paper proposes a method to couple biopolymer assembly behavior with a phase-separated aqueous two-phase system (ATPS) to construct a hierarchical structure on protein-polysaccharide hydrogels. Based on the phase separation of Artemisia sphaerocephala Krasch polysaccharide (ASKP) and whey protein isolate (WPI) fibrils, a bi-continuous hydrogel was fabricated through spontaneous iron-facilitated molecular chelation. The impact of WPI fibrils under ultrasonic treatment for varying durations on gel structurization and physicochemical properties was also exploited. Shorter length of WPI fibrils facilitated stepwise cross-linking gelation during the oxidation of ferrous ions to ferric ions. Specifically, ultrasonic treatment for 6 min and 8 min promoted the accumulation of shorter fibrils, resulting in the formation of a more intensive clockwise-oriented hierarchical network. Compared to the hydrogels with longer linear aggregates without the ultrasonic treatment, the incorporation of the shorter aggregates resulting from ultrasonic treatment for 6 min and 8 min significantly increased the gel's water holding capacity and mechanical properties. Additionally, the fibrils that underwent ultrasonic treatment for 6 min resulted in a more stable three-dimensional network with optimal elastic performance. This study presents a new water-in-water emulsion-templated strategy for structural regulation and stabilization of multiform hydrogel.

Thermally-resilient, phase-invertible, ultra-stable all-aqueous compartments by pH-modulated protein colloidal particles

The essence of compartmentalization in cells is the inspiration behind the engineering of synthetic counterparts, which has emerged as a significant engineering theme. Here, we report the formation of ultra-stable water-in-water (W/W) emulsion droplets. These W/W droplets demonstrate previously unattained stability across a broad pH spectrum and exhibit resilience at temperatures up to 80℃, overcoming the challenge of insufficient robustness in dispersed droplets of aqueous two-phase systems (ATPS). The exceptional robustness is attributed to the strong anchoring of micelle-like casein colloidal particles at the PEO/DEX interface, which maintains stability under varying environmental conditions. The increased surface hydrophobicity of these particles at high temperatures contributes to the formation of thermally-stable droplets, enduring temperatures as high as 80℃. Furthermore, our study illustrates the adaptable affinity of micelle-like casein colloidal particles towards the PEO/DEX-rich phase, enabling the formation of stable DEX-in-PEO emulsions at lower pH levels, and PEO-in-DEX emulsions as the pH rises above the isoelectric point. The robust nature of these W/W emulsions unlocks new possibilities for exploring various biochemical reactions within synthetic subcellular modules and lays a solid foundation for the development of novel biomimetic materials.

In situ extraction and enrichment of multiple amino acids by integrated mechanochemical extraction and aqueous two-phase system

A highly efficient and environment-friendly extraction technology was established to extract and enrich multiple amino acids (AAs) in situ by deep eutectic solvent (DES)-assisted mechanochemical extraction (MCE) and aqueous two-phase system (ATPS). Firstly, the DES was prepared from betaine and ethylene glycol in a 1:2 M ratio. Then, a series of conditions were evaluated by single-factor and multifactor experiments to select the optimum experimental parameters. The highest extraction yields of AAs were obtained by DES-assisted MCE and ATPS enrichment, including that the amount of DES was 0.97 mL, the milling time was 4.88 min, the amount of salt was 8 g, and the solid-to-liquid ratio was 0.055 g mL−1. After extracting, the diluted AA extracts were analyzed by a high-performance liquid chromatography-fluorescence detector after pre-column derivatization with 6-aminoquinolyl-N-hydroxysccinimidyl carbamate. The methodological validation demonstrated the proposed method exhibited satisfactory limits of detection ranged from 0.11 to 0.82 ng mL−1, reliable interday precision (%RSD) of 0.014–0.067 % for retention time and 1.29–3.34 % for the peak area, acceptable reproducibility with %RSD below 4.1 %, and good %recovery between 90.3 % and 122.5 % at three spiked concentrations. The surface morphology of raw, only ground, and DES-ground sample powders was tested by scanning electron microscopy to further explore the changes in the physicochemical properties of samples. By comparing with other AAs extraction methods, the developed method is not only eco-friendly, but greatly improves the yield of free AAs extracted from chrysanthemum.

Abstract 3624: A low-cost kit for gentle, effective and timely extracellular vesicle (GET EVs) isolation: Accelerating development of RNA-based liquid biopsies for neuroendocrine neoplasms

Abstract Extracellular vesicles (EVs) are cell fragments released by all cells, making them promising platforms for cancer biomarker development. Effective isolation of ultra-pure EVs for downstream analyses is key for EV-based liquid biopsy development. However, current EV isolation methods such as ultracentrifugation (UC) and other commercialized EV isolation kits are lackluster because of damage to EVs, length of time required, and low EV recovery rates. In response, we developed an “GET EV” kit which is based on the principles of lipid energy states, also known as the aqueous two-phase system (ATPS) to isolate EVs from any media. Nanoscale flow cytometry (nFC) analysis revealed that ATPS has greater EV enrichment capability (21.4 × vs. 10.9 × times fold enrichment) and higher EV recovery efficiency (97.6% vs. 69.3% recovery) than UC. Other EV isolation kits were tested and were inferior to ATPS in terms of EV recovery and EV-RNA recovery. EV subpopulations as determined by EV biomarkers (CD9, CD63, CD81, etc.) were confirmed by nFC. “Omics” studies (small RNA sequencing and proteomics) also confirmed the presence of expected EV markers. We developed an RNA-based liquid biopsy for Neuroendocrine Neoplasms (NENs) by quantitating the presence of EVs containing miR-375, an established biomarker for NENs. Using the GET EVs kit, we isolated EVs from healthy volunteer and patient plasma samples of NEN patients. RT-qPCR demonstrated enriched expression of miR-375 in EVs isolated from Lung and GI NEN patient plasma samples. Our study establishes the use of GET EV kits to efficiently, rapidly, and gently isolate EVs from small plasma samples (250 uL). This is a low-cost innovation that will finally enable the development of other RNA-based Liquid Biopsies for other disease sites with significant diagnostic and prognostic implications. Citation Format: Boyang Su, Morteza Jeyhani, Xiaojing Yang, Jina Nanayakkara, Reese Wunsche, Neil Renwich, Scott Tsai, Hon Leong. A low-cost kit for gentle, effective and timely extracellular vesicle (GET EVs) isolation: Accelerating development of RNA-based liquid biopsies for neuroendocrine neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3624.

Recent insight into the advances and prospects of microbial lipases and their potential applications in industry.

Enzymes play a crucial role in various industrial sectors. These biocatalysts not only ensure sustainability and safety but also enhance process efficiency through their unique specificity. Lipases possess versatility as biocatalysts and find utilization in diverse bioconversion reactions. Presently, microbial lipases are gaining significant focus owing to the rapid progress in enzyme technology and their widespread implementation in multiple industrial procedures. This updated review presents new knowledge about various origins of microbial lipases, such as fungi, bacteria, and yeast. It highlights both the traditional and modern purification methods, including precipitation and chromatographic separation, the immunopurification technique, the reversed micellar system, the aqueous two-phase system (ATPS), and aqueous two-phase flotation (ATPF), moreover, delves into the diverse applications of microbial lipases across several industries, such as food, vitamin esters, textile, detergent, biodiesel, and bioremediation. Furthermore, the present research unveils the obstacles encountered in employing lipase, the patterns observed in lipase engineering, and the application of CRISPR/Cas genome editing technology for altering the genes responsible for lipase production. Additionally, the immobilization of microorganisms' lipases onto various carriers also contributes to enhancing the effectiveness and efficiencies of lipases in terms of their catalytic activities. This is achieved by boosting their resilience to heat and ionic conditions (such as inorganic solvents, high-level pH, and temperature). The process also facilitates the ease of recycling them and enables a more concentrated deposition of the enzyme onto the supporting material. Consequently, these characteristics have demonstrated their suitability for application as biocatalysts in diverse industries.

Extraction and separation of astaxanthin with the help of pre-treatment of Haematococcus pluvialis microalgae biomass using aqueous two-phase systems based on deep eutectic solvents

The microalgae Haematococcus pluvialis are the main source of the natural antioxidant astaxanthin. However, the effective extraction of astaxanthin from these microalgae remains a significant challenge due to the rigid, non-hydrolyzable cell walls. Energy savings and high-efficiency cell disruption are essential steps in the recovery of the antioxidant astaxanthin from the cysts of H. pluvialis. In the present study, H. pluvialis microalgae were first cultured in Bold's Basal medium under certain conditions to reach the maximum biomass concentration, and then light shock was applied for astaxanthin accumulation. The cells were initially green and oval, with two flagella. As the induction time increases, the motile cells lose their flagellum and become red cysts with thick cell walls. Pre-treatment of aqueous two-phase systems based on deep eutectic solvents was used to decompose the cell wall. These systems included dipotassium hydrogen phosphate salt, water, and two types of deep eutectic solvents (choline chloride–urea and choline chloride–glucose). The results of pre-treatment of Haematococcus cells by the studied systems showed that intact, healthy cysts were significantly ruptured, disrupted, and facilitated the release of cytoplasmic components, thus facilitating the subsequent separation of astaxanthin by liquid–liquid extraction. The system containing the deep eutectic solvent of choline chloride–urea was the most effective system for cell wall degradation, which resulted in the highest ability to extract astaxanthin. More than 99% of astaxanthin was extracted from Haematococcus under mild conditions (35% deep eutectic solvent, 30% dipotassium hydrogen phosphate at 50 °C, pH = 7.5, followed by liquid–liquid extraction at 25 °C). The present study shows that the pre-treatment of two-phase systems based on deep eutectic solvent and, thus, liquid–liquid extraction is an efficient and environmentally friendly process to improve astaxanthin from the microalgae H. pluvialis.